Avaliação da Ação Antimicrobiana

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AVALIAÇÃO DA AÇÃO ANTIMICROBIANA DO 7 
EXTRATO PIROLENHOSO NEUTRALIZADO E BIDESTILADO 8 
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GIL SANDER PRÓSPERO GAMA 14 
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Macaíba/RN 33 
Fevereiro de 2023 34 
 
Nº 104 
 
 
 MINISTÉRIO DA EDUCAÇÃO 
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE 
PRÓ-REITORIA DE PÓS-GRADUAÇÃO 
UNIDADE ACADÊMICA ESPECIALIZADA EM CIÊNCIAS AGRÁRIAS - UAECIA 
ESCOLA AGRÍCOLA DE JUNDIAÍ - EAJ 
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FLORESTAIS 
 
 
 
 
GIL SANDER PRÓSPERO GAMA 
 
 
 
 
 
 
 
 
 
AVALIAÇÃO DA AÇÃO ANTIMICROBIANA DO EXTRATO PIROLENHOSO 
NEUTRALIZADO E BIDESTILADO 
 
 
Dissertação apresentada ao Programa de Pós-
Graduação em Ciências Florestais da Universidade 
Federal do Rio Grande do Norte, como parte das 
exigências para obtenção do título de Mestre em Ciências 
Florestais (Tecnologia e Utilização de Produtos Florestais 
– Energia da Biomassa Florestal). 
 
 
 
Orientador: 
Prof. Dr. Alexandre Santos Pimenta 
 
Coorientador: 
Prof. Dr. Francisco Marlon Carneiro Feijó 
 
 
 
 
 
Macaíba/RN 
Fevereiro de 2023 
 
 Universidade Federal do Rio Grande do Norte - UFRN 
Sistema de Bibliotecas - SISBI 
Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Rodolfo Helinski - Escola Agrícola de Jundiaí - EAJ - 
Macaiba 
 
 Gama, Gil Sander Próspero. 
 Avaliação da ação antimicrobiana do extrato pirolenhoso 
neutralizado e bidestilado / Gil Sander Próspero Gama. - 2023. 
 95f.: il. 
 
 Dissertação (mestrado) - Universidade Federal do Rio Grande do 
Norte, Unidade Acadêmica Especializada em Ciências Agrárias, 
Programa de Pós-Graduação em Ciências Florestais. Macaíba, RN, 
2023. 
 Orientador: Prof. Dr. Alexandre Santos Pimenta. 
 Coorientador: Prof. Dr. Francisco Marlon Carneiro Feijó. 
 
 
 1. Pirólise da madeira - Dissertação. 2. Antisséptico - 
Dissertação. 3. Produtos naturais - Dissertação. I. Pimenta, 
Alexandre Santos. II. Feijó, Francisco Marlon Carneiro. III. 
Título. 
 
RN/UF/BSPRH CDU 674 
 
 
 
 
 
 
Elaborado por Elaine Paiva de Assunção - CRB-15/492 
 
 
 
 
 
 
 
 
 
 
 
 
 
 i 
AVALIAÇÃO DA AÇÃO ANTIMICROBIANA DO EXTRATO PIROLENHOSO 
NEUTRALIZADO E BIDESTILADO 
 
Gil Sander Próspero Gama 
Dissertação julgada para obtenção do título de Mestre em Ciências Florestais (Área de 
Concentração em Ciências Florestais - Linha de Pesquisa: Tecnologia e Utilização de 
Produtos Florestais) e aprovada pela banca examinadora em 16 de fevereiro de 2023. 
Banca Examinadora 
 
 
 
 
Prof. Dr. Alexandre Santos Pimenta 
EAJ/UFRN 
Presidente 
 
 
 
Profª. Dra. Tatiane Kelly Barbosa de Azevedo 
EAJ/UFRN 
Examinador interno 
 
 
Prof. Dr. Rafael Rodolfo de Melo 
Universidade Federal Rural do Semiárido - UFERSA 
Examinador interno 
 
 
 
 
 
 
 
 
 
Prof. Dr. Francisco Marlon Carneiro Feijó 
Universidade Federal Rural do Semiárido - UFERSA 
Examinador Externo 
 
 
 
 
Prof. Dr. Ananias Francisco Dias Júnior 
Universidade Federal do Espírito Santo UFES 
Examinador Externo 
 
Macaíba/RN 
Fevereiro de 2023 
 
ii 
 
iii 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AO SENHOR DA MINHA VIDA: “Deus meu e Rei 
meu”, a quem eu devo cada respirar e cada conquista. 
Ao único que é digno de receber a honra, a glória e o 
poder. Rei eterno e real que derrama sobre mim a sua 
infinita bondade e misericórdia. 
 
DEDICO 
 
 
iv 
 
AGRADECIMENTOS 
__________________________________________________________________________ 
 
Agradeço 
 
Ao meu orientador Prof. Dr. Alexandre Santos Pimenta e ao coorientador Prof. Dr. 
Francisco Marlon Carneiro Feijó, por terem visto em mim a capacidade de trabalhar em 
parceria com eles nas suas pesquisas e terem me concedido a chance de aperfeiçoar a 
vivência científica e acadêmica. Por sempre estarem disponíveis a me orientar e por serem 
exemplos de profissionais que terei a honra de levar para a vida. 
À Escola Agrícola de Jundiaí, Universidade Federal do Rio Grande do Norte - UFRN, 
por me proporcionar a oportunidade de cursar o mestrado e fundamentar minha carreira 
acadêmica. 
Ao Laboratório de Microbiologia Veterinária (LAMIV) da Universidade Federal Rural 
do Semiárido – UFERSA, pela parceria e apoio durante a realização da pesquisa. 
Ao Laboratório de Microscopia Eletrônica (CPVSA) da Universidade Federal Rural do 
Semiárido – UFERSA, pela parceria e apoio durante a realização da pesquisa. 
Ao Laboratório de Morfofisiologia Animal Aplicada (LABMORFA) da Universidade 
Federal Rural do Semiárido – UFERSA, pela parceria e apoio durante a realização da 
pesquisa. 
Ao Instituto Nacional de Metrologia, Qualidade e Tecnologia – INMETRO, mais 
precisamente às pesquisadoras Thays V. C. Monteiro e Maíra Fasciotti, pela parceria e 
apoio durante a realização da pesquisa. 
À minha família que é a minha base forte em todas as situações, se mostrando 
sempre meus auxiliadores, tanto na vida pessoal, quanto na acadêmica: meu pai Petérson 
H. F. Gama, minha mãe Juçária P. F. Gama e ao meu irmão Éricson P. Gama. 
De maneira geral, a todos os meus amigos que de alguma forma fazem parte desta 
trajetória, por todo apoio, incentivo, orientação e dedicação a mim concedidos. 
A Deus acima de tudo, por me dar o dom da vida e proporcionar cada uma das 
oportunidades que me trouxeram até aqui. 
 
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de 
Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. 
 
 
 
 
 
v 
 
RESUMO GERAL 
__________________________________________________________________________ 
 
AVALIAÇÃO DA AÇÃO ANTIMICROBIANA DO EXTRATO PIROLENHOSO 
NEUTRALIZADO E BIDESTILADO 
O extrato pirolenhoso (EP) é um coproduto gerado no processo de pirólise da biomassa 35 
vegetal que pode apresentar uma gama de aplicações. Dentre estas aplicações, tem-se 36 
como agente antimicrobiano. No entanto, existem afirmações que limitam a ação 37 
antimicrobiana que o EP desempenha apenas à fração de ácidos orgânicos presente em 38 
sua composição química, gerando dúvidas quanto à sua eficiência como um conjunto. Com 39 
isso, o objetivo desta pesquisa foi avaliar a atividade antimicrobiana do EP, testar a 40 
influência do seu pH nesta atividade, avaliar a composição química deste produto e registrar 41 
imagens de microscopia eletrônica de varredura (MEV) das células microbianas. As 42 
avaliações antimicrobianas, para a determinação da Concentração Inibitória Mínima (CIM), 43 
foram realizadas in vitro pelo método de microdiluição em caldo. Para as Concentrações 44 
Bactericida e Fungicida Mínimas (CBM e CFM) utilizou-se o método de crescimento em ágar 45 
BHI. A composição química do EP e seu concentrado foi determinada por cromatografia 46 
gasosa e espectrometria de massas e as imagens MEV foram obtidas com o auxílio de um 47 
microscápio eletrônico (Pfeiffer Vacuum – D-35614 Assiar). As análises estatísticas foram 48 
realizadas por meio de análise de regressão utilizando o software R (versão 4.1.3). Os 49 
resultados obtidos demonstraram que mesmo em pH neutro o EP se mostrou eficaz como 50 
antimicrobiano, provando que a sua ação não se limita apenas à uma classe de compostos, 51 
mas sim ao seu conjunto. No entanto, o aumento da neutralização influenciou nas 52 
concentrações de EP requeridas para a inibição dos microrganismos avaliados. Ambos os 53 
EPs, obtidos de B. vulgaris e E. urograndis, desempenharam papel antimicrobiano 54 
satisfatório, inibindo o desenvolvimento de todos os microrganismos testados. As imagens 55 
de MEV demonstraram que o EP infuencia na morfologia da parede celular dos 56 
microrganismos, resultando em alterações em sua estrutura. Conclui-seque esses produtos 57 
são promissores ao desenvolvimento de alternativas antimicrobianas eficazes. No entanto, 58 
estudos posteriores são necessários para firmar o seu uso, ressaltando a necessidade de 59 
testes in vivo. 60 
Palavras-chave: pirólise da madeira, vinagre de madeira, resistência microbiana, produtos 61 
naturais, antisséptico 62 
 
 
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GENERAL ABSTRACT 
__________________________________________________________________________ 
 
EVALUATION OF THE ANTIMICROBIAL ACTION OF NEUTRALIZED AND DOUBLE-
DISTILLATE WOOD VINEGAR 
Wood vinegar (WV) is a co-product generated in the pyrolysis process of plant biomass that 63 
can have a range of applications. Among these applications, it is used as an antimicrobial 64 
agent. However, there are statements that limit the antimicrobial action that EP performs only 65 
to the fraction of organic acids present in its chemical composition, raising doubts as to its 66 
efficiency as a whole. Thus, the objective of this research was to evaluate the antimicrobial 67 
activity of WV, test the influence of its pH on this activity, evaluate the chemical composition 68 
of this product and record images of scanning electron microscopy (SEM) of microbial cells. 69 
Antimicrobial evaluations to determine the Minimum Inhibitory Concentration (MIC) were 70 
performed in vitro by the broth microdilution method. For the Minimum Bactericidal and 71 
Fungicide Concentrations (MBC and MFC) the agar growth method was used. The chemical 72 
composition of WV and its concentrate was determined by gas chromatography and mass 73 
spectrometry and the SEM images were obtained with the aid of an electron microscope 74 
(Pfeiffer Vacuum – D-35614 Assiar). Statistical analyzes were performed using regression 75 
analysis using the R software (version 4.1.3). The results obtained showed that even at 76 
neutral pH, WV proved to be effective as an antimicrobial, proving that its action is not limited 77 
to a single class of compounds, but to all of them. However, the increase in neutralization 78 
influenced the WV concentrations required for the inhibition of the evaluated microorganisms. 79 
Both WV obtained from B. vulgaris and E. urograndis played a satisfactory antimicrobial role, 80 
inhibiting the development of all tested microorganisms. SEM images demonstrated that WV 81 
influences the morphology of the cell wall of microorganisms, resulting in alterations in its 82 
structure. It is concluded that these products are promising for the development of effective 83 
antimicrobial alternatives. However, further studies are needed to confirm its use, highlighting 84 
the need for in vivo tests. 85 
Keywords: wood pyrolysis, wood vinegar, microbial resistance, natural products, antiseptic 86 
 
 
 
 
 
vii 
 
SUMÁRIO 
________________________________________________________________________ 
 
Página 
1. INTRODUÇÃO GERAL .................................................................................................. 1 
2. OBJETIVO GERAL ........................................................................................................ 4 
3. REVISÃO DE LITERATURA .......................................................................................... 6 
3.1. RELATOS HISTÓRICOS DO USO DE EPB E DERIVADOS ...................................... 6 
3.2. EXTRATO PIROLENHOSO: PROPRIEDADES QUÍMICAS E FÍSICAS ..................... 7 
3.3. MÉTODOS, EQUIPAMENTOS DE RECUPERAÇÃO E PURIFICAÇÃO .................... 8 
3.4. BIOATIVIDADE DO EP ............................................................................................ 10 
3.5. OUTRAS APLICAÇÕES RELACIONADAS AO EP................................................... 12 
LITERATURA CITADA ........................................................................................................ 17 
4. CAPÍTULO 1. EFFECT OF PH ON THE ANTIBACTERIAL AND ANTIFUNGAL 
ACTIVITY OF WOOD VINEGAR (PYROLIGNEOUS EXTRACT) FROM EUCALYPTUS ... 27 
RESUMO ......................................................................................................................... 28 
ABSTRACT ...................................................................................................................... 29 
INTRODUCTION .............................................................................................................. 30 
MATERIAL AND METHODS ............................................................................................. 31 
RESULTS ......................................................................................................................... 33 
DISCUSSION ................................................................................................................... 40 
CONCLUSIONS ............................................................................................................... 44 
ACKNOWLEDGEMENTS ................................................................................................. 44 
REFERENCES ..................................................................... Erro! Indicador não definido. 
5. CAPÍTULO 2. ANTIMICROBIAL ACTIVITY AND CHEMICAL PROFILE OF 
PYROLIGNEOUS ACIDS FROM BAMBOO AND EUCALYPTUS ...................................... 50 
RESUMO ......................................................................................................................... 51 
ABSTRACT ...................................................................................................................... 52 
INTRODUCTION .............................................................................................................. 53 
MATERIAL AND METHODS ............................................................................................. 55 
RESULTS ......................................................................................................................... 58 
CONCLUSIONS ............................................................................................................... 71 
ACKNOWLEDGEMENTS ................................................................................................. 72 
REFERENCES ................................................................................................................. 72 
6. CONSIDERAÇÕES FINAIS .......................................................................................... 80 
7. ANEXOS....................................................................................................................... 82 
 
 
viii 
 
LISTA DE FIGURAS 
__________________________________________________________________________ 
 
CAPÍTULO 1 
Figure 1. Graphs representing the effect of the pH of WV on the growth of Pseudomonas 
aeruginosa, Salmonella enteritides, Staphylococcus aureus, Streptococcus agalactiae, and 
Candida albicans as a function of the concentration at zero time (A) and 24 hours (B) after 
incubation ............................................................................................................................ 36 
 
CAPÍTULO 2 
Figure 2. Absorbances of microorganisms’ cultures as a function of the concentration of 
eucalyptus (orange curves) and bamboo (blue curves) vinegar; A1, B1, C1, D1, E1, and F1 – 
at the moment of inoculation – 0 h; and A2, B2, C2, D2, E2, and F2 – 24 h after inoculation 
(48 h for C. albicans)............................................................................................................ 60 
Figure 3. Total ion chromatograms of Eucalyptus urograndis (A) and Bambusa vulgaris (B) 
carbonization vinegar ........................................................................................................... 62 
Figure 4. SEM micrographs of Staphylococcus aureus before (A) and after 24-h exposure 
(B) to bamboo vinegar. Magnification: 30.000 X ................................................................... 69 
Figure 5. SEMmicrographs of Streptococcus agalactiae before (A) and after 24-h exposure 
(B) to bamboo vinegar. Magnification: 13,400 X ................................................................... 70 
Figure 6. SEM micrographs of Candida albicans before (A), after 24 h (B), and 48 h (C) of 
exposure to bamboo vinegar. Magnification: 5,000 and 7,410 X (arrows in the micrographs 
indicate cell budding) ........................................................................................................... 70 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ix 
 
LISTA DE TABELAS 
__________________________________________________________________________ 
 
CAPÍTULO 1 
Table 1. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations 
(MBC) values against the microorganisms according to increasing pH levels ...................... 34 
Table 2. Regression models that were adjusted to explain the behavior of absorbances from 
cultures of Pseudomonas aeruginosa, Salmonella enteritides, Staphylococcus aureus, 
Streptococcus agalactiae, and Candida albicans after 24 hours of incubation at each pH level 
according to WV concentration ............................................................................................ 36 
Table 3. Results of the identity test applied to the regression models adjusted for the 
absorbances of the microbial cultures after 24 hours of incubation (A): Pseudomonas 
aeruginosa, Salmonella enteritides, Staphylococcus aureus, and Streptococcus agalactiae; 
(B): Candida albicans .......................................................................................................... 39 
 
CAPÍTULO 2 
Table 4. Values of MIC, MBC, and MFC (%) for the microorganisms subjected to the action 
of Eucalyptus urograndis and Bambusa vulgaris vinegar ..................................................... 58 
Table 5. Regression models to predict the behavior of microorganisms' growth as a function 
of the eucalyptus and bamboo vinegar concentrations ......................................................... 60 
Table 6. Annotated compounds in the carbonization vinegar of Eucalyptus urograndis and 
Bambusa vulgaris ................................................................................................................ 63 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
LISTA DE ABREVIATURAS 
__________________________________________________________________________ 
 
AP – Ácido Pirolenhoso 
CIM – Concentração Inibitória Mínima 
EPB – Extrato Pirolenhoso Bruto 
EP – Extrato Pirolenhoso 
LPB – Líquido Pirolenhoso Bruto 
MBC – Minimum Bactericidal Concnetration 
MEV – Microscopia Eletrônica de Varredra 
MFC – Minimum Fungicidal Concentration 
MIC – Minimum Inhibitory Concentration 
PA – Pyroligneous acid 
PBS – Solução Tampão Fosfato de Sódio 
PE – Pyroligneos extract 
SEM – Scanning Electron Microscopy 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Introdução 
_____________________________ 
 
1 
 
1. INTRODUÇÃO GERAL 
__________________________________________________________________________ 
 
 
A pirólise da madeira é descrita como processo de degradação térmica que ocorre 87 
na presença restrita ou ausência completa de oxigênio (AGUIRRE et al., 2020). Conforme a 88 
pirólise está ocorrendo são gerados produtos sólidos, líquidos e gasosos, sendo a 89 
temperatura do processo o principal fator que controla estes resultados (SOUZA et al., 90 
2012). Atualmente, o principal produto resultante da pirólise é o carvão vegetal, 91 
correspondente à fração sólida (GREWAL et al., 2018). 92 
A porção líquida originada é referente ao líquido pirolenhoso bruto (LPB). Este 93 
líquido, também chamado de extrato pirolenhoso bruto (EPB), é formado da fração 94 
condensável dos vapores da fumaça desprendida do material que está sendo pirolisado 95 
(SOUZA et al., 2012). Basicamente, estes vapores são oriundos da quebra de moléculas 96 
como as hemiceluloses, celuloses e as ligninas (LI et al., 2017). 97 
O EPB é composto por três porções, o alcatrão, o extrato pirolenhoso (EP) e os 98 
óleos leves (SENA et al., 2014). O EP é um produto que vem sendo estudado há décadas e 99 
tem sua utilização cultural estabelecida há muito tempo e isto pode ser atribuído à sua 100 
composição química e propriedades físicas (CAMPOS, 2007; TIILIKKALA et al., 2010; 101 
SURESH et al., 2019). 102 
Quimicamente, é um produto extremamente complexo, devido à grande variedade 103 
de compostos presentes em sua constituição, podendo chegar a mais de duzentas 104 
substâncias diferentes (SCHNITZER et al., 2015; ARAÚJO et al., 2017). Outra característica 105 
que chama a atenção dos pesquisadores é a sua acidez, pois apresenta caráter ácido, com 106 
pH variando entre 2.5 e 3.6 (AUBIN; ROY, 1990; RAHMAT et al., 2014; PIMENTA et al., 107 
2018). Esta acidez é associada à elevada concentração de ácidos orgânicos presentes no 108 
mesmo, principalmente o ácido acético (SIPILÄ et al., 1998; THEAPPARAT et al., 2018). 109 
Devido a esta composição química particular, este produto tem sido aplicado para 110 
diversos fins desde tempos muito antigos (DORAN, 1932; KURLANSKY, 2002; CAMPOS, 111 
2007; TIILIKKALA et al., 2010). Por sua variabilidade de aplicações, o interesse neste 112 
bioproduto tem ganhado cada vez mais força, tendo diversas pesquisas focadas na sua 113 
ação antimicrobiana (CHEN et al., 2015; ARAÚJO et al., 2017; PAN et al., 2017; SOUZA et 114 
al., 2018; AGUIRRE et al., 2020; MALIANG et al., 2020). 115 
Este fato ocorre porque a sua aplicação como um possível antimicrobiano se 116 
mostra muito importante, pois, atualmente, há uma busca por alternativas de tratamento 117 
para doenças causadas por microrganismos, isto porque, o aumento no surgimento de 118 
resistência bacteriana aos fármacos antibióticos tem tomado proporções preocupantes 119 
 
2 
 
(WORLD HEALTH ORGANIZATION, 2020). Este fato é atribuído ao uso indiscriminado 120 
destes fármacos e, adicionado a isto, um agravante para este quadro é o uso de antibióticos 121 
não se resumir apenas à medicina humana, mas também serem amplamente utilizados em 122 
outras áreas como a medicina veterinária (ZIMERMAN, 2010; PALMA et al., 2020). 123 
Com isso, diz-se que o EP pode ser uma importante alternativa para esta finalidade, 124 
pois com a variedade de compostos presentes em sua composição química e cada um 125 
deles podendo apresentar um mecanismo de ação diferente, o desenvolvimento de 126 
resistência pelos microrganismos seria dificultado (SURESH et al., 2019). No entanto, 127 
existem especulações na literatura de que a atividade antimicrobiana do EP é referente 128 
apenas à presença do ácido acético na sua constituição, com o intuito de afirmar que esta 129 
atividade biológica não pode ser atribuída ao todo, ou seja, à ação dos demais componentes 130 
encontrados no mesmo (MEDEIROS; GASPAROTTO, 2021). Sendo assim, faz-se 131 
necessário investigar a influência do pH na ação antimicrobiana deste produto. 132 
Outra questão tem chamado a atenção de pesquisadores: a dificuldade de 133 
armazenamento e transporte do EP. Há em sua constituição, cerca de 80 a 88% de água 134 
(YATAGAI et al., 2002), isto torna o seu armazenamento instável e dificultado, além de 135 
aumentar os custos de transporte, devido a requerer mais espaço que o necessário. Liu et 136 
al. (2021) relatam a este respeito e testaram uma metodologia visando amenizar estes 137 
empecilhos sem que a bioatividade do EP fosse afetada. Com o exposto, nota-se a 138 
necessidade da aplicação de processos que reduzam a quantidade de água presente neste 139 
produto. 140 
Vale ressaltar também que é importante que as emissões dos gases poluentes 141 
lançados na atmosfera pela pirólise da lenha sejam atenuadas e que haja um 142 
gerenciamento estratégico dos processosque utilizam essa matéria prima e dos coprodutos 143 
a partir dela gerados (GREWAL et al., 2018). Dentre estes coprodutos está o EP, logo, com 144 
isso, a sua utilização como antimicrobiano classifica-se como uma alternativa de mitigação 145 
das emissões poluentes. 146 
Com o exposto, torna-se explícita a necessidade da comprovação científica das 147 
aplicações antimicrobianas do EP oriundo da produção do carvão vegetal e de desenvolver 148 
uma metodologia viável que vise reduzir a quantidade de água no mesmo sem afetar 149 
negativamente a sua bioatividade, ressaltando que a sua utilização poderá trazer benefícios 150 
como, atenuação das emissões de gases poluentes para a atmosfera e o desenvolvimento 151 
de um novo antimicrobiano para reverter um cenário preocupante a nível mundial, além de 152 
agregar valor à cadeia produtiva do carvão. 153 
 
 
 
3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Objetivo Geral 
_____________________________ 
 
4 
 
2. OBJETIVO GERAL 
__________________________________________________________________________ 
 
A presente pesquisa tem como objetivo avaliar as atividades antibacteriana e 154 
antifúngica de extratos pirolenhosos de duas espécies vegetais: Bambusa vulgaris (bambu) 155 
e Eucalyptus urograndis (clone I144); Avaliar a composição química desses produtos; 156 
Investigar o efeito da neutralização de um dos EPs frente a sua ação antimicrobiana e; 157 
Registrar imagens de microscopia eletrônica de varredura das células microbianas após a 158 
exposição ao EP. 159 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revisão de Literatura 
_____________________________ 
 
6 
 
3. REVISÃO DE LITERATURA 
__________________________________________________________________________ 
 
 
3.1. Relatos históricos do uso de EPB e derivados 160 
 
O extrato pirolenhoso (EP) já teve diversas maneiras de utilização ao decorrer da 161 
história. Há relatos do uso do alcatrão pelos antigos egípcios na calafetação de 162 
embarcações e em alguns princípios do embalsamento (BALAT et al., 2009). Campos 163 
(2007) relata que, no século XVII, a madeira já era destilada na Europa, a fim de se obter 164 
alcatrão. Este autor também menciona a utilização milenar do extrato pirolenhoso na China 165 
e na Índia para o tratamento de enfermidades. 166 
Já no século XIX, nos anos de 1813, houve uma grande produção e utilização do EP 167 
para aplicação no processo de coloração de tecidos de linho na Inglaterra (CAMPOS, 2007). 168 
Ainda no século XIX, em meados de 1862, um tipo de aplicação peculiar foi registrada. 169 
Devido à escassez de sal em alguns países durante a guerra civil americana, houve um 170 
problema para a conservação de carnes, de modo que, famílias detentoras de suprimentos 171 
de sal o mantinham guardado como se fossem joias. Com isso, nessas regiões começaram 172 
a aparecer alguns boatos de que a utilização do EP serviria para conservar bacon e outros 173 
tipos de carne, notícia divulgada em um jornal do Alabama (KURLANSKY, 2002). 174 
Os relatos datando do século XX trazem como o primeiro livro a respeito do EP, em 175 
1945, o exemplar denominado de “Fabricação e utilização do extrato pirolenhoso”, obra de 176 
autoria de Tatsujiro Fakuda, o qual relatava a respeito do uso eficiente do pirolenhoso em 177 
culturas de arroz (ALMEIDA, 2012). 178 
No ano de 1932 já eram realizadas pesquisas comparando a eficiência do EP com 179 
outros produtos para a desinfecção de solo para a agricultura. Doran (1932) traz em seu 180 
estudo relatos sobre a ótima qualidade do EP como eficiente desinfetante do solo. Este 181 
autor relata vantagens desta fração do EPB quando comparado ao formaldeído e ácido 182 
acético. O mesmo expõe que o EP não prejudicou a germinação de sementes de beterraba, 183 
pepino e alface, mesmo quando sua aplicação ocorreu apenas um dia antes da semeadura, 184 
demonstrando a vantagem frente ao formaldeído, pois para este, deve haver um tempo 185 
maior de espera para realizar a semeadura para que a germinação das sementes não seja 186 
afetada. Ele ainda menciona que no solo tratado com EP houve um aumento no peso seco 187 
das plantas, além da diminuição do custo de tratamento do solo por unidade de área. 188 
Analisando o contexto atual, existem diversas pesquisas e formas de aplicações 189 
deste extrato como, herbicida, adjuvante de herbicida, inseticida, desenvolvimento de 190 
plantas, aumento na disponibilidade de nutrientes no solo e melhoria das características 191 
 
7 
 
deste, industrial, antioxidante, entre outros (TRINDADE et al., 2014; SEO et al., 2015; 192 
CAMPOS, 2017; SOUZA et al., 2018; MALLIANG et al., 2020). Na Tailândia existem testes 193 
verificando a eficiência do EPB de bambu e derivados na ação antifúngica e como 194 
preservativo de madeira (THEAPPARAT et al., 2015). 195 
Em Quebec, Canadá, pesquisadores avaliam a atividade antibacteriana e antifúngica 196 
do EP proveniente de madeira macia composta por partículas de madeira de pinho e de 197 
abeto (SURESH et al., 2019). Em zonas rurais da Tailândia a extração do EPB da produção 198 
de carvão de madeira e de bambu para uso em fogões de barro pode ser utilizada para a 199 
proteção de sementes para a agricultura (CHALERMSAN; PEERAPAN, 2009). No Japão o 200 
uso deste produto é bastante aceito como agroquímico orgânico (GREWAL et al., 2018). Na 201 
China é testado como alternativa para melhorar a saúde do solo (MALIANG et al., 2020). Na 202 
Malásia é avaliado como promotor de crescimento de plantas (MAHMUD et al., 2016). 203 
O interesse voltado para este coproduto se dá porque, além das suas propriedades 204 
químicas e físicas e suas potencialidades de uso, a captação deste produto durante a 205 
produção do carvão evita que os gases poluentes sejam lançados como fumaça na 206 
atmosfera, no entanto, em algumas regiões, ainda existem empecilhos jurídicos e 207 
econômicos a respeito da sua utilização (DAROIT et al., 2013). Porém, é fato que o 208 
gerenciamento de maneira estratégica da biomassa vegetal e os resíduos oriundos dos seus 209 
processos de utilização é uma alternativa de mitigação das emissões poluentes (GREWAL 210 
et al., 2018). 211 
3.2. Extrato pirolenhoso: propriedades químicas e físicas 212 
 213 
O EP, também chamado de vinagre de madeira, é um produto de coloração 214 
amarelada ou marrom transparente, referente à porção aquosa (entre 60 a 75%) do EPB 215 
resultante da condensação da fumaça produzida no processo de pirólise da madeira 216 
(CAMPOS, 2007; SENA et al., 2014; SEO et al., 2015; SOUZA et al., 2018; SURESH et al., 217 
2019). Basicamente, ele se origina da quebra de algumas moléculas que estão presentes na 218 
composição do material vegetal que está sendo pirolisado, sendo algumas delas a lignina, a 219 
hemicelulose e a celulose (LI et al., 2017). 220 
De forma mais específica, diz-se que o EP é um ácido carboxílico que apresenta uma 221 
mistura originada de diversos compostos que são oriundos de inúmeras reações 222 
termoquímicas que ocorrem durante a pirólise (CAMPOS, 2017). Em sua maioria, ele é 223 
constituído por água, cerca de 80% do seu total, no entanto, sua constituição química é 224 
extremamente complexa, pois nele podem ser encontrados mais de duzentos compostos 225 
orgânicos diferentes como, fenóis, cetonas, álcoois, metanol, butanol, derivados de 226 
piranfuranos, derivados de seringol, cresóis, guaiacol, fenol, formaldeído, maltou, cetonas, 227 
 
8 
 
os ácidos valérico, propiônico e fórmico, ácido metílico, ácido acético, dentre outros 228 
(TIILIKKALA et al., 2010; SCHNITZER et al., 2015; ARAÚJO et al., 2017; SILVA et al., 2017; 229 
SURESH et al., 2019). Segundo Sipilä et al. (1998) e Theapparat et al. (2018), alguns dos 230 
constituintes mais expressivosdo EP são o ácido acético e o fórmico, com destaque para o 231 
ácido acético, podendo apresentar valores entre 3.0 e 7.4%. 232 
Souza et al. (2012) relatam em sua pesquisa a presença do guaiacol e seringol na 233 
constituição química do EP. Estes autores mencionam que estas duas substâncias fazem 234 
parte do grande grupo dos metoxifenóis, sendo estes produtos oriundos da degradação da 235 
lignina, juntamente com os fenóis. Em um estudo utilizando o EP de um clone de eucalipto 236 
(Eucalyptus urograndis) foi demonstrada a presença de 93 substâncias, sendo que os 237 
compostos fenólicos foram o maior grupo encontrado, além das cetonas, aldeídos, piranos, 238 
furanos e ésteres. De forma mais específica, os autores destacaram altos níveis de 239 
guaiacol, fenol, furfural e cresol, além de citar duas substâncias que merecem atenção por 240 
supostamente serem cancerígenas, sendo o N-nitrosodimetilamina e o fenol (PIMENTA et 241 
al., 2018). 242 
A composição química do EP pode apresentar algumas semelhanças com a química 243 
da fração do EPB referente ao alcatrão. Souza et al. (2012) avaliaram os constituintes do 244 
alcatrão de Eucalyptus sp. por meio de técnicas de GC-FID e de GC-MS. Nos resultados 245 
obtidos, foram determinadas cerca de 65 substâncias orgânicas, sendo a maior parte delas 246 
fenóis, metoxifenóis e grupos cetônicos, dos quais se destacou o seringol. Além destes, 247 
foram encontrados alcoóis e cerca de 44% de ésteres de ácido carboxílico. 248 
As aplicações do EP nas mais diversas áreas do conhecimento estão relacionadas 249 
não só à sua composição química, mas também à sua viscosidade, pH, densidade, entre 250 
outros (CAMPOS, 2007; SURESH et al., 2019). De acordo com Pimenta et al., (2018), este 251 
produto apresenta coloração amarelada, densidade de 1,032 g cm-3 e pH de 2,85. Outro 252 
estudo demonstrou o pH de 3,6, densidade de 1,021 g mL-1 e coloração amarelo-253 
amarronzado (RAHMAT et al., 2014). No fim, as características intrínsecas de cada um 254 
destes produtos podem variar de acordo com a espécie utilizada (DIAS JÚNIOR et al., 255 
2018). 256 
3.3. Métodos, equipamentos de recuperação e purificação 257 
 258 
Como relatado nos tópicos anteriores, o método de produção do EP está referido ao 259 
processo de pirólise. Este processo tem dois regimes de aquecimento definidos: a pirólise 260 
rápida e a lenta, sendo que quando se deseja obter um maior rendimento do EPB e, 261 
consequentemente, do EP como produto final da pirólise, temperaturas mais elevadas e 262 
 
9 
 
menores tempos de residência, ou seja, pirólise rápida são os mais indicados (BALAT et al., 263 
2009). 264 
Em seu estudo, Bridgwater e Coulson (2007) trazem comparações dos rendimentos 265 
gravimétricos do EPB nestes dois regimes. Os autores demonstraram que os rendimentos 266 
em pirólise rápida foram consideravelmente superiores à lenta, apresentando valores de 267 
75% de rendimento em comparação a cerca de 50% em regime lento. 268 
A coleta do EPB é realizada por meio de tubos recuperadores instalados nos fornos de 269 
pirólise. Nestes fornos, a fumaça produzida no processo da degradação da madeira é 270 
conduzida para o tubo recuperador, onde os vapores condensáveis se condensam, 271 
precipitam e são coletados (SANTOS et al., 2011). 272 
Na literatura podem ser encontrados alguns relatos de tubos de recuperação para 273 
diferentes realidades. Pimenta et al. (2018) realizou a coleta do EPB em laboratório, onde a 274 
pirólise foi processada em uma mufla, sendo que os autores acoplaram a ela um dispositivo 275 
de tubo projetado para condensar os vapores. Neste processo, o tubo de recuperação foi 276 
mantido resfriado por água a fim de sustentar a temperatura em torno de 25 °C, 277 
proporcionando condições de condensação. O rendimento obtido por eles foi de 42.4% de 278 
EPB. 279 
Já em campo, Gonçalves et al. (2010) confeccionaram um tubo de chapas de zinco 280 
acoplado a um forno de alvenaria do tipo rabo quente com capacidade aproximada de 12.4 281 
m3 de madeira. Nesta pesquisa, o tubo media 8 metros de comprimento e não apresentava 282 
sistema de resfriamento artificial como no estudo de Pimenta et al. (2018). Os resultados 283 
mostraram um rendimento de 2.01% na coleta do extrato, no entanto, os autores 284 
recomendam a utilização de resfriadores externos para o aumento da eficiência de coleta, 285 
devido ao baixo rendimento obtido. 286 
Após a sua captação, reações químicas entre as partes componentes do EPB 287 
acontecem, a chamada polimerização, por este motivo este produto deve ficar em repouso 288 
por cerca de três a seis meses para que os componentes se estabilizem com o término das 289 
reações (CAMPOS, 2007). Segundo Araújo et al. (2017) este tempo de repouso pode variar. 290 
Ao final do repouso torna-se possível notar a diferenciação das três fases componentes do 291 
EPB, o alcatrão, o EP e os óleos leves (ALVES, 2007). 292 
O processo de refinamento do extrato pirolenhoso passa por diversas fases para 293 
gerar diferentes níveis de purificação, industrial e purificado (farmacêutico). Inicialmente, o 294 
extrato pirolenhoso bruto deve ser submetido à decantação. Nesta fase é separado o extrato 295 
ácido do alcatrão insolúvel. Por sua vez, a porção correspondente ao extrato ácido é 296 
composta por EP e alcatrão solúvel. Sendo assim, o extrato ácido passa por mais uma 297 
etapa, a destilação simples, onde há a separação destas duas frações (SOUZA et al., 2012). 298 
 
10 
 
Nesta fase, este produto já é recomendado para aplicação na agricultura, pois já está livre 299 
de alcatrão e impurezas (CAMPOS, 2007). 300 
Para um maior refinamento do EP, a fim de gerar maior segurança da ausência de 301 
algum resíduo do alcatrão, faz-se o processo de bidestilação a vácuo, sob 1.0 mm HG a 100 302 
°C, gerando um extrato pirolenhoso purificado, de baixa toxicidade, tornando-o um composto 303 
de constituição similar à de fármacos fitoterápicos, adquirindo um grau de purificação 304 
farmacêutico (PIMENTA et al., 2018; SOARES et al., 2020). 305 
 
3.4. Bioatividade do EP 
 
Atualmente, vários pesquisadores têm buscado alternativas terapêuticas no controle 306 
de doenças causadas por microrganismos. Diversos patógenos têm desenvolvido 307 
resistência aos antimicrobianos já existentes, pois de acordo com Berquó et al. (2004), é 308 
prática comum que o tratamento dessas doenças seja basicamente realizado através deste 309 
medicamentos. 310 
Além do consumo severo de antimicrobianos pelo homem, a sua aplicação na 311 
criação animal é um forte agravante para este quadro, pois o aumento da resistência 312 
microbiana está relacionado ao uso indiscriminado destes fármacos e é nítido o aumento 313 
das taxas de resistência quando há um consumo maior destes medicamentos (ZIMERMAN, 314 
2010; PALMA et al., 2020; ZIMERMAN, 2010). 315 
Isto porque, o uso de antimicrobianos de forma inadequada, seja no consumo 316 
humano ou na alimentação animal, caracteriza-se como causador de um aumento na 317 
disseminação de microrganismos multirresistentes a fármacos antibióticos, sendo que uma 318 
porção destes microrganismos pode resistir até mesmo aos antimicrobianos mais potentes 319 
(NACHMAN, 2016). 320 
Um antimicrobiano pode ser descrito como uma substância que apresenta ação de 321 
inibir o crescimento microbiano (ABAS et al., 2018). Substâncias de origem vegetal se 322 
mostram uma importante alternativa para esta aplicação, pois têm sua utilização 323 
consolidada na medicina popular para o tratamento de diversas enfermidades desde tempos 324 
muito antigos (COSTA, 2015). 325 
Por este motivo, devido a sua constituição química variada, o EP se mostra como um 326 
produto de interesse para este fim. Este interesse está relacionado com a presença de 327 
substâncias como, compostos fenólicos, carbonilas e ácidos orgânicos, substâncias que têm 328 
asua bioatividade comprovada (ABAS et al., 2018). 329 
Estas constatações abrem um leque promissor de aplicações do EP nesta área de 330 
pesquisa em todo o mundo. Avaliações utilizando EPs provenientes das madeiras de 331 
 
11 
 
Eucalyptus urograndis e Mimosos tenuiflora frente a vinte e uma cepas bacterianas, 332 
incluindo gram-positivas e gram-negativas, demonstraram que ambos foram capazes de 333 
inibir o crescimento bacteriano, mesmo na menor concentração testada no estudo, 20% 334 
(ARAÚJO et al., 2017). 335 
Soares et al. (2020) relatam resultados semelhantes utilizando as mesmas espécies 336 
vegetais. Os autores avaliaram as concentrações de 20, 15 e 10% dos EPs. Os resultados 337 
demonstraram a ação dos mesmos para todas as cepas testadas na concentração de 20%, 338 
porém, frente a cepa de Staphylococcus aureus, a inibição também ocorreu a 15%. 339 
Suresh et al. (2019) avaliaram a sua ação antimicrobiana em duas condições, com a 340 
sua acidez normal e com o pH neutralizado. Em ambas as situações foram obtidos 341 
resultados favoráveis à atividade do EP frente a cepas gram-positivas e gram-negativas, 342 
porém, os valores da concentração inibitória mínima foram relativamente menores frente a 343 
todas as cepas avaliadas com o produto no seu estado neutralizado. 344 
Abas et al. (2018) investigaram a ação antimicrobiana deste produto utilizando discos 345 
de antibióticos. As cepas empregadas nas avaliações foram Bacillus cereus, Staphylococcus 346 
aureus, Lactobacillus plantarum e Escherichia coli. Os resultados obtidos demonstraram alto 347 
potencial antimicrobiano, apresentando zonas de inibição do crescimento microbiano 348 
variando entre 29 e 79%. Outros autores relatam esta aplicação do EP (SOUZA et al., 2018). 349 
Além da sua atividade antimicrobiana frente a cepas bacterianas, tem-se também 350 
avaliado a sua utilização no controle de outros microrganismos como os fungos. Testes 351 
usando EP de Enterolobium contortisiliquum foram aplicados para realizar a desinfecção de 352 
sementes de Schizolobium amazonicum e demonstraram potencial para o controle de 353 
fungos como Fusarium sp., Rhizoctonia sp. e Aspergillus sp. (MACEDO et al., 2019). 354 
 Araújo et al. (2017) trazem o efeito antifúngico do EP de Eucalyptus urograndis e 355 
Mimosos tenuiflora frente a Candida albicans e Cryptococcus neoformans. Trabalhos 356 
realizados utilizando cepas de Candida sp. também têm mostrado resultados promissores. 357 
Ibrahim et al. (2013) executaram testes com EPs concentrados de Rhizophora apiculata e 358 
relataram ação destes frente a quatro cepas de C. albicans. 359 
No entanto, Almeida (2012) relata que o EP a ser empregado para o controle fúngico 360 
deve ser escolhido com um maior grau de atenção pois, em seu trabalho, os resultados 361 
demonstraram que há uma maior eficiência antifúngica para o seu extrato obtido em escala 362 
laboratorial, em comparação à industrial, e atribui este fato à presença de compostos que 363 
tenham maior ligação com componentes ligados ao alcatrão, mencionando compostos como 364 
corilon, 2-metoxi 4-metilfenol, 1,6 - dimetoxifenol, etc. 365 
Outra atividade referida ao EP é a antiviral. Avaliações realizadas frente ao vírus da 366 
encefalomiocardite demonstram esta atividade. No estudo, foram utilizados EPs de madeira 367 
 
12 
 
dura, macia e de bambu, onde todos estes extratos foram capazes de desinfestar de forma 368 
significativa o vírus (LI et al., 2017; LI et al., 2018). Os autores ainda relatam que a 369 
quantidade de grupos hidroxila presente nos compostos fenólicos do extrato pirolenhoso 370 
interferem significativamente na ação antiviral e demonstram que o composto catecol e seus 371 
derivados foram os que se sobressaíram nos testes, em comparação aos demais compostos 372 
fenólicos presentes nos extratos. 373 
Esta ação também é relatada por Marumoto et al. (2012). Na sua pesquisa in vitro, 374 
com o mesmo vírus, foi observada a ação antiviral do EP de bambu, atribuindo a maior 375 
atividade ao fenol. No estudo, os autores ainda fizeram a mistura deste composto fenólico 376 
com o ácido acético, o que potencializou significativamente a atividade antiviral do fenol, 377 
sugerindo que esta combinação se mostrou uma boa alternativa para a inativação do vírus. 378 
Além das potencialidades já mencionadas, a atividade antioxidante também é 379 
registrada. Ao realizar a aplicação do EP de Rhizophora apiculata e seus compostos 380 
isolados, Loo et al. (2008) avaliaram a sua capacidade na eliminação dos radicais livres, no 381 
seu potencial de redução do molibdênio e no seu poder antioxidante redutor férrico, 382 
resultando em dados promissores para esta aplicação. 383 
 
3.5. Outras aplicações relacionadas ao EP 
 
A literatura tem demonstrado diversos usos do EPB nas mais diferentes áreas de 384 
aplicação. Na agricultura, como um todo, ele é amplamente utilizado (TIILIKKALA et al., 385 
2010). No entanto, sua aplicação não deve ocorrer na sua forma bruta, sem um refinamento 386 
prévio, sendo assim, é necessário que ele passe por um processo de purificação para a 387 
eliminação do alcatrão solúvel e insolúvel, processo este que dá origem ao EP e que pode 388 
ser feito por meios industriais ou artesanais, pela sua decantação (SILVEIRA et al., 2010). 389 
Após o refinamento são feitas diluições adequadas para cada caso específico, visando 390 
adequá-lo para o uso (SOUZA et al., 2012). 391 
Aguirre et al. (2020) avaliaram, em laboratório, seu efeito herbicida em diferentes 392 
concentrações (10, 5, 2 e 1%) frente a plântulas de ervas daninhas. Os resultados 393 
demonstraram mortalidade total das plântulas há apenas dois dias após a aplicação em 394 
todas as concentrações, exceto a de 1%. Nesta concentração a taxa de mortalidade foi mais 395 
lenta, no entanto, o estudo revela o efeito promissor deste produto como herbicida mesmo 396 
em baixas concentrações. 397 
Já em campo, ao se misturar o extrato pirolenhoso a subconcentrações de herbicidas 398 
químicos, há uma potencialização destes herbicidas, reduzindo a quantidade necessária de 399 
produto químico aplicado ao solo, expondo a função adjuvante do EP (SEO et al., 2015). Os 400 
 
13 
 
relatos deste produto agindo de forma eficiente no controle de ervas daninhas estão 401 
presentes em diversos estudos (TIILIKKALA et al., 2010). 402 
A aplicação do EP ao solo interfere na acidez deste solo, aumentando o pH na 403 
camada de 0-20 cm, porém em camadas mais profundas não foram notificadas 404 
modificações significativas (TOGORO et al., 2014). Estes autores ainda mencionam que o 405 
teor de material orgânico deste solo não foi influenciado com a sua aplicação. 406 
Um estudo expõe a eficiência do EP de bambu para melhorar o pH do solo e traz 407 
relatos do emprego de EPs de outras origens interferindo na atividade enzimática do mesmo 408 
(MALIANG et al., 2020). No estudo realizado por Koc et al. (2018) a melhora da atividade 409 
enzimática de alguns solos também foi relatada. Além disso, ele pode ser um poderoso 410 
desinfetante de solo (DORAN, 1932). 411 
Tem sido amplamente avaliado como estimulante do crescimento de plantas. Quando 412 
adicionado a fertilizantes orgânicos tem demonstrado resultados mais eficientes do que o 413 
uso de fertilizantes químicos no cultivo de mudas de Dypsis lutescens, resultando em 414 
plantas mais altas, com maior número de brotações e de folhas (WANDERLEY et al., 2012). 415 
Nos trabalhos realizados com as orquídeas Cattleya loddigesii, Cattleya intermedia e 416 
Miltonia clowesii foi observado aumento na altura das plantas, no número de raízes, de 417 
pseudobúlbos, da massa fresca, maiores áreas foliares, além de aumentar os teores de 418 
nitrogênio foliar (SCHNITZER et al., 2010; SCHNITZER et al., 2015). Silva et al. (2017), 419 
utilizando a espécie Oeceoclades maculata, encontraram resultados promissoresde 420 
desenvolvimento da mesma empregando 50 mL de solução de 2 a 3 mL L-1 de EP no 421 
substrato de fixação. 422 
No cultivo do quiabo, a adição de EP a fertilizantes promoveu maior número de 423 
folhas, raízes mais compridas, maior altura da planta e aumento no peso dos frutos e no 424 
diâmetro foliar (MAHMUD et al., 2016). 425 
Já alguns estudos voltados para a área florestal trazem a aplicação deste produto na 426 
produção de mudas de Pinus elliottii promovendo maior desenvolvimento radicular e foliar 427 
(PORTO et al.,2007) e no aumento da taxa de germinação sementes de Eugenia 428 
dysenterica quando tratadas com baixas concentrações de EP (JOSÉ et al., 2016). Pesenti, 429 
(2021) avaliou seu efeito alelopático gerando controle germinação de Eragrostis plana e 430 
Bidens pilosa, com eficiência de 87 a 100%, respectivamente. As doses aplicadas neste 431 
estudo foram de 17.5% para B. pilosa e 20% para E. plana. 432 
Os usos deste produto no controle de pragas e doenças também estão sendo 433 
amplamente difundidos. A aplicação de EP puro e associado a inseticidas frente a Anticarsia 434 
gemmatalis e Pseudoplusia includens apresenta efeito semelhante ao controle feito com 435 
inseticidas químicos puros (PETTER et al., 2013). A ingestão de folhas de milho embebidas 436 
 
14 
 
com este produto causa mortalidade de Spodoptera frugiperda em sua fase larval 437 
(TRINDADE et al., 2014). Análises in vitro demonstram a ação antifúngica contra fungos 438 
patogênicos da soja (PIETA, 2017). 439 
Na proteção de sementes os efeitos atribuídos ao EP são promissores. Ele 440 
apresentou capacidade de diminuir o ataque do gorgulho Collosobruchus maculates 441 
Frabricius à sementes de feijão caupi, minimizando a postura de ovos e a perfuração das 442 
sementes causada por este inseto (CHALERMSAN e PEERAPAN, 2009). Os autores ainda 443 
fizeram a comparação entre três tipos de EP frente a estas variáveis, o extrato cru, 444 
centrifugado e um comercial. Os resultados expuseram que o EP centrifugado se mostrou o 445 
mais eficiente na redução da perfuração das sementes e da postura dos ovos. 446 
Seu efeito repelente foi também relatado por Rahmat et al. (2014), onde quando 447 
misturado a grãos de milho armazenados em recipiente fechado causaram a mortalidade de 448 
gorgulhos. Os autores atribuem o ocorrido ao fato deste produto repelir os insetos, 449 
deixando-os sem alimento e, consequentemente, levando-os à morte. Uma pesquisa 450 
avaliando a repelência do produto contra mosquitos que se alimentam no corpo humano 451 
mostrou eficácia frente a Culex pipiens pallens e Aedes togoi (KIARIE-MAKARA et al., 452 
2010). 453 
No entanto, um estudo relata um efeito inverso no controle de formigas cortadeiras. 454 
Quando mudas de Eucalyptus sp. foram pulverizadas com concentrações de 1 e 2% de EP 455 
não houve a inibição do ataque das formigas, ao contrário, apresentou-se um aumento no 456 
forrageamento das mesmas (SOUZA-SILVA et al., 2005). Nestas concentrações, este 457 
extrato causa atratividade a estes insetos, isto é sugerido à melhor condição nutricional que 458 
as mudas apresentam, demonstrando que as formigas selecionam mudas mais nutritivas 459 
para suprir as necessidades das colônias (SOUZA-SILVA; ZANETTI, 2007). 460 
Outra área de aplicação que vem sendo estudada é a de culturas hidropônicas. A 461 
adição de 0,25 mL.L-1 de EP em solução hidropônica para a cultura do alface mostrou-se 462 
eficiente para ajustar o pH da solução a um ponto ótimo para esta cultura. Nesta 463 
concentração as plantas absorveram água e nutrientes equiparadamente com a solução 464 
tratada com ácido nítrico, insumo usado comumente neste tipo de cultivo (CHEN et al., 465 
2015). 466 
Além disso, tem sua aplicação como produto defumante e aromatizante, conhecido 467 
como fumaça líquida há muito tempo na indústria alimentícia para conservar e defumar 468 
comida (CAMPOS, 2017). Usado também como antisséptico, aplicado frente a bactérias 469 
causadoras de mastite, reduzindo o número de células bacterianas (SOARES et al., 2020). 470 
Tem efeito nematicida frente a nematoides das galhas e do inhame (FARIAS et al., 2020; 471 
 
15 
 
SANTOS et al., 2017). Está sendo empregado no estudo como fotoprotetor em experimento 472 
com a sua mistura com a quitosana (PORTO et al., 2018). 473 
Pesquisas têm o avaliado como promotor de crescimento e bem estar-animal, 474 
visando introduzir este produto na sua alimentação e assim baratear custos e diminuir o uso 475 
de antibióticos para criação de animais confinados, por exemplo. Diógenes et al., (2019) 476 
avaliaram níveis crescentes de dosagens de EP em ração oferecida a codornas e 477 
monitoraram o desempenho em relação ao ganho de peso dos animais, taxa de conversão 478 
alimentar e consumo da ração e recomendaram a adição da dosagem de 2,5% deste extrato 479 
à alimentação das codornas. Os autores relataram uma diminuição do consumo de ração 480 
nesta dosagem, ou seja, menores quantidades de ração são suficientes para o aumento do 481 
peso dos animais. 482 
 
 
 
 
 
 
 
 
 
 
 
 
16 
 
 
 
 
 
 
 
 
 
 
 
 
 
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agroecologia.org.br/index.php/rbagroecologia/article/download/15449/10664. 774 
 
WANDERLEY, C.S.; FARIA, R.T.; VENTURA, M.U. Chemical fertilization, organic fertilization 775 
and pyroligneous extract in the development of seedlings of areca bamboo palm (Dypsis 776 
lutescens). Acta Scientiarum, v.34, n.2, p.163-167, 2012. Disponível em: 777 
http://doi.org/10.4025/actasciagron.v34i2.12488. 778 
 779 
WORLD HEALTH ORGANIZATION. Antibiotic resistance, 2020. Disponível em: 780 
https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance. 781 
 
YATAGAI, M.; NISHIMOTO, M.; HORI, K.; OHIRA, T.; SHIBATA, A. Termiticidal activity of 782 
wood vinegar, its components and their homologues. J Wood Sci, n.4, p.338-342, 2002. 783 
Disponível em: https://doi.org/10.1007/BF00831357. 784 
 785 
ZIMERMAN, R.A. Uso indiscriminado de antimicrobianos e resistência microbiana. 3. 786 
ed. Brasília: Editora MS, 2010. 787 
 
 
 
 
 
 
 
 
 
 
26 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Capítulo 1 
_____________________________ 
 
27 
 
4. CAPÍTULO 1 
__________________________________________________________________________ 
 
 
 
 
 
EFFECT OF pH ON THE ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF WOOD 
VINEGAR (PYROLIGNEOUS EXTRACT) FROM EUCALYPTUS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
28 
 
EFEITO DO pH NA ATIVIDADE ANTIBACTERIANA E ANTIFÚNGICA DO 
VINAGRE DE MADEIRA (EXTRATO PIROLENHOSO) DE EUCALIPTO 
 
Gil Sander Próspero Gama1, Alexandre Santos Pimenta1, Francisco Marlon Carneiro Feijó2, Caio Sergio Santos2, Renato 
Vinicius Oliveira Castro3, Tatiane Kelly Barbosa de Azevedo1, Lúcio César Dantas de Medeiros1 
 
*Artigo aceito para publicação. Link para as normas da revista no anexo I 
 
RESUMO 788 
O presente trabalho teve como objetivo avaliar o efeito da neutralização progressiva na 789 
atividade antimicrobiana do extrato pirolenhoso (EP) de eucalipto. Amostras de madeira 790 
foram carbonizadas a uma taxa de aquecimento de 0.7 °C min-1 até a temperatura final de 791 
450 °C. Os líquidos brutos da carbonização foram deixados em repouso e a fração aquosa foi 792 
separada. A fração aquosa correspondente ao EP bruto foi destilada obtendo o EP purificado. 793 
Alíquotas de EP purificado foram progressivamente neutralizadas do pH 2.5 até 7.5, 794 
respectivamente, 2,5, 3,0, 3,5, 4,0, 4,5, 5,0, 5,5, 6,0, 6,5, 7,0, e 7,5. Com o método da micro-795 
diluição em caldo, o efeito antimicrobiano das amostras neutralizadas em cada pH foi 796 
avaliado contra Pseudomonas aeruginosa (ATCC 27853), Salmonella enteritidis 797 
(ATCC 13076), Staphylococcus aureus (ATCC 25923), Streptococcus agalactiae (CEPA 798 
CLINICA) e Candida albicans (ATCC 10231). As concentrações inibitórias mínimas e as 799 
concentrações bactericida e fungicida mínimas foram determinadas. Os resultados foram 800 
analisados por regressão e foram ajustados os modelos estatísticos. Foi detectada uma perda 801 
progressiva da atividade antimicrobiana do EP com a neutralização. Entretanto, mesmo em 802 
pH neutro e ligeiramente alcalino, a atividade inibitória ao crescimento microbiano se 803 
manteve em certa extensão. 804 
 805 
Palavras-chave: carbonização de madeira de eucalipto; extrato pirolenhoso de eucalipto; 806 
bactericida e fungicida natural 807 
 
 
 
 
 
 
 
 
29 
 
 
EFFECT OF PH ON THE ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF 
WOOD VINEGAR (PYROLIGNEOUS EXTRACT) FROM EUCALYPTUS 
 
Gil Sander Próspero Gama1, Alexandre Santos Pimenta1, Francisco Marlon Carneiro Feijó2, Caio Sergio Santos2, Renato 
Vinicius Oliveira Castro3, Tatiane Kelly Barbosa de Azevedo1, Lúcio César Dantas de Medeiros1 
 
ABSTRACT 808 
The study aimed to assess the effect of progressive neutralization on the antimicrobial 809 
properties against bacteria and yeasts of wood vinegar obtained from the pyrolysis of 810 
Eucalyptus urograndis wood. Wood samples were carbonized at a heating rate of 0.7 °C min-1 811 
until a final temperature of 450 °C. The raw pyrolysis liquids were left to settle and the 812 
aqueous fraction was separated. Then, the aqueous fraction (raw wood vinegar – WV) was 813 
purified to yield the WV. WV samples were collected and neutralized from pH 2.5 until 7.5 814 
(2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5. Through the broth microdilution method, 815 
the antimicrobial effect of the neutralized samples at each pH was assessed on 816 
Pseudomonas aeruginosa (ATCC 27853), Salmonella enteritidis (ATCC 13076), 817 
Staphylococcus aureus (ATCC 25923), Streptococcus agalactiae (CEPA CLINICA), and 818 
Candida albicans (ATCC 10231). The minimum inhibitory concentration (MIC) and 819 
minimum bactericidal (and fungicidal) concentrations were determined. Results were 820 
submitted to regression analysis, and statistical models were fitted. A progressive decrease in 821 
the antimicrobial activity was determined as pH increased. Nevertheless, even when neutral 822 
and slightly alkaline pH values are reached, the inhibitory activity remained at a certain level. 823 
Higher pH values of the WV were associated with lower antimicrobial activity. However, its 824 
activity remained even at neutral and slightly alkaline pH values. 825 
 826 
Keywords: wood vinegar and pH effect; antibacterial activity; antifungal activity 827 
 
 
 
 
 
 
30 
 
INTRODUCTION 828 
Wood vinegar (WV), pyroligneous acid, pyroligneous extract, and water-soluble liquid 829 
smoke are expressions referring to the same product, namely the aqueous fraction separated 830 
through the settling of the raw pyrolysis liquids (Sena et al. 2014). WV has a complex 831 
chemical composition, which can reach up to 200 compounds, among them phenols, ketones, 832 
furans, aldehydes, pyrans, alcohols, and organic acids (Schnitzer et al. 2015; Araújo et al. 833 
2017; Dias Júnior et al. 2018; Pimenta et al. 2018; Suresh et al. 2019). WV is essentially an 834 
acidic product with a pH ranging usually from 2.5 to 3.6 (Aubin and Roy, 1990; Rahmat et al. 835 
2014; Pimenta et al. 2018), depending on the chemical composition of the pyrolyzed raw 836 
material. This acidity is due to the presence of organic acids, the most common being acetic 837 
and formic acids. The most usual organic acid in WV is acetic acid, with concentrations of 3.0 838 
to 7.4% (Sipilä et al. 1998; Theapparat et al. 2018). 839 
Due to its particular chemical composition, preservative, and medicinal properties, 840 
WV has been employed for several purposes since ancient times (Tiilikkala et al. 2010). There 841 
are millenary reports of its use in the treatment of diseases in China and India (Campos 2007); 842 
salt substitute in 1862 to preserve meats (Kurlansky 2002); soil disinfection (Doran, 1932), 843 
among other applications. This broad variety of applications has led to increasing interest in 844 
this wood-pyrolysis bioproduct by researchers around the world, with several studies focused 845 
on its antimicrobial action and other properties (Araújo et al. 2017; Souza et al. 2018; Chen et 846 
al. 2015; Maliang et al. 2020). The role of phenols and other compounds on the antimicrobial 847 
activity of WV hasbeen well established in recent works, such as Suresh et al. (2019) and 848 
Suresh et al. (2020), for instance. In these works, the authors assessed WV in both acidic and 849 
neutral forms and demonstrated that even after neutralization, the product had antimicrobial 850 
activity, albeit weaker, but not absent. More than that, since after neutralization, the acetic 851 
acid is no longer present in WV, Suresh et al. (2019) highlighted that the antimicrobial effect 852 
cannot be attributed to the presence of this compound alone, and instead must be attributed to 853 
the combined action with several other compounds. 854 
However, the previous research works involving the antimicrobial activity of 855 
neutralized WV were carried out directly with the acidic and neutral versions, without 856 
verifying what happens to that activity during progressive neutralization. Thus, from the 857 
original acidic pH to neutrality, there is a gap in information about the antimicrobial activity 858 
of WV in the pH range from 2.5 to 7.0. In this context, the present work aimed to assess the 859 
 
31 
 
effect of increasing pH (from the original pH of 2.5 until 7.0) on the antimicrobial activity of 860 
wood vinegar (pyroligneous extract) from eucalyptus urograndis – clone I144 on 861 
Pseudomonas aeruginosa, Salmonella enteritides, Staphylococcus aureus, Streptococcus 862 
agalactiae, and Candida albicans. 863 
MATERIAL AND METHODS 864 
 865 
Production and purification of WV 866 
Wood samples from Eucalyptus urograndis trees (usual denomination of hybrids of 867 
Eucalyptus urophylla x Eucalyptus grandis in Brazil), clone I144, were collected. These 868 
samples were collected from 8-year-old plantations in the experimental area of the Science 869 
Unit Agrárias, Agricultural School of Jundiaí, Federal University of Rio Grande do Norte 870 
(coordinates of 05° 51' 30" S and 35° 21' 14" W). Tree selection, harvesting, and wood sample 871 
collection followed the procedures described by Carneiro et al. (2013). The wood samples 872 
consisted of 3 cm thick disks, and before carbonization, they were cut into wedges and oven-873 
dried at 103 °C (+ 2 °C) under forced ventilation until constant weight. The carbonization 874 
runs were performed in a laboratory muffle furnace (Labor SP-1200, SP LABOR, São Paulo, 875 
Brazil) with cry wood samples weighing about 500 g in each run. Twenty runs were 876 
conducted. In each run, the wood samples were placed inside an externally-heated steel 877 
container and submitted to a heating rate of 0.7 °C min-1 from room temperature to 450 °C. 878 
The liquid fraction from the carbonization was recovered using a water-cooled steel 879 
condenser (25 °C). The composite sample of the raw liquids was decanted and the aqueous 880 
supernatant was separated and bi-distilled until 100 – 103 °C to obtain the purified WV. The 881 
purified WV was vacuum filtered through a 0.2 μm filter (MF-Millipore, Merck, Darmstadt, 882 
Germany). 883 
Neutralization of WV 884 
20 mL aliquots from the purified WV resulting from the previous step were obtained 885 
with two replicates of each one. The first ones were kept at their original pH of 2.5. Then, two 886 
by two, the other aliquots were increasingly neutralized to obtain pH values of 3.0, 3.5, 4.0, 887 
4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5. For neutralization, a 2 mol L-1 NaOH solution was employed 888 
and the final pH of the aliquots was monitored with a pHmeter (PG 2000, Gehaka, São Paulo, 889 
SP, Brazil). Thus, 11 types of samples of WV with different pH values were obtained. 890 
 
32 
 
In vitro evaluation of the antimicrobial activity 891 
The assays were carried out by the broth microdilution method with 96-well 892 
microplates, according to the procedures of the CLSI (2012) routine. For the assessment tests, 893 
five microorganisms were employed: four bacterial strains, P. aeruginosa (ATCC 27853), S. 894 
enteritidis (ATCC 13076), S. aureus (ATCC 25923), and S. agalactiae (CEPA CLINICA), and 895 
a strain of the yeast C. albicans (ATCC 10231). All the microorganisms were kept in BHI 896 
(brain heart infusion, Kasvi, São José dos Pinhais-PR, Brazil) under refrigeration, and just 897 
before each assay, they were cultivated in a bacteriological oven (Fanem model 50, 898 
Guarulhos, SP, Brazil) at 37 °C (± 1 °C) for 24 hours with the same culture media. Then the 899 
microbial inocula were prepared. The adjustment of the concentration of bacterial and fungal 900 
cells in the culture media was achieved by employing a spectrophotometer (Biospectro SP-22, 901 
Labmais, Curitiba, PR, Brazil) at the wavelength of 530 nm with reading values from 0.08 to 902 
0.1, corresponding to 0.5 in the MacFarland scale (density of 1.5 x 108 cells mL-1). 903 
Then in each well of the microplates, 100 µL of BHI culture media was added. After 904 
that, serial dilutions were performed for each pH in the BHI medium, as follows: in the first 905 
well, 100 µL of WV was added until reaching a dilution of 50%. From that point onward, a 906 
constant volume of 100 µL was collected from the first well and added to the following one to 907 
reach 25% dilution. The procedure was repeated until reaching a concentration of 0.78123%. 908 
The 100 µL volume extracted from the last well was discarded. Therefore, seven 909 
concentrations of WV at each pH value were obtained: 50, 25, 12.5, 6.25, 3.125, 1.5625, and 910 
0.78123% (always half of the previous concentration starting from a dilution of 50%). For 911 
each concentration and each pH, three replicates were conducted. This way, 231 wells for 912 
observation were obtained (7 concentrations x 11 pH x 3 replicates, or 77 experimental 913 
treatments x 3 replicates). 914 
After the dilutions, 0.5 µL of each microbial inoculum was added to each well. Next, 915 
the microplates were incubated in a bacteriological oven at 37 °C (± 1 °C) for 24 hours. After 916 
that time, the tubes were read visually and the first triplicates of each experimental treatment 917 
that were completely translucid were considered to establish the minimum inhibitory 918 
concentration (MIC). The visual reading was employed since the colorimetric method usually 919 
applied using resazurin was inefficient. After the visual assessment, the microplates were read 920 
in an ELISA microplate reader (model 660, URIT Medical Electronic, Nanshan, Shenzhen, 921 
China) to determine the absorbances, a way to quantify the microbial growth according to the 922 
 
33 
 
concentration and pH. For each experimental treatment, three replicates were read. The 923 
positive control for the readings was achieved with chlorhexidine (Vic Pharma by Shülke, 924 
Taquaritinga, SP, Brazil) at 0.2%. The minimum bactericidal concentration (MBC) and 925 
minimum fungicidal concentration (MFC) were determined from the MIC value measured for 926 
each combination of microorganism and pH levels, where the solution of the MIC well and 927 
those of higher concentration were inoculated in Petri dishes containing BHI culture medium. 928 
The Petri dishes were incubated in a bacteriological oven at 37 °C (± 1 °C) for 24 hours. After 929 
this period, the presence or absence of microbial colonies was assessed. 930 
Statistical analysis 931 
The experimental data from the readings of absorbance were subjected to regression 932 
analysis by employing the R software (version 4.1.3) at the moment of the inoculation (zero 933 
hours) and after 24 hours. For each type of microorganism, at each pH level, a regression 934 
model was fitted to describe and predict the behavior of absorbance (Y) as a function of WV 935 
concentration (X). The best statistical prediction models were selected based on the following 936 
decision criteria: high correlation coefficient (R) between experimental and estimated values, 937 
the significance of the model parameter, biological realismof the model, and root-mean-938 
square deviation (RMSE). Among several models assessed, the logistic model [Y= β1 ln(X) + 939 
β0 + ε, recommended by Gujarati and Porter (2009)] was the best to explain the behavior of 940 
the absorbances after 24 hours. The data on absorbances were graphically plotted on zero time 941 
only to demonstrate their pattern of behavior after inoculation. For the absorbance after 24 942 
hours of incubation of the microbial cultures, a specific model was adjusted for each 943 
combination of pH level and concentration. After the adjustment of the equations, the curves 944 
of the factor pH for the same microorganism were compared to each other using the model 945 
identity test, according to the routine described by Regazzi (1993, 1996, 1999) and previously 946 
applied in a similar experiment (Araújo et al. 2017). 947 
RESULTS 948 
Antimicrobial activity: determination of MIC, MBC, and MFC 949 
Table 1 contains the results obtained for the MIC at each pH for the five 950 
microorganisms. The pattern observed was a decrease in the antimicrobial effect as the pH got 951 
closer to neutral. The results obtained for MBC and MFC, also displayed in Table 1 (numbers 952 
in italics), follow the same trend as the MIC. At lower pH levels, the concentrations required 953 
to inhibit the microbial growth were lower and as the pH approached neutral, the 954 
 
34 
 
concentrations needed to achieve the same effect were higher. The value of MIC, MBC and 955 
MFC of chlorhexidine (positive control) was constant, presenting a value of 0.003%. 956 
Table 1. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations 
(MBC) values against the microorganisms according to increasing pH levels 
Microorganism 
WV concentration (%) 
pH 
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 
P. aeruginosa 3.12 3.12 3.12 6.25 6.25 12.5 12.5 25 25 50 25 
 12.5 6.25 6.25 12.5 12.5 12.5 25 50 50 50 50 
S. enteritidis 3.12 3.12 3.12 6.25 6.25 6.25 25 25 25 50 25 
 3.12 6.25 6.25 25 12.5 12.5 25 50 50 >50 50 
S. aureus 6.25 6.25 6.25 6.25 12.5 12.5 25 50 50 >50 50 
 6.25 6.25 6.25 12.5 12.5 25 25 50 >50 >50 >50 
S. agalactiae 6.25 6.25 6.25 6.25 12.5 12.5 25 50 50 >50 >50 
 6.25 6.25 12.5 12.5 25 25 25 50 50 >50 >50 
C. albicans 3.12 3.12 3.12 6.25 6.25 12.5 25 25 25 25 25 
 3.12 3.12 3.12 6.25 6.25 25 25 25 25 50 50 
Where: *In Table body, for each microorganism, the first line corresponds to the MIC and the numbers in italics are the 
MBC; **For Candida albicans, the numbers in italics are the minimum fungicidal concentrations; ***The sign > is 
employed to suggest the possibility that a concentration higher than 50% can result in inhibition 
Antimicrobial activity: behavior of the absorbances 957 
Figure 1 contains the graphs based on the models fitted from the regression analysis of 958 
the absorbances, each one corresponding to a single microorganism. In all components 959 
displayed in Figure 1, graphs identified with the letter A are representations of the behavior of 960 
the absorbances of the culture media at the 0 hours (just after the inoculation) and after 24 961 
hours of incubation (B). Although the MIC, MBC, and MFC values could not be determined 962 
from the graphs in Figure 1, the knowledge of the absorbances was still a valuable tool to 963 
quantify precisely the interaction between pH, WV concentration, and microbial growth, as 964 
will be discussed in the next section. In the graphs marked with the B letter, at the same pH, 965 
higher absorbances are associated with lower efficacy of the WV. 966 
 
35 
 
 
 
 
 
 
36 
 
 
Figure 1. Graphs representing the effect of the pH of WV on the growth of Pseudomonas 
aeruginosa, Salmonella enteritides, Staphylococcus aureus, Streptococcus agalactiae, and 
Candida albicans as a function of the concentration at zero time (A) and 24 hours (B) after 
incubation 
In Table 2, the regression models that were adjusted to each microorganism are 967 
presented. As can be observed in the figures, as the concentration decreased and the pH 968 
increased, the absorbances rose. A higher value of absorbance indicates that the culture 969 
medium became more turbid due to the higher concentration of microbial cells that 970 
developed. For the models that were adjusted to explain the effect of pH in the growth of P. 971 
aeruginosa (Table 2), a low value of the coefficient of correlation was determined to the pH 972 
of 2.5, which implicates a relatively high dispersion of the experimental data having the 973 
regression as reference. However, a trend of behavior is reflected by the regression line and 974 
for the other pH values, this pattern is confirmed since the values of R2 for the same type of 975 
model are higher with a maximum value of 0.8348 for the pH of 5.5. 976 
Table 2. Regression models that were adjusted to explain the behavior of absorbances from 
cultures of Pseudomonas aeruginosa, Salmonella enteritides, Staphylococcus aureus, 
Streptococcus agalactiae, and Candida albicans after 24 hours of incubation at each pH level 
according to WV concentration 
 Pseudomonas aeruginosa 
pH Regression Model R2 
2.5 Y = – 0.266 ln X + 0.9922 0.5539 
3.0 Y = – 0.287 ln X + 1.0194 0.6047 
3.5 Y = – 0.284 ln X + 1.0201 0.6416 
4.0 Y = – 0.344 ln X + 1.2765 0.8074 
4.5 Y = – 0.358 ln X + 1.2777 0.7514 
5.0 Y = – 0.384 ln X + 1.3839 0.7885 
5.5 Y = – 0.379 ln X + 1.4791 0.8348 
6.0 Y = – 0.346 ln X + 1.5663 0.8170 
6.5 Y = – 0.323 ln X + 1.5487 0.7152 
7.0 Y = – 0.292 ln X + 1.5721 0.7252 
7.5 Y = – 0,337 ln X + 1,4743 0.8003 
 Salmonella enteritides 
2.5 Y = – 0.063 ln X + 0.4273 0.3095 
 
37 
 
3.0 Y = 0.0369 ln X – 0.0014 0.2296 
3.5 Y = – 0.099 ln X + 0.5175 0.6156 
4.0 Y = – 0.136 ln X + 0.6351 0.7253 
4.5 Y = – 0.164 ln X + 0.6957 0.7816 
5.0 Y = – 0.224 ln X + 0.8705 0.8319 
5.5 Y = – 0.243 ln X + 0.9836 0.9399 
6.0 Y = – 0.213 ln X + 0.9241 0.8866 
6.5 Y = – 0.253 ln X + 1.1261 0.9470 
7.0 Y = – 0.223 ln X + 1.1013 0.9591 
7.5 Y = – 0.217 ln X + 0.9850 0.9623 
 Staphylococcus aureus 
2.5 Y = – 0.014 ln X + 0.1428 0.1089 
3.0 Y = – 0.094 ln X + 0.4339 0.6699 
3.5 Y = – 0.109 ln X + 0.4865 0.7822 
4.0 Y = – 0.140 ln X + 0.5964 0.8711 
4.5 Y = – 0.121 ln X + 0.5055 0.7285 
5.0 Y = – 0.146 ln X + 0.5980 0.8722 
5.5 Y = – 0.144 ln X + 0.6379 0.9417 
6.0 Y = – 0.154 ln X + 0.7425 0.8401 
6.5 Y = – 0.158 ln X + 0.7783 0.9051 
7.0 Y = – 0.110 ln X + 0.6978 0.8046 
7.5 Y = – 0.114 ln X + 0.5974 0.9420 
 Streptococcus agalactiae 
2.5 Y = – 0.100 ln X + 0.5120 0.5393 
3.0 Y = – 0.098 ln X + 0.4993 0.6694 
3.5 Y = – 0.114 ln X + 0.5509 0.7903 
4.0 Y = – 0.122 ln X + 0.5932 0.8116 
4.5 Y = – 0.130 ln X + 0,5996 0.8130 
5.0 Y = – 0.155 ln X + 0.7264 0.8039 
5.5 Y = – 0.150 ln X + 0.7993 0.7223 
6.0 Y = – 0.089 ln X + 0.7560 0.3874 
6.5 Y = – 0,100 ln X + 0.7058 0.6428 
7.0 Y = – 0.064 ln X + 0.7066 0.5442 
7.5 Y = – 0,080 ln X + 0.6972 0.5923 
 Candida albicans 
2.5 Y = – 0.014 ln X + 0.0513 0.2943 
3.0 Y = – 0.066 ln X + 0.3515 0.5429 
3.5 Y = – 0.084 ln X + 0.3891 0.7021 
4.0 Y = – 0.056 ln X + 0.2616 0.2572 
4.5 Y = – 0.106 ln X + 0.4449 0,5773 
5.0 Y = – 0.142 ln X + 0.6274 0.7734 
5.5 Y = – 0.149 ln X + 0.6668 0.7814 
6.0 Y = – 0.083 ln X + 0.5174 0,3000 
6.5 Y = – 0.123 ln X + 0.6511 0.5792 
7.0 Y = – 0.146 ln X + 0.7763 0.7761 
7.5 Y = – 0.158 ln X + 0.7951 0.7770 
*Y = absorbance; X = WV concentration; R2 = coefficient of determination 
Regarding the models that were adjusted to predict the growth of S. enteritides 977 
(Table 2) under the effect of variable concentrations of WV in different pH, for the values of 978 
pH of 2.5 and 3.0, the coefficients of correlation were low, 0.3095 and 0.2296, respectively. 979 
From pH 3.0 ahead, the values of R2 were higher reaching 0.9623 at pH 7.0. For S. 980 
enteritides, as a general trend, the values of R2 from pH of 5.5 until7.5 were the highest 981 
among the microorganisms assessed, which represents good power of predictability of growth 982 
pattern by the logistic model. 983 
 
38 
 
For the growth of S. aureus, the value of R2 at the pH 2.5 was the lowest among all the 984 
microorganisms that were assessed with a value of 0.1089 (Table 2). However, from this point 985 
ahead, the values of the coefficient of correlation were higher than 0.700, except at the pH of 986 
3.0 where the value was 0.6699. Once again, some variation in the growth of the 987 
microorganism at these values of pH (and included in the error of the adjustment) resulted in 988 
lower values of R2. As displayed in Table 2, the growth of S. agalactiae was best explained by 989 
the logistic model in the pH range of 3.5 to 5.5, which is reflected by the higher values of the 990 
coefficient of correlation in this range. Despite presenting good coefficients of correlation in 991 
general, the logistic model was not so effective to explain the growth of the yeast C. albicans 992 
at the pH values of 2.5, 4.0, and 6.0. At these points, the R2 had low values which shows a 993 
variable response of the microorganism to the action of WV as pH was varying. 994 
Reported in Table 3 are the results of the identity test of the regression models. As a 995 
general trend, the identity tests demonstrated that as pH increased, the quality of the WV as an 996 
antimicrobial changed, most likely due to the differential neutralization of its phenolic 997 
components. This difference in the quality of WV in each pH was positively determined by 998 
the statistical dissimilarity among the models compared through the identity test. 999 
 
 
 
 
 
 
 
39 
 
Table 3. Results of the identity test applied to the regression models adjusted for the absorbances of the 
microbial cultures after 24 hours of incubation (A): Pseudomonas aeruginosa, Salmonella enteritides, 
Staphylococcus aureus, and Streptococcus agalactiae; (B): Candida albicans 
A B 
Comparisons 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Comparisons 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 
2.5 ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ 2.5 ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ 
3.0 ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ 3.0 ≠ = ≠ ≠ ≠ ≠ ≠ ≠ ≠ 
3.5 ≠ ≠ ≠ ≠ ≠ ≠ ≠ ≠ 3.5 = ≠ ≠ ≠ ≠ ≠ ≠ ≠ 
4.0 ≠ ≠ ≠ ≠ ≠ ≠ ≠ 4.0 ≠ ≠ ≠ ≠ ≠ ≠ ≠ 
4.5 ≠ ≠ ≠ ≠ ≠ ≠ 4.5 ≠ ≠ ≠ ≠ ≠ ≠ 
5.0 ≠ ≠ ≠ ≠ ≠ 5.0 ≠ ≠ ≠ ≠ ≠ 
5.5 ≠ ≠ ≠ ≠ 5.5 ≠ ≠ ≠ ≠ 
6.0 ≠ ≠ ≠ 6.0 ≠ ≠ ≠ 
6.5 ≠ ≠ 6.5 ≠ ≠ 
7.0 ≠ 7.0 ≠ 
Where: symbol for different (≠): the statistical modes differ statistically; equal symbol (=): the models do not differ statistically 
 
 
 
 
40 
 
DISCUSSION 1000 
Antimicrobial activity: determination of MIC, MBC, and MFC 1001 
The data displayed in Table 1 corroborate the results of the positive antimicrobial 1002 
effects achieved by several researchers listed in the review published by Tiilikkala et al. 1003 
(2010), and more recently by Souza et al. (2018). Other studies have demonstrated the 1004 
antibacterial and antifungal properties of WV from varied lignocellulosic sources (Ibrahim et 1005 
al. 2013; Araújo et al. 2013; Abas et al. 2018, Souza et al. 2018). The experimental results 1006 
presented here corroborate the results reported by Velmurugan et al. (2009) and Suresh et al. 1007 
(2019), who assessed the antimicrobial effect of different types of WV both in their original 1008 
acid form and after neutralization. Both those studies described the antimicrobial effect of 1009 
WV before and after neutralization and found a decrease in the activity but not the 1010 
disappearance of the biological effect when pH became neutral. 1011 
As can be observed in Table 1, the results demonstrated that as the neutralization 1012 
increased and the pH approached 7.0, the concentration of WV required for microbial 1013 
inhibition increased. Even at neutral pH, the WV still had antimicrobial activity, although 1014 
needing higher concentrations for this purpose. For P. aeruginosa, the MIC of WV at pH 2.5 1015 
was 3.12% and increased to 50% at neutral pH. When the pH reached 7.5, the MIC returned 1016 
to the same value of 25% observed for pH 6.5. The same relationship between MIC and pH 1017 
was determined for S. enteritides. For C. albicans, the MIC at pH 2.5 was 3.12% and 1018 
increased to 25% at pH 5.5, after which it remained constant until the pH reached 7.5. The 1019 
pattern of inhibition as a function of the concentration and pH of the WV is different for each 1020 
microorganism. As commented by Suresh et al. (2019), the activity of neutralized WV 1021 
indicates that the antibacterial property of the product is due to its complex chemical 1022 
composition, and not the presence of acetic acid. Citing other authors, Suresh et al. (2019) 1023 
highlighted that the inhibition of the WV against microorganisms, especially fungi, is caused 1024 
by the antioxidative property of the phenolic compounds. In this respect, previous studies 1025 
have reported that the inhibition of lipid oxidation caused by phenolics is enhanced at acidic 1026 
pH. 1027 
However, for S. aureus and S. agalactiae, a different pattern of inhibition was 1028 
observed. For the first microorganism, when the pH was equal to 7.0 (neutral), no inhibition 1029 
in the growth of the culture was observed. When the pH became slightly alkaline (7.5), the 1030 
MIC was 50%, which is the same concentration required to inhibit completely the culture 1031 
 
41 
 
growth at pH 6.5. In the case of S. agalactiae, for both pH levels, 7.0 and 7.5, a WV 1032 
concentration of 50% was not enough to inhibit the microbial growth. Is important to 1033 
highlight that both microorganisms required higher WV concentration to inhibit growth 1034 
starting with the original pH, which was 2.5. For the other three microorganisms, the initial 1035 
MIC value was 3.12%. There are several compounds (phenolics and ketones) in WV’s 1036 
chemical composition that have antimicrobial activity, so most likely they interact differently 1037 
with one or another microorganism due to differences in cell wall structure and composition. 1038 
These differences among microorganisms combined with the degree of response to the action 1039 
of one or another compound in the chemical composition of WV probably explain why the 1040 
product does not inhibit the microbial growth with the same inhibition results at the same 1041 
concentration. In the present study, S. aureus and S. agalactiae were the most resistant species 1042 
to the inhibitory effect of WV, while C. albicans was the most sensitive one (Table 1). 1043 
The work from Suresh et al. (2019) reported a decrease in the effect of the product 1044 
after neutralization but not complete disappearance, the same trend reported in the present 1045 
work. Still, based on their experimental data, those authors stated categorically that the 1046 
antimicrobial effect of the WV even after neutralization indicated that the antibacterial 1047 
property of the product was due to its complex chemical composition rather than the large 1048 
presence of acetic acid. In their experiments assessing the antimicrobial action on Escherichia 1049 
coli, Enterobacter aerogenes, P. aeruginosa, Listeria monocytogenes, and Enterococcus 1050 
faecalis, the authors found a loss of activity ranging from 10 to 24%, with the degree of loss 1051 
varying according to the species. 1052 
Antimicrobial activity: behavior of the absorbances 1053 
The pattern of increasing values of absorbance in the graphs in Figure 1 reflects the 1054 
intensity of the microbial growth, which means more cells could develop in the medium, 1055 
making it more turbid and preventing light from passing through (Cunha and Vieira 2014). On 1056 
the right side of the figures, the different pH levels are identified by colors that are the same 1057 
as the plottedcurves. In Table 2, the regression models fitted to explain the effect of the pH of 1058 
WV on each microorganism according to the concentration are displayed. The models can 1059 
predict the behavior of the absorbance (Y) as a function of WV concentration (X). 1060 
From the curves displayed in Figure 1, it is possible to verify that at lower pH levels, 1061 
the concentrations of WV required to achieve the antimicrobial effect were lower, 1062 
corroborating the experimental data on MIC described previously. As the pH of WV 1063 
 
42 
 
increased, the requirement for higher concentrations of the product to inhibit microbial growth 1064 
also increased, reflected by higher absorbance values when WV low concentrations were not 1065 
effective. Another trend that corroborates the behavior of the data displayed in Table 1 is that 1066 
the absorbances at pH of 7.0 were always higher than those at pH 7.5. That fact indicates that 1067 
the statement that the antimicrobial properties are not solely related to the effect of acetic acid 1068 
is correct (Suresh et al. 2019). The acetic acid present in a given aqueous solution is 1069 
completely converted to sodium acetate at pH 7.0 and remains that way at alkaline pH levels. 1070 
Consequently, there is no possibility of its being responsible for the antimicrobial effect of 1071 
WV found in this work, at both pHs of 7.0 and 7.5. The same pattern was found by other 1072 
authors cited previously. 1073 
Therefore, the pH of the WV undoubtedly influences its antimicrobial activity but does 1074 
not cause it alone. The chemical composition of the WV reported in the literature shows a 1075 
product composed of around 200 compounds (Schnitzer et al. 2015; Araújo et al. 2017; 1076 
Pimenta et al. 2018). Among these components, alcohols, furans, ketones, organic acids, 1077 
phenols, and pyrans are the most representative and abundant (Theapparat et al. 2018). The 1078 
phenolic compounds are the main group with which antimicrobial properties are closely 1079 
associated, a fact is long proven by scientific reports (Abas et al. 2018). Also, Suresh et al. 1080 
(2019) highlighted that fact and also mentioned that each component has a different mode of 1081 
action. Because of this, according to the authors, WV is even more interesting for use as an 1082 
antimicrobial agent, since it is unlikely the microorganisms will develop any mechanism of 1083 
resistance against all the components of the product at the same time. 1084 
In the present work, what varies regarding a particular microorganism are the pH of 1085 
the WV and the concentration of this product. Thus, for each microorganism, regression 1086 
models were fitted, one model for each pH level, where the independent variable is the 1087 
absorbance and the dependent one is the WV concentration. This way, for each 1088 
microorganism subjected to the action of WV, the models fitted for each pH could be 1089 
compared to detect differences in the antimicrobial action depending on pH. The importance 1090 
of this comparison is not only to determine the effectiveness of the WV itself at each pH level 1091 
but also to provide information about the interaction between the microorganism and the WV 1092 
at a given pH when the concentration is varied. 1093 
Then, as displayed in Table 3 (A), the quality of WV changed as pH increased, since 1094 
the comparison among the models was different from one pH to another. In other words, for 1095 
 
43 
 
each microorganism, the variation of pH could result in a different effect of WV and 1096 
concomitantly a specific type of interaction. Something in the WV was decreasing and 1097 
making it weaker, so as the pH of the WV increased, higher concentrations of the product 1098 
were required to continue inhibiting the growth of the five microorganisms in the culture 1099 
medium. This pattern corroborates the results of Setiawati et al. (2019), cited previously, who 1100 
observed that changes in the composition occur with different pH levels. A slightly different 1101 
pattern was observed in Table 3 (B), where the identity test, when applied to the models fitted 1102 
for C. albicans, determined that the effect of WV at pH of 3.0 and 3.5 was equal to that at pH 1103 
4.0, probably meaning the presence of an interaction of the inhibitory product with this 1104 
microorganism. Nevertheless, for all other pH levels, the regression models were different 1105 
from each other. 1106 
As commented by Pimenta et al. (2018), the partial loss of the antimicrobial activity 1107 
can probably be attributed to the reaction of the sodium hydroxide with the phenolic 1108 
compounds, turning them into salts, a type of chemical change that deactivates their hydroxyl 1109 
groups, which are responsible for the antiseptic properties presented by most of them in the 1110 
pristine form. Phenols are acids with pKa around 10.0, so they are weak acids. There are at 1111 
least 20 types of phenols in WV’s chemical composition (Araújo et al. 2017; Pimenta et al., 1112 
2018), and since each compound is different, as the pH increases, their hydroxyl groups are 1113 
not equally neutralized because of the substituent groups that are present in the aromatic ring. 1114 
This way as the pH increases, most likely different chemical species are generated in aqueous 1115 
media, ones more available than others to exert an antimicrobial effect. 1116 
The results of Setiawati et al. (2019) and the results of the present work corroborate 1117 
the points raised in the previous paragraph. The cited authors, when evaluating neutralized 1118 
WV, found some change in the percentage of phenolic compounds in the chemical 1119 
composition of the product obtained from durian wood. According to them, in the acidic 1120 
version, the main compound was guaiacol, while in the neutralized product, pyrocatechol was 1121 
the prevailing substance. The explanation presented by the authors was that in the neutralized 1122 
form, alkyl groups in the para position (carbon 4) of phenolics accept electrons and that 1123 
behavior decreases the ionization of the compounds due to the addition of NaOH in WV. The 1124 
changes in the proportion of phenolic compounds in the neutral version of WV explain why 1125 
the product becomes less effective when compared to the acidic versions in terms of the 1126 
power to inhibit the growth of microorganisms. These results were expected to a certain 1127 
 
44 
 
extent because according to Brown et al. (1997), acid and basic solutions can differ greatly 1128 
from each other in their chemical properties so products obtained from the reaction after 1129 
neutralization do not have the same characteristics as the original solution. However, since 1130 
WV is a solution containing many kinds of compounds, even with its acid fraction completely 1131 
neutralized, there are still other compounds preserving the bioactive characteristic of the 1132 
product. 1133 
Further research should be performed to understand the specific chemical species of 1134 
WV that prevail as inhibitors at each pH level when increasing neutralization as carried out. 1135 
This could enable predicting the behavior of the product when employed in varied 1136 
applications as a natural antibiotic or parasiticide. For example, if the product is employed as 1137 
an additive for animal feed, the WV after being swallowed will find strong acidic conditions 1138 
in the digestive tract of poultry and swine. In this condition, the inhibitory power of WV on 1139 
microorganisms is maximized. However, if the intention is to use the product to compose drug 1140 
formulations for external uses such as ointments and creams, or to deter parasites like ticks, 1141 
the importance of the pH in the final use may be important to maximize the action of the 1142 
product. 1143 
CONCLUSIONS 1144 
 1145 
The eucalyptus WVmaintained its antimicrobial effect even at neutral and slightly 1146 
alkaline pH, which runs counter to the claims in other studies that the inhibitory action of the 1147 
product versus microorganisms is related just to the presence of acetic acid in its chemical 1148 
composition. Our experimental data indicate the potential of the possible antibacterial and 1149 
antifungal use of WV. However, it is important to highlight that all the experiments reported 1150 
here were conducted in vitro so other research works should investigate the behavior of WV 1151 
under in vivo conditions. Another important aspect is the degree of purification of the AP for 1152 
such applications. The model identity test was a valuable tool to detect different responses of 1153 
microorganisms at each pH level, validating the attribution of the bioactive action of WV to 1154 
the set of components as a whole and not only to a single compound. 1155 
ACKNOWLEDGEMENTS 1156 
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de 1157 
Nível Superior – Brasil (CAPES) – Finance Code 001 and CNPq (National Research Council, 1158 
Brazil). 1159 
 
45 
 
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pyroligneous acid from oil palm fiber. Journal of Applied Pharmacology Sciences 2018; 1162 
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Araújo ES, Pimenta AS, Feijó FMC, Castro, RVO, Fasciotti M, Monteiro TVC, Lima KMG. 1164 
Antibacterial and antifungal activities of pyroligneous acid from the wood of Eucalyptus 1165 
urograndis and Mimosa tenuiflora. Journal of Applied Microbiology 2017; 124(1):85-96. 1166 
Aubin H, Roy C. Study on the corrosiveness of wood pyrolysis oils. Fuel Science and 1167 
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Brown TL, Lemay Jr HE, Bursten BE, Murphy C, Woodward P, Langford S, Sagatys D, 1169 
George A. Chemistry: The Central Science, 3rd ed. 1997, Pearson, Australia. 1170 
Campos AD. Técnicas para produção de extrato rodução pirolenhoso para uso agricola. 1171 
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do Norte. Revista Árvore 2013; 37(6):1-12. 1176 
Chen J, Wu JH, Si HP, Lin KY. Effects of adding wood vinegar to nutrient solution on the 1177 
growth, photosynthesis, and absorption of mineral elements of hydroponic lettuce. Journal of 1178 
Plant Nutrition 2015; 39(4):456-462. 1179 
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Cunha AHN, Vieira JA. Detecção da bactéria Escherichia coli em águas residuárias 1183 
empregando sistema em fluxo por turbidimetria. Revista Mirante 2014, 7(2). 1184 
Dias Jr AF, Andrade CR, Protásio TP, Melo ICNA, Brito JO, Trugilho PF. Pyrolysis and wood 1185 
by-products of species from the Brazilian semi-arid region. Scientia Forestalis 2018; 1186 
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Doran WL. Acetic acid and pyroligneous acid in comparison with formaldehyde as soil 1188 
disinfectants. Journal of Agricultural Research 1932, 44(7):71-578. 1189 
Gujarati DN, Porter DC. Basic Econometrics, 50nd ed., Editora McGraw-Hill 2009; Boston, 1190 
MA, USA. 1191 
Ibrahim D, Kassim J, Sheh-Hong L, Rusli W. Efficacy of pyroligneous acid from Rhizophora 1192 
apiculata on pathogenic Candida albicans. Journal of Applied Pharmacology Science 2013; 1193 
3(7)007-013. 1194 
Kurlansky M. Salt: a world history. 6th ed., Ed Pinguin Books 2014; Los Angeles, CA, USA. 1195 
Lebois M, Connil N, Onno B, Prévost H, Dousset X. (2004) Effects of divercin V41 1196 
combined to NaCl content, phenol (liquid smoke) concentration and pH on Listeria 1197 
monocytogenes ScottA growth in BHI broth by an experimental design approach. Journal of 1198 
Applied Microbiology 2004; 96(5)931-937. 1199 
Li R, Narita R, Nishimura H, Marumoto S, Yamamoto SP, Ouda R, Yatagai M, Fujita T, 1200 
Watanabe T. Antiviral activity of phenolic derivatives in pyroligneous acid from hardwood, 1201 
softwood, and bamboo. Sustainable Chemical Engineering 2017; 6(1):119-126. 1202 
Maliang H, Tang L, Lin H, Chen A, Ma J. Influence of high-dose continuous applications of 1203 
pyroligneous acids on soil health assessed based on pH, moisture content, and three 1204 
hydrolases. Environmental Science and Pollution Research 2020; 27(20):15426-15439. 1205 
Medeiros LCD, Pimenta AS, Braga RM, Carnaval TKA, Medeiros Neto PN, Melo DMA. 1206 
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Eucalyptus urograndis and Mimosa tenuiflora. Revista Árvore 2020, 43(4):e430408. Doi: 1208 
http://dx.doi.org/10.1590/1806-90882019000400008 1209 
Pimenta AS, Fasciotti M, Monteiro TVC, Lima KMG. Chemical composition of pyroligneous 1210 
acid obtained from eucalyptus GG100 clone. Molecules 2018; 23(2):426. 1211 
Rahmat B, Pangesti D, Natawijaya D, Sufyadi D. Generation of wood-waste vinegar and its 1212 
effectiveness as a plant growth regulator and pest insect repellent. Bioresources 2014, 1213 
9(4):6350-6360. 1214 
Regazzi AJ. Test to verify the identity of regression models and equality of some parameters 1215 
in an orthogonal polynomial model. Revista Ceres 1993; 40:176-195 1216 
 
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Regazzi AJ. Test to verify identity of regression models. Pesquisa Agropecuária Brasileira 1217 
1996; 31:1-17. 1218 
Regazzi AJ. Test to verify the identity of regression models and the equality of parameters in 1219 
the case of experimental designs. Revista Ceres 1999; 46:383-409. 1220 
Schnitzer JA, Su MJ, Ventura MU, Faria RT. Doses de extrato pirolenhoso no cultivo de 1221 
orquídea. Revista Ceres 2015; 62(1):101-106. 1222 
Sena MFM, Andrade AM, Thode Filho S, Santos FR, Pereira LF. Potencialidades do extrato 1223 
pirolenhoso: práticas de caracterização. Revista Eletrônica em Gestão, Educação e Tecnologia 1224 
Ambiental 2014, 18(14):41-44. 1225 
Setiawati E, Annisia W, Soedarmanto H, Iskandar T. Characterization of neutralized wood 1226 
vinegar derived from durian wood (Durio zibethinus) and its prospect as pesticide in acidic 1227 
soil. International Seminar and Congress of Indonesian Soil Science Society 2019; IOP 1228 
Conference Series: Earth and Environmental Science, Bogor, Java Occidental, Indonesia. 1229 
Sipilä K, Kuoppala E, Fagernaés L, Oasmaa A. Characterization of biomass-based flash 1230 
pyrolysis oils. Biomass and Bioenergy 1998; 14(2):103-113. 1231 
Soares WNC, Lira GPO, Santos CS, Dias GN, Pimenta AS, Pereira AF, Benício LDM, 1232 
Rodrigues GSO, Amora SSA, Alves ND, Feijó FMC. Pyroligneous acid from Mimosa 1233 
tenuiflora and Eucalyptus urograndis as an antimicrobial in dairy goats. Journal of Applied 1234 
Microbiology 2020; 131(2):604-614. 1235 
Souza JLS, Guimarães VBS, Campos AD, Lund RG. Antimicrobial potential of pyroligneous 1236 
extracts – a systematic review and technological prospecting. Brazilian Journal of 1237 
Microbiology 2018; 49(1):128-139. 1238 
Suresh G, Pakdel H, Roussi T, Brar SK, Fliss I, Roy C. In vitro evaluation of antimicrobial 1239 
efficacy of pyroligneous acid from softwood mixture. Biotechnology Research and Innovation 1240 
2019; 3(1):47-53. 1241 
Suresh G, Pakdel H, Roussi T, Brar SK, Diarra M, Roy C. Evaluation of pyroligneous acid as 1242 
a therapeutic agent against Salmonella in a simulated gastrointestinal tract of poultry. 1243 
Brazilian Journal of Microbiology 2020; 51:1309-1316. 1244 
 
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Theapparat Y, Chandumpai A, Leelasuphakul W, Faroongsarng D. Physicochemistry and 1245 
utilization of wood vinegar from carbonization of tropical biomass waste. IntechOpen2018; 1246 
Open Access Books (Chapter 8). 1247 
Thurette J, Membre JM, Ching LH, Tailiez R, Catteau M. Behavior of Listeria spp. in smoked 1248 
fish products affected by liquid smoke, NaCl concentration, and temperature. Journal of. Food 1249 
Protection 1998; 61(11):1475–1479. 1250 
Tiilikkala K, Fagernas L, Tiilikkala J. History and use of wood pyrolysis liquids as biocide 1251 
and plant protection product. Open Agriculture Journal 2020; 4, 111-118. 1252 
Velmurugan N, Han SS, Lee YS. Antifungal activity of neutralized wood vinegar with water 1253 
extracts of Pinus densiflora and Quercus serrata sawdust. International Journal of 1254 
Environmental Research 2009; 3(2):1735-6835. 1255 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
49 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Capítulo 2 
_____________________________ 
 
50 
 
5. CAPÍTULO 2 
__________________________________________________________________________ 
 
 
 
 
 
 
ATIVIDADE ANTIMICROBIANA E PERFIL QUÍMICO DE ÁCIDOS PIROLENHOSOS DE 
BAMBU E EUCALIPTO 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
51 
 
ATIVIDADE ANTIMICROBIANA E PERFIL QUÍMICO DE ÁCIDOS 
PIROLENHOSOS DE BAMBU E EUCALIPTO 
 
Gil Sander Próspero Gama1, Alexandre Santos Pimenta1, Francisco Marlon Carneiro Feijó2, Caio Sérgio dos 
Santos2, Bruno Caio Chaves Fernandes3, Moacir Franco de Oliveira2, Elias Costa de Souza4, Thays V. C. 
Monteiro5, Maíra Fasciotti5, Tatiane Kelly Barbosa de Azevedo1, Rafael Rodolfo de Melo2, Ananias Francisco 
Dias Júnior6 
 
*Artigo submetido para publicação. Link para as normas da revista no anexo I 
 
RESUMO 1256 
 1257 
A resistência microbiana a drogas é um problema de saúde pública; portanto, há uma busca por alternativas para 1258 
substituir produtos convencionais com agentes naturais. Um dos potenciais agentes antimicrobianos é o vinagre 1259 
(ácido pirolenhoso) derivado da carbonização de matérias-primas lignocelulósicas. Os objetivos do presente 1260 
trabalho foram avaliar a ação antibacteriana e antifúngica de dois tipos de vinagre de carbonização, um de 1261 
madeira de Eucalyptus urograndis e outra da biomassa de Bambusa vulgaris, e determinar seu perfil químico. O 1262 
efeito antimicrobiano foi avaliado contra Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella 1263 
enteritidis, Escherichia coli, Streptococcus agalactiae e Candida albicans. A concentração inibitória mínima e o 1264 
poder bactericida e fungicida mínimo concentrações foram determinados. Micrografias dos microorganismos 1265 
antes e depois da exposição a ambos os tipos de vinagre foram obtidos por microscopia eletrônica de varredura. 1266 
O perfil químico do vinagre de eucalipto e bambu foi realizado por cromatografia gasosa e espectrometria de 1267 
massa (GC/MS). Ambos os tipos de vinagre apresentaram significativa atividade antimicrobiana, tendo o vinagre 1268 
de bambu uma maior eficiência. Ambos os extratos pirolenhosos estudados parecem promissores para o 1269 
desenvolvimento de antimicrobianos naturais, devido à sua eficiência contra patógenos. Análises GC/MS 1270 
demonstraram que os perfis químicos de ambos os tipos de vinagre eram semelhantes, mas com algumas 1271 
diferenças significativas. O componente majoritário do vinagre de eucalipto foi o furfural (17,2%), enquanto o 1272 
do vinagre de bambu foi o fenol (15,3%). O vinagre de bambu apresentou teor mais expressivo de ácidos 1273 
orgânicos. Micrografias de microorganismos tiradas após a exposição aos dois tipos de vinagre apresentaram 1274 
diversas modificações nas células. 1275 
 1276 
Palavras-chave: madeira de eucalipto; biomassa de bambu; vinagre de carbonização; ácido pirolenhoso; 1277 
processo de carbonização 1278 
Pontos chave: 1279 
1. Ácidos pirolenhosos (PA) de eucalipto e bambu possuem atividade antimicrobiana 1280 
2. O PA de bambu tem um efeito antimicrobiano mais forte do que o de eucalipto 1281 
3. O furfural é o principal componente do PA do eucalipto, e o fenol do bambu 1282 
4. PA de bambu e eucalipto promoveram alterações nas células dos microrganismos 1283 
 
 
 
 
 
 
 
 
 
52 
 
ANTIMICROBIAL ACTIVITY AND CHEMICAL PROFILE OF PYROLIGNEOUS 
ACIDS FROM BAMBOO AND EUCALYPTUS 
 
Gil Sander Próspero Gama1, Alexandre Santos Pimenta1, Francisco Marlon Carneiro Feijó2, Caio Sérgio dos 
Santos2, Bruno Caio Chaves Fernandes3, Moacir Franco de Oliveira2, Elias Costa de Souza4, Thays V. C. 
Monteiro5, Maíra Fasciotti5, Tatiane Kelly Barbosa de Azevedo1, Rafael Rodolfo de Melo2, Ananias Francisco 
Dias Júnior6 
 
ABSTRACT 1284 
 
Microbial resistance to drugs is a public health problem; therefore, there is a search for alternatives to replace 1285 
conventional products with natural agents. One of the potential antimicrobial agents is vinegar (pyroligneous 1286 
acid) derived from the carbonization of lignocellulosic raw materials. The objectives of the present work were to 1287 
evaluate the antibacterial and antifungal action of two kinds of vinegar from carbonization, one of Eucalyptus 1288 
urograndis wood and another of Bambusa vulgaris biomass, and determine their chemical profile. The 1289 
antimicrobial effect was assessed against Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella 1290 
enteritidis, Escherichia coli, Streptococcus agalactiae, and Candida albicans. The minimum inhibitory 1291 
concentration and the minimum bactericidal and fungicidal concentrations were determined. Micrographs of the 1292 
microorganisms before and after exposure to both kinds of vinegar were obtained by scanning electron 1293 
microscopy. The chemical profile of the eucalyptus and bamboo vinegar was carried out by gas chromatography 1294 
and mass spectrometry (GC/MS). Both types of vinegar presented significant antimicrobial activity, with 1295 
bamboo vinegar having a higher efficiency. Both studied pyroligneous extracts seem promising for developing 1296 
natural antimicrobials due to their efficiency against pathogens. GC/MS analyses demonstrated that the chemical 1297 
profiles of both kinds of vinegar were similar but with some significant differences. The major component of the 1298 
eucalyptus vinegar was furfural (17.2%), while the one of bamboo vinegar was phenol (15.3%). Bamboo vinegar 1299 
had a more expressive content of organic acids. Micrographs of microorganisms taken after exposure to both 1300 
kinds of vinegar displayed several modifications in the cells. 1301 
Key-words: eucalyptus wood; bamboo biomass; carbonization vinegar; pyroligneous acid; carbonization 1302 
process 1303 
Key points: 1304 
1. Pyroligneous acids (PA) from eucalyptus and bamboo have antimicrobial activity 1305 
2. Bamboo PA has a stronger antimicrobial effect than eucalyptus one 1306 
3. Furfural is the main component of eucalyptus PA, and phenol of the bamboo one 1307 
4. Bambo and eucalyptus PA promoted alterations in the microorganisms’ cells 1308 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
53 
 
INTRODUCTION 1309 
 
In Brazil, eucalyptus species and clones are the prevalent material that constitutes the basis of the planted forests 1310 
for charcoal-making, mainly due to their fast growth, genetic plasticity, and high volumetric productivity (Silva 1311 
and Ataíde 2019). There are, in the country, 9.55 million hectares of planted forests, 78% of which are 1312 
represented by plants of the genus Eucalyptus and its clones (7.47 million hectares), intended for the productive 1313 
sector in general (Ibá 2021). Clones from the hybrid Eucalyptus urophylla x Eucalyptus grandis (commonly so-1314 
called Eucalyptus urograndis in Brazil) are some of the most used plant materials for charcoal production 1315 
(Marchesan et al. 2019; Meira et al. 2021). These materials have favorable characteristics for this use, such as a 1316 
considerable content of lignin (31.08%) (Silva and Ataíde 2019).In addition to the features of wood, charcoal 1317 
made from this material has good quality, a satisfactory yield, low ash content, high calorific value, and good 1318 
mechanical strength (Marchesan et al. 2019). Silva et al. (2018) mention in their review that among the 1319 
Eucalyptus urograndis clones for charcoal-making, clones I144 and GG100 stand out due to their good-quality 1320 
wood characteristics and high productivity. 1321 
Bamboo is a term employed to define a group of woody grasses initially distributed in the tropics in hot and 1322 
humid regions (Truong and Le 2014). Brazil has the most remarkable diversity of bamboo species in Latin 1323 
America, with species that can reach up to 40 m in height, a diameter of 30 cm, and productivities from 25 to 30 1324 
t ha-1 year-1 (Hernandez-Mena et al. 2014). Bamboo species are versatile, used in varied applications, and, in 1325 
some countries, qualified as high-value species, contributing positively to the local economy as an income source 1326 
for the populations (Lin et al. 2019). Among these applications, its use as an alternative source of biomass for 1327 
charcoal production stands out, being widely studied for this purpose (Asada et al. 2002; Truong and Le 2014; 1328 
Partey et al. 2017). This happens because bamboo biomass has properties similar to those of wood used in 1329 
charcoal production, consisting mainly of cellulose, hemicellulose, and lignin (Hernandez-Mena et al. 2014), 1330 
generating charcoal with fixed carbon between 55 and 59%, with a calorific value around 23.1 MJ kg-1 and 1331 
gravimetric yields from 32 to 34%, depending on the carbonization system used (Tippayawong et al. 2010). 1332 
Brazil is the largest producer of charcoal in the world, representing 12% of world production (IBÁ 2021). 1333 
Carbonization is described as a process of chemical decomposition, by heat, of the molecules of the 1334 
lignocellulosic raw material in an atmosphere with a controlled presence of oxygen or in the absence of it (Silva 1335 
and Ataíde 2019). However, approximately 70% of the wood destined for charcoal production turns into vapors 1336 
(smoke) during the carbonization process (Dias Jr. et al. 2018). It results in significant energy and economic loss 1337 
since most of the money invested in the raw material goes away as smoke to the surrounding ambient, generating 1338 
air pollution. Searching for solutions that benefit both the productive sector and the environment becomes very 1339 
important. Finding ways to take advantage of the released smoke is a way to reverse this scenario and, at the 1340 
same time, mitigate air pollution from the carbonization process. The recovery of raw pyrolysis liquids followed 1341 
by a proper destination for them is an option to accomplish such a destination that can decrease roughly 40% of 1342 
greenhouse gas emissions and generates a good product for several applications (Silva and Ataíde 2019). 1343 
Wood vinegar, pyroligneous acid, also known as water-soluble liquid smoke, is an aqueous product that comes 1344 
from the recovery of the liquefaction of part of the carbonization smoke after passing through adequate devices 1345 
(Araújo et al. 2017; Soares et al. 2020). This aqueous fraction has an acidic character and results mainly from the 1346 
cleavage of the chemical bonds of cellulose, hemicelluloses, and lignin (Li et al. 2017; Pimenta et al. 2018). 1347 
Since pyroligneous acid can be converted into a product with several end-uses, its recovery and employment are 1348 
essential to decrease air pollution and add value to the carbonization process by increasing the economic return 1349 
of the charcoal production chain. The primary use of carbonization vinegar is the addition to foods of several 1350 
types to give them the smoked flavor (Burdock 2010, Montazeri et al. 2013). Carbonization vinegar from 1351 
different sources has been attracting the attention of researchers worldwide due to the biological effects and 1352 
varied chemical composition, which results in a product that can be addressed to several end-uses (Tiilikkala et 1353 
al. 2010; Araújo et al. 2017; Pimenta et al., 2018). On the chemical composition of carbonization vinegar, over 1354 
200 chemical compounds could be identified (Schnitzer et al. 2015; Araújo et al. 2017), with the product being 1355 
increasingly employed in agriculture applications such as an herbicide, seeds protector, and growth promoter for 1356 
 
54 
 
plants and animals (Chalermsan and Peerapan 2009; Tiilikkala et al. 2010; Mahmud et al. 2016); as nematicide 1357 
(Charehgani 2020); and still acting as an insecticide, antioxidant, antiviral, anti-inflammatory, antibacterial, and 1358 
antifungal agent (Harada t al. 2013; Li et al. 2017; Souza et al. 2018; Suresh et al. 2019) among several others 1359 
uses. Also, a distilled-type bamboo vinegar has been patented as an anti-allergy valuable agent for inhibiting 1360 
Type I allergies such as allergic rhinitis, hay fever, allergic conjunctivitis, atopic dermatitis, allergic asthma, 1361 
urticaria, food allergy, and anaphylaxis (Imamura and Watanabe 2007). 1362 
Microorganisms such as Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis, and Staphylococcus 1363 
aureusare part of those that have developed resistance mechanisms to conventional drugs over time. They are 1364 
the major causes of diseases in humans and animals (Kaper et al. 2004; Cabassi et al. 2017; Quan et al. 2019). 1365 
For instance, illnesses caused by strains of E. coli include infections of the intestinal and urinary tracts, 1366 
hemorrhagic colitis, hemolytic uremic syndrome, diarrhea, and meningitis (Kaper et al. 2004; Fairbrother and 1367 
Nadeau 2006). Cattle and other ruminants are one of the most significant sources of these pathogens transmitted 1368 
to humans through the consumption of water contaminated by the feces of these animals or through direct 1369 
contact with them. (Fairbrother and Nadeau 2006; Meng et al. 2012). In its turn, some diseases caused by P. 1370 
aeruginosa are otitis in cattle, mastitis in dairy herds, and endometritis in horses, as well as infections in 1371 
domestic animals, such as those that occur in the urinary tract of dogs (Salomonsen et al. 2013; Haenni et al. 1372 
2015). In humans, the most severe infections include meningitis, pneumonia, malignant otitis, and 1373 
endophthalmitis, among others (Bodey et al. 1983; Klockgether et al. 2017); Contagion by S. enteritidis happens 1374 
through the consumption of contaminated food, mainly eggs, and causes infections such as gastroenteritis 1375 
(Gantois et al. 2009; Quan et al. 2019). As for infections with S. aureus, according to Tong et al. (2015), they 1376 
can range from mild to severe infections, of which endocarditis and osteoarticular infections, in addition to skin 1377 
and lung infections, can be mentioned. One of the biggest problems this species can generate in farm animals is 1378 
the high economic loss caused by mammary mastitis affecting dairy cows (Saeed et al. 2022). As noted, also 1379 
mastitis can be caused by more than one microorganism (and it is not limited to those mentioned here). This 1380 
disease has resulted in significant economic losses in infected producing sites (Guimarães et al. 2017; Romero et 1381 
al. 2018; Azooz et al. 2020). Still, results attained by Feijó et al. (2022) reported the successful employment of 1382 
wood vinegar from Mimosa tenuiflora as a post-surgical antiseptic in cats subjected to 1383 
ovariosalpingohysterectomy. The authors observed a significant improvement in the animals’ healing compared 1384 
to those from the control group treated with chlorhexidine. An antiseptic that effectively prevents mastitis in 1385 
dairy animals could be developed by having eucalyptus wood vinegar based on the results reported by Soares et 1386 
al. 2020. The new product was recentlypatented (Pimenta et al. 2022). 1387 
Nowadays, there is a seek for therapeutic alternatives against microorganisms that developed resistance to 1388 
conventional drugs. Such resistance is a severe public health issue, as commented by Laxminarayan et al. (2013), 1389 
and it has resulted mainly due to the indiscriminate use of conventional products, which accelerated the 1390 
resistance process (Costa and Silva Jr. 2017, Palma et al. 2020). Antimicrobial drugs were discovered about 100 1391 
years ago, and since then, they have been widely used in several areas, such as human medicine, veterinary 1392 
medicine, and animal production (Kovanda et al. 2019). Then, the efforts to discover new antimicrobial products 1393 
are intended to come from natural and environmentally appropriate sources. This direction is a function of the 1394 
problem of microbial resistance and also the fact that the use of antimicrobials is essential for humanity. 1395 
Therefore, several studies have evaluated carbonization kinds of vinegar as promising for developing a natural 1396 
antimicrobial alternative (Tiilikkala et al. 2010; Araújo et al. 2017; Souza et al. 2018; Chukeatirote and Jenjai 1397 
2018; Suresh et al. 2019; Desvita et al. 2022). With this, it is justified to evaluate the antibacterial and antifungal 1398 
potential of species frequently used in charcoal production, aiming to combine the productivity of charcoal with 1399 
a possible solution to the issue of microbial resistance and the reduction of greenhouse gas emissions. 1400 
In the context discussed above, the present work aims: 1401 
- To determine the antimicrobial activity of two kinds of vinegar from the carbonization of Eucalyptus 1402 
urograndis wood and Bambusa vulgaris grown in Northeastern Brazil; 1403 
- To attain the chemical profile of both types of vinegar and establish a comparison between their 1404 
composition; 1405 
 
55 
 
- By using scanning electron microscopy to observe what modifications brought about the action of 1406 
bamboo and eucalyptus vinegar on the microorganisms’ cells that inhibited their growth; and 1407 
- Through a brief literature review, compare the results of antimicrobial activity and chemical profiling 1408 
with published data and highlight similarities and differences between the assessed types of vinegar with those 1409 
whose properties and chemical composition have been described in previous research works. 1410 
 1411 
MATERIAL AND METHODS 1412 
 1413 
Collection of samples of woody material 1414 
Wood samples from Eucalyptus urophylla x Eucalyptus grandis hybrid (clone I144) were collected from 8-year-1415 
old plantations in the experimental area of the Agricultural Sciences Unit, Universidade Federal do Rio Grande 1416 
do Norte (05° 51' 30” S and 35° 21' 14” W), municipality of Macaíba, Rio Grande do Norte State, Brazil. 1417 
Samples of bamboo (Bambusa vulgaris) were acquired from 3-year-old commercial plantations in Timon, 1418 
Maranhão state, Brazil (05° 5' 42'' S and 42° 50' 13'' W). One hundred 3.0-cm wood disks were collected 1419 
following the procedures established by Carneiro et al. (2013) and divided into four wedges each. The bamboo 1420 
samples consisted of 100 sections from the stems measuring 10 cm long. The bamboo samples were collected at 1421 
0, 25, 50, 75, and 100% of the stems’ commercial height. Both samples were oven-dried (Sterilifer, model SX 1422 
cr/80, São Paulo, Brazil).at 103 + 2 °C for 48 hours until absolute dryness. 1423 
Carbonization process, production, and refining of eucalyptus and bamboo vinegar 1424 
For the carbonization runs, the dry samples of each material were placed separately in a steel container inside a 1425 
laboratory muffle equipped with a condensation apparatus to collect the total pyrolysis liquids. The condensation 1426 
device was water-cooled to maintain its temperature around 25 – 30 °C, providing conditions for the 1427 
condensation of vapors from the carbonization bed. For each type of woody material, 15 carbonization runs were 1428 
carried out with about 500 g of plant material each. After each was concluded, the liquid products were mixed to 1429 
make one composite sample of vinegar from each woody material at the end of the experiment. The 1430 
carbonization process was carried out from the ambient temperature until reaching 450 °C, with a heating rate of 1431 
0.7 °C min-1 , totalizing 8 hours. Composite samples of eucalyptus and bamboo vinegar were distilled under a 1432 
20 mmHg vacuum until 100 – 103 °C to remove tar and heavy oils. After the distillation, the products were 1433 
stored in amber bottles, previously sanitized with boiling water, and refrigerated at 6 °C for further procedures. 1434 
Gas chromatography/mass spectrometry (GC/MS) analyses 1435 
For the GC/MS analyses, the protocols reported by Pimenta et al. (2018) were employed. For both types of WV 1436 
samples, 5 mL aliquots were taken, and 1.5 mL of ammonium hydroxide (Caledon, Ammonia Solution UN 1437 
2672, Canada) was added to each one. After this addition, the organic fraction of each sample was subjected to 1438 
liquid-liquid extraction with 1 mL of ethyl acetate (HPLC grade, Merck, São Paulo, SP, Brazil). After this step, 1439 
the samples were promptly analyzed. The GC/MS analyses were carried out in a SHIMADZY QP 2010 gas 1440 
chromatography/mass spectrometer equipped with a DB-Wax 52 CB (Agilent, 30 m length, 0.25 mm diameter, 1441 
0.25 μm film thickness). The injector was kept at 250 °C, and 1 µL of each sample was injected with a 1:10 split 1442 
ratio. The oven was programmed with an initial temperature of 50 °C for 2 min, and after this, a heating rate of 2 1443 
°C min-1 was applied until reaching 240 °C, keeping the final temperature for 2 min. Helium was used as carrier 1444 
gas with a flow rate of 3 mL min-1 . The total time for each chromatography run was 99 min. The acquisition of 1445 
the mass spectra was carried out from m/z 50 to 650 Da. The electronic ion Source (EI) and the MS interface 1446 
were maintained at 250 °C. The solvent cut-off time was 3 min. Major (>1.5% area, in bold in Table 3) and 1447 
minor (~0.2%) compounds were detected and identified based on their characteristic electron ionization mass 1448 
 
56 
 
spectra (EI, 70 eV) compared to those from the NIST library. All reported chemical compounds had a mass 1449 
spectral similarity of at least 85%. The acetic acid content was determined quantitatively by GC/MS according to 1450 
the procedure IPT number 4596, revision 13 (Instituto de Pesquisas Tecnológicas – São Paulo, SP, Brazil) using 1451 
acetic acid (Sigma-Aldrich, São Paulo, SP, Brazil) as external standard. The samples’ water contents were 1452 
determined using a Karl-Fischer compact titrator V10S (Mettler-Toledo, São Paulo, SP, Brazil). The organic 1453 
fraction was obtained by subtracting the water content plus the acetic acid content from the total weight of the 1454 
samples. 1455 
Determination of the minimum inhibitory concentration (MIC) 1456 
For the microbiological assays, bacterial strains of Staphylococcus aureus (ATCC 25923), Pseudomonas 1457 
aeruginosa (ATCC 27853), and Salmonella enteritidis (ATCC 13076) were used. Two clinical strains of 1458 
Escherichia coli, one previously isolated from an emu, were employed in the assays with a strain of 1459 
Streptococcus agalactiae isolated from cows with diagnosed subclinical bovine mastitis. Still, a standard fungal 1460 
strain of Candida albicans (ATCC 10231) was used. The microorganisms were maintained in BHI broth (Brain 1461 
Heart Infusion – Kasvi) under refrigeration. The sensitivity of the microorganisms was evaluated in vitro to 1462 
determine the MIC. The assays were performed by the broth microdilution method using 96-well microplates 1463 
following the M07-A9 Methodology for Sensitivity Testing to Antimicrobial Agents by Dilutionfor Bacteria for 1464 
Aerobic Growth from the Clinical and Laboratory Standards Institute (CLSI, 2012). All assays had chlorhexidine 1465 
0.2% (Vic Pharma by Schülke, Taquaritinga, SP, Brazil) as the conventional reference product. Before each 1466 
assay, the strains were cultivated in BHI broth in a bacteriological oven (Fanem, São Paulo, SP, Brazil) at 37 ± 1 1467 
°C for 24 hours for bacterial strains and 48 hours for fungal strains. Subsequently, the process of preparing the 1468 
microbial inocula was carried out. For this, aliquots of the culture from the last 24 and 48 hours were removed 1469 
and transferred to new test tubes containing the same sterile culture medium. The adequacy of the concentration 1470 
of microbial cells in the inoculum was carried out with the aid of a spectrophotometer (Biospectro, model SP-22, 1471 
São Paulo, SP, Brazil) at a wavelength of 530 nm, where the reference value for absorbance reading was 1472 
between 0.08 and 0.1, corresponding to the scale MacFarland number 0.5 (density 1.5 x 108 cells per mL). 1473 
Then, 100 µL of sterile BHI culture medium was added to each well of the microplates. Subsequently, serial 1474 
dilutions of each EP were performed, adding 100 µL of EP to the first microwell of each triplicate row, resulting 1475 
in the first dilution. Thus, the first microwell now contained 200 µL. Subsequently, a 100 µL was removed from 1476 
the first well and added to the second, and so on, constantly dropping the eucalyptus or bamboo vinegar in a 1477 
concentration by half in each dilution. The assessed concentrations of the vinegar started from 50% until 1478 
0.78125%, encompassing 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78123% of each one. After the dilutions, a 1479 
volume of 0.5 µL of the corresponding microbial inoculum was added to each microwell. Immediately after the 1480 
inoculations, the microplates were read in an Elisa plate reader spectrophotometer (URIT Medical Electronic) at 1481 
630 nm to base record the absorbance data (optical density) at the time of inoculation (0h). Then, the microplates 1482 
were incubated in a bacteriological oven at 37 ± 1 °C for 24 hours for bacterial strains and 48 hours for fungal 1483 
strains. After this time, a visual reading was performed to determine the minimum inhibitory concentration 1484 
(MIC), considering the triple microwells completely translucent as the decisionmaking criterion to establish this 1485 
parameter. After this first visual reading, the plates were read in a spectrophotometer to acquire the absorbances 1486 
 
57 
 
after the incubation period (24 and 48 hours), providing quantitative data on the microbial behavior under the 1487 
effect of the type of vinegar and at the different concentrations of each one. 1488 
Determination of the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) 1489 
for bacterial and fungal strains, respectively, was carried out from microwells where there was no visible 1490 
microbial growth (MIC and higher concentrations). Thus, an aliquot of 50 μL was removed from each well and 1491 
seeded in a Petri dish containing a BHI culture medium. For all microorganisms, the Petri dishes were incubated 1492 
at 37 ± 1 °C for 24 h, except those with the Candida albicans culture, which were incubated for 48 h. After this, 1493 
the absence or presence of colonies growth was observed to define the minimum bactericidal and fungicidal 1494 
concentrations. All tests to determine MIC, MBC, and MFC were performed in triplicate, each with three 1495 
replicates. 1496 
Analysis by scanning electron microscopy (SEM) 1497 
The previously adjusted and incubated inoculums were used in this step. 1 mL of each of these inoculums was 1498 
added to 1 mL of vinegar aqueous solution in the amount of twice the MIC corresponding to each 1499 
microorganism. In addition, another 1 mL of inocula was added to the same culture medium without the vinegar 1500 
to obtain healthy microorganisms’ cells for comparison purposes. Then, both were incubated in a bacteriological 1501 
oven at 37 °C for 24 hours (C. albicans for 48 h). 1502 
After incubation, the culture media were taken to the Laborline centrifuge (Omega P.I.C. Microprocess system) 1503 
at 3000 rpm for 10 minutes to form the processing pellets. Then, the sample preparation steps were followed, 1504 
according to the procedures reported by Mares (1989) and Ibrahim et al. (2013), with some modifications 1505 
explained hereafter. For fixation, a fixative solution containing 25% glutaraldehyde, 0.2 M sodium phosphate 1506 
buffer solution (PBS), and water (1.2, 5.0, and 3.8 mL, respectively) was used. The pelletized material was 1507 
exposed to the fixative for 12 hours. After this time, each pellet was washed three times in PBS solution (0.1 M) 1508 
for 30 min. 1509 
Post-fixation was performed using osmium solution (1.0 g diluted in 100 mL of PBS) for 30 minutes. 1510 
Subsequently, the material was washed with PBS thrice for 5 minutes. The dehydration step was performed with 1511 
ethyl alcohol solution in different percentages ranging from 15 to 100% (15, 25, 30, 50, 70, and 100%). In 1512 
percentages from 15 to 70%, the pelleted cells were exposed for 10 minutes. With pure alcohol (100%), the 1513 
exposure time was only 5 minutes. The physical method, air drying carried out drying; that is, the microbial cells 1514 
were exposed to air at room temperature until the material was dehydrated. Then, after all these steps, the 1515 
samples were glued onto carbon tapes fixed on stubs and, subsequently, metalized by being exposed to a layer of 1516 
9 nm gold (Quorum Technologies – Q150R) and later taken for observations. in a scanning electron microscope 1517 
(Pfeiffer Vacuum – D-35614 Assiar) at an accelerated voltage rate of 30 KV to produce images saved in TIFF 1518 
format. 1519 
Statistical analyses 1520 
Experimental data of absorbances versus concentrations of both types of vinegar were subjected to regression 1521 
analyses using the R software, and statistical models were fitted for each microorganism. The best statistical 1522 
 
58 
 
prediction models were selected based on the following decision criteria: high correlation coefficient (R) 1523 
between experimental and estimated values, the significance of the model parameter, biological realism of the 1524 
model, and root-mean-square deviation (RMSE) as recommended by Gujarati and Porter (2009). A correlation 1525 
coefficient closer to 1 and a lower RMSE indicate the best model estimation power. 1526 
RESULTS 1527 
 
Determination of the minimum inhibitory concentrations (MIC, MBC, and MFC) 1528 
In Table 4, the values for minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), 1529 
and minimum fungicidal concentration (MFC) determined for each microorganism are displayed. These values 1530 
referring to the positive control (made with chlorhexidine) were constant, being 0.039%. Both types of vinegar 1531 
from eucalyptus and bamboo were efficient as antimicrobial agents, inhibiting the growth of all tested strains (E. 1532 
coli, P. aeruginosa, S. enteritidis, S. aureus, S. agalactiae, and C. albicans). Compared with the other 1533 
microorganisms, C. albicans was more resistant to both products, with MIC values of 12.5 and 6.25%, for 1534 
eucalyptus and bamboo vinegar, respectively. 1535 
Table 4. Values of MIC, MBC, and MFC (%) for the microorganisms subjected to the action of Eucalyptus 
urograndis and Bambusa vulgaris vinegar 
Microorganisms 
Type of vinegar 
Eucalyptus urograndis Bambusa vulgaris 
MIC MBC MIC MBC 
Escherichia coli 6.25 25 3.125 3.125 
Pseudomonas aeruginosa 6.25 12.5 3.125 3.125 
Salmonella enteritidis 6.25 6.25 3.125 3.125 
Staphylococcus aureus 6.25 12.5 1.562 3.125 
Streptococcus agalactiae 6.25 6.25 1.562 6.25 
Candida albicans* 12.5 - 6.25 12.5 
Where:MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; MFC: minimum inhibitory 
concentration; *: in C. albicans it is representing that in place of the initials MBC one must have the initials MFC; -: no 
inhibition observed in the assessed concentration 
Graphs containing the curves demonstrating the pattern of inhibition of microorganisms under the effect of the 1536 
two types of vinegar are presented in Figure 2. The respective regression models fitted to predict the inhibition 1537 
for each microorganism at different concentrations under the effect of both types of vinegar are shown in Table 1538 
5. To highlight, the models were adjusted based on experimental data so the inhibition behavior for each 1539 
microorganism subjected to the effect of each type of vinegar could be adequately predicted. These graphs were 1540 
fitted by recording absorbances (optical density) at the time of inoculation (0 h) and after incubation (24 h for 1541 
bacteria and 48 h for C. albicans) of each microorganism subjected to the effect of the kinds of vinegar assessed 1542 
in this study. The shapes of the curves are similar, which means that the inhibition pattern of both types of 1543 
vinegar is the same. However, the inhibition promoted by bamboo vinegar occurs at lower concentrations when 1544 
compared to eucalyptus vinegar. 1545 
 
59 
 
 
 
60 
 
Figure 2. Absorbances of microorganisms’ cultures as a function of the concentration of eucalyptus (orange 
curves) and bamboo (blue curves) vinegar; A1, B1, C1, D1, E1, and F1 – at the moment of inoculation – 0 h; and 
A2, B2, C2, D2, E2, and F2 – 24 h after inoculation (48 h for C. albicans) 
Table 5. Regression models to predict the behavior of microorganisms' growth as a function of the eucalyptus 
and bamboo vinegar concentrations 
Type of vinegar Regression Models R2 
 Escherichia coli 
Eucalyptus Y = – 0.3609 ln X + 1.1893 0.7658 
Bamboo Y = – 0.2009 ln X + 0.6561 0.7560 
 Pseudomonas aeruginosa 
Eucalyptus Y = – 0.3556 ln X + 1.1463 0.7968 
Bamboo Y = – 0.2203 ln X + 0.6869 0.7647 
 Salmonella enteritidis 
Eucalyptus Y = – 0.3225 ln X + 1.0631 0.8027 
Bamboo Y = – 0.2262 ln X + 0.6924 0.7626 
 Staphylococcus aureus 
Eucalyptus Y = – 0.2617 ln X + 0.8822 0.7891 
Bamboo Y = – 0.1905 ln X + 0.6094 0.6926 
 Streptococcus agalactiae 
Eucalyptus Y = – 0.3693 ln X + 1.2378 0.7497 
Bamboo Y = – 0.2652 ln X + 0.7921 0.7617 
 Candida albicans 
Eucalyptus Y = – 0.1582 ln X + 0.5774 0.8614 
Bamboo Y = – 0.1405 ln X + 0.5231 0.5762 
*Where: Y = absorbance; X = vinegar concentration; R2 = coefficient of determination 
 
From the interpretation of the graphs, higher vinegar concentrations resulted in lower absorbance data which 1546 
means a higher inhibitory effect, as expected. At the very moment of inoculation, the behavior of absorbances 1547 
follows a constant pattern with values between 0.04 and 0.09 (Figure 2 – A1, B1, C1, D1, E1, and F1). However, 1548 
after incubation, it is possible to notice the inhibitory effect that both kinds of vinegar had by observing the 1549 
curves displaying the behavior of microorganisms' growth as a function of the inhibitory agents' action (Figure 2 1550 
– A2, B2, C2, D2, E2, and F2). The curves registered the gradual decrease in cell density in the medium after the 1551 
microorganisms were exposed to increasing concentrations of antimicrobial agents. 1552 
 
61 
 
Similar inhibition results were found by Araújo et al. (2017) using two kinds of vinegar from Mimosa tenuiflora 1553 
and Eucalyptus urograndis (clone GG100) wood. In the present work, lower concentrations of bamboo vinegar 1554 
were required compared to eucalyptus. For bamboo, MIC values varied between 1.562 and 6.25%, unlike the 1555 
values found for eucalyptus, which were between 6.25 and 12.5%. A similar pattern was observed for MBC and 1556 
CFM results. The discussion regarding the difference in action between the two types of vinegar is presented in 1557 
the next topic. Other authors obtained similar results to those found in the present work when they evaluated 1558 
bamboo and eucalyptus vinegar, corroborating the data of the current research (Wang et al. 2012; Harada et al. 1559 
2013; Araújo et al. 2017; Rattanawut et al. 2017; Soares et al. 2020). However, the present work’s novelty is 1560 
comparing the antimicrobial effect of eucalyptus and bamboo grown in Northeastern Brazil, establishing a 1561 
relationship between the inhibition patterns with the chemical composition of each type of vinegar. 1562 
In graphs displayed in Figure 2, the absorbances record the density of cells present in a given medium and, this 1563 
way, the less dense the medium is, the lower the absorbance value will be, due to the lower proliferation of 1564 
microbial cells in the solution, resulting in a lower optical density. The results followed the same pattern 1565 
achieved by Cunha and Vieira (2014) and Gama et al. (2022). In Figure 2, in addition to presenting the microbial 1566 
behavior subjected to the effect of both types of carbonization vinegar at different concentrations, a comparison 1567 
between the antimicrobial activity of the two agents can be inferred. For all of the microorganisms, the bamboo 1568 
vinegar had lower absorbance values corresponding to a higher antimicrobial effect, corroborating the MIC data 1569 
presented in Table 4. This difference in antimicrobial activity may be closely related to the chemical constitution 1570 
of each type of product. Differences in the expression of the antimicrobial effect of kinds of vinegar produced 1571 
from different lignocellulosic materials are common and reported by several authors (Velmurugan et al. 2009; 1572 
Araújo et al. 2017; Souza et al. 2018; Morales et al. 2019; Soares et al. 2020). 1573 
As an illustrative example and to provide a basis of comparison, carbonization vinegar from cocoa husks 1574 
(Theobroma cacao) resulted in zones of inhibition between 6 and 6.12 mm when tested against C. albicans 1575 
(Desvita et al. 2022). However, when the same microorganism was exposed to the vinegar of Dimocarpus 1576 
longan, the zone of inhibition was 17.56 ± 0.01 mm (Chukeatirote and Jenjai 2018). In the study by Araújo et al. 1577 
(2017), the diameter values ranged from 8.3 to 25 mm when Mimosa tenuiflora vinegar was assessed against the 1578 
same microorganism. This behavior is expected since each plant species, and each microorganism has its genetic 1579 
peculiarities. Because of that, in terms of antimicrobial effects, no generalizations can be made for any 1580 
carbonization vinegar. The correct strategy is to evaluate one by one and verify how intense its impact is on this 1581 
or that microorganism. Also, Chai et al. (2013) proved such property of furfural by testing its effect on 1582 
Salmonella sp. and Bacillus subtilis, demonstrating inhibition in different levels. Another research work reports a 1583 
strong inhibitory potential of furfural against bacteria (strains of S. aureus, Proteus mirabilis, and Klebsiella 1584 
pneumonia) and fungi (Wani et al., 2016). As Yang et al. (2020) commented, natural products containing 1585 
furfural usually had an excellent antimicrobial effect on E. coli, C. albicans, and S. aureus. This information 1586 
reasserts the potential of vinegar from the carbonization of varied lignocellulosic sources as antimicrobial agents. 1587 
The models adjusted and presented in Table 2 are essential considering practical applications such as developing 1588 
antiseptics. Bearing in mind the inhibition pattern as a function of the concentration of a given vinegar, antiseptic 1589 
formulations can be tailored and directed for specific uses. This strategy was successfully employed by Pimenta 1590 
et al. (2022) to tailor an antiseptic formulation to be used in the post-milking asepsis of dairy animals to prevent 1591 
mastitis. Beforepatenting the new antiseptic, the authors also performed assays in field conditions for two years 1592 
with goats and cows to certify the product’s performance against mastitis-causing pathogens. However, the 1593 
primary laboratory assays were mandatory before the final formulation could be tailored together with 1594 
surfactants and other adjuvants to achieve the best adherence and fixedness of the product on the animal’s teats. 1595 
GC/MS analyses 1596 
The total ion chromatograms attained for both types of vinegar are displayed in Figure 3. There are some 1597 
differences between the chemical composition of the two products regarding the presence or absence of 1598 
compounds and the intensity of the peaks as a percentage (Table 6). A total of 116 compounds were annotated, 1599 
and among them, the phenols, furans, aldehydes, and organic acids stood out. Among the total compounds 1600 
 
62 
 
identified, 53 are common to both types of vinegar (blue boxes in Table 6), 33 occur exclusively in eucalyptus 1601 
vinegar (green boxes), and 31 solely in bamboo (orange boxes). In the same table, compounds with exclusive 1602 
occurrence in one or other types of vinegar were marked with a green box. For both products, the phenolic 1603 
compounds and furfural are the main components. However, the eucalyptus vinegar had furfural as the majority 1604 
compound with a peak area of 17.2%, while in bamboo vinegar, the area was 4.9%. Bamboo vinegar had phenol 1605 
as its primary component, with an area of 15.3%, while in the eucalyptus vinegar, the value was 8.5%. 1606 
 
Figure 3. Total ion chromatograms of Eucalyptus urograndis (A) and Bambusa vulgaris (B) carbonization 
vinegar 
 
63 
 
Table 6. Annotated compounds in the carbonization vinegar of Eucalyptus urograndis and Bambusa vulgaris 
 Type of carbonization vinegar 
 Eucalyptus urograndis Bambusa vulgaris 
Annotated 
Pick # 
Compound 
RT 
(min) 
Area 
(%) 
OC 
RT 
(min) 
Area 
(%) 
OC 
1 2,3-Pentanedione 4.470 0.1 4.466 0.1 
2 2-Propen-1-ol 5.770 0.0 * * * 
3 Pyridine 7.790 1.0 7.816 1.8 
4 Cyclopentanone, 2-methyl- 8.069 0.1 * * * 
5 Pyridine, 2-methyl- 8.995 0.2 9.063 0.2 
6 Pyridine, 2,6-dimethyl- * * * 10.254 0.1 
7 3(2H)-Furanone, dihydro-2-methyl- * * * 11.086 0.2 
8 Pyridine, 2-ethyl- * * * 11.789 0.1 
9 1,4-Dioxin, 2,3-dihydro- 12.094 0.1 12.076 0.1 
10 Pyridine, 3-methyl- 12.221 0.1 12.297 0.2 
11 N-Nitrosodimethylamine 12.877 0.2 12.846 0.3 
12 2-Cyclopenten-1-one, 2,3-dimethyl- 14.339 0.1 * * * 
13 3-Pentanol * * * 14.726 0.1 
14 2-Cyclopenten-1-one 15.200 1.9 15.193 0.8 
15 4-Hexen-3-ol, 2-methyl- 15.381 0.2 * * * 
16 2-Furanmethanol, tetrahydro- * * * 15.385 0.2 
17 2-Hydroxy-3-pentanone * * * 15.612 0.1 
18 Cyclopentanol, 3-methyl- 15.636 0.1 * * * 
19 2-Cyclopenten-1-one, 2-methyl- 15.809 1.5 15.812 0.4 
20 1-Hydroxy-2-butanone 16.510 1.0 16.495 3.4 
21 Butanoic acid, 2-hydroxy-, methyl ester 16.894 0.0 * * * 
22 2H-Pyran-3(4H)-one, dihydro- 17.928 0.1 * * * 
23 4-Hydroxy-3-hexanone * * * 18.265 0.1 
 
64 
 
24 2-Cyclohexen-1-one 19.100 0.1 * * * 
25 Butanoic acid, 2-propenyl ester * * * 19.578 0.1 
26 2-Cyclopenten-1-one, 2,3-dimethyl- 19.966 0.3 19.982 0.1 
27 2-Furanmethanol, tetrahydro- 20.192 1.0 * * * 
28 Acetic acid * * * 20.884 12.5 
29 Furfural 21.468 17.2 21.402 4.9 
30 2-Cyclopenten-1-one, 2,3,4-trimethyl- 22.541 0.2 * * * 
31 2-Furanmethanol, tetrahydro- * * * 23.017 0.2 
32 2,5-Hexanedione 23.390 0.3 23.369 0.2 
33 Ethanone, 1-(2-furanyl)- 23.503 1.5 23.485 0.8 
34 2-Cyclopenten-1-one, 3-methyl- 23.663 2.4 23.637 1.2 
35 2-Cyclopenten-1-one, 2,3-dimethyl- 24.813 1.4 24.801 0.8 
36 2,3-Pentanedione 25.330 0.6 * * * 
37 2-Butanone, 1-(acetyloxy)- 25.568 0.5 * * * 
38 1-Buten-3-yne, 1-(1,1-dimethylethoxy)-, (Z)- * * * 25.572 0.2 
39 Propanoic acid 26.413 0.7 26.149 5.8 
40 Bicyclo[2.2.2]octane, 2-methyl- 26.654 0.2 * * * 
41 Bicyclo[3.3.1]nonane * * * 26.685 0.2 
42 2-Furancarboxaldehyde, 5-methyl- 27.301 4.4 27.281 0.9 
43 3,6-Heptanedione * * * 27.437 0.2 
44 Pyridine, 3-methoxy- 27.540 0.4 27.515 0.5 
45 1,3-Dimethyl-1-cyclohexene * * * 27.669 0.1 
46 Methyl 2-furoate 27.677 0.3 * * * 
47 2(3H)-Furanone, dihydro-5-methyl- * * * 28.993 0.3 
48 Furan, tetrahydro-2,5-dimethyl- 29.018 0.2 * * * 
49 Cyclohexane, (1-methylethylidene)- 29.241 0.2 29.236 0.2 
50 2-Acetyl-5-methylfuran 29.435 0.2 29.454 0.1 
51 2-Cyclopenten-1-one, 3-ethyl- 29.634 0.5 29.621 0.4 
52 Butyrolactone 30.027 0.8 30.077 2.2 
53 2-Furanone, 2,5-dihydro-3,5-dimethyl 31.166 1.0 31.147 0.6 
54 Butanoic acid 31.339 0.6 31.252 1.8 
 
65 
 
55 Bicyclo[2.2.2]octane, 1,2,3,6-tetramethyl- 33.060 0.1 * * * 
56 3-Decen-1-ol, acetate, (Z)- 33.381 0.1 * * * 
57 Pentanoic acid, 3-methyl- 33.555 0.1 33.506 0.2 
58 Bicyclo[4.1.0]heptan-3-one, 4,7,7-trimethyl-, [1R-(1.alpha.,4.beta.,6.alpha.)]- * * * 34.035 0.1 
59 2(5H)-Furanone, 3-methyl- 34.822 0.5 34.784 0.4 
60 4-Hepten-3-one, 4-methyl- 35.161 0.2 35.148 1.2 
61 3-Nonen-1-ol, (Z)- * * * 35.374 0.1 
62 E-1,9-Hexadecadiene * * * 35.825 0.1 
63 Benzene, 1,2-dimethoxy- 35.970 0.1 * * * 
64 E,Z-2,13-Octadecadien-1-ol * * * 36.595 0.1 
65 2(5H)-Furanone 36.894 0.1 36.840 0.1 
66 Pentanoic acid 37.240 0.2 37.187 0.2 
67 1,4-Cyclooctanedione * * * 38.482 0.1 
68 Crotonic acid * * * 39.157 0.3 
69 2,3-Dimethoxytoluene 40.224 0.2 * * * 
70 Cyclopentane, 1-acetyl-1,2-epoxy- 40.377 0.2 40.343 0.5 
71 4-Methyloctanoic acid * * * 40.581 0.1 
72 3-Heptenoic acid 40.618 0.1 * * * 
73 2-Methylcyclopropanecarboxylic acid * * * 41.020 0.2 
74 1,2-Cyclopentanedione, 3-methyl- 41.294 3.3 41.242 2.3 
75 Cyclopentanone, 2-methyl-3-(1-methylethyl)- * * * 41.571 0.0 
76 2-Hydroxy-3-propyl-2-cyclopenten-1-one 42.496 0.2 42.461 0.2 
77 Phenol, 2-methoxy- 42.748 9.4 42.720 9.5 
78 Phenol, 2-methoxy-4-methyl- 43.217 0.4 * * * 
79 Cycloheptanone, 2-ethyl- * * * 43.523 0.3 
80 Cyclopentanone, 2-methyl-3-(1-methylethyl)- 43.550 0.3 * * * 
81 2-Pentenoic acid * * * 44.248 0.3 
82 2-Cyclopenten-1-one, 3-ethyl-2-hydroxy- 44.566 1.0 44.525 1.0 
83 Benzyl oxy tridecanoic acid * * * 45.235 0.1 
84 Phenol, 2,6-dimethyl- 45.567 0.2 * * * 
85 2-Heptenal, 2-propyl- 46.242 0.1 46.211 0.1 
 
66 
 
86 2-Methoxy-5-methylphenol 46.759 0.5 * * * 
87 Phenol, 2-methoxy-4-methyl- 47.488 4.4 47.462 2.2 
88 Maltol * * * 47.812 1.5 
89 2-Hydroxy-3-propyl-2-cyclopenten-1-one * * * 48.348 0.5 
90 1H-Inden-1-one, 2,3-dihydro- 49.085 0.3 49.073 0.2 
91 Phenol 50.403 8.5 50.327 15.3 
92 1H-Pyrrole-2-carboxaldehyde 50.826 0.1 50.769 0.2 
93 Phenol, 4-ethyl-2-methoxy- 51.041 1.8 51.023 1.7 
94 Benzene, 1,2,3-trimethoxy-5-methyl- 52.166 0.3 * * * 
95 Phenol, 2-ethyl- 53.660 0.2 53.625 0.1 
96 Phenol, 2,3-dimethyl- 53.820 0.5 * * * 
97 Phenol, 4-methyl- 53.996 2.3 53.936 3.5 
98 Phenol, 3-methyl- 54.335 3.3 54.291 1.3 
99 Phenol, 2-methoxy-4-propyl- 54.703 0.3 54.687 0.2 
100 Phenol, 2,3-dimethyl- 57.030 0.2 56.994 0.1 
101 Phenol, 2-methoxy-3-(2-propenyl)- 57.327 0.1 57.302 0.1 
102 Phenol, 2-(1-methylethyl)-, methylcarbamate * * * 57.840 0.1 
103 Phenol, 3-(1-methylethyl)- 57.877 0.1 * * * 
104 Phenol, 4-ethyl- * * * 58.142 5.8 
105 Phenol, 3,4-dimethyl- 58.188 0.8 * * * 
106 Phenol, 3-ethyl- 58.500 0.3 58.451 0.1 
107 1,2,3-Trimethoxybenzene 58.906 0.1 * * * 
108 Phenol, 2,4,5-trimethyl- 59.670 0.1 * * * 
109 Phenol, 2,4-dimethyl- 60.162 0.4 60.134 0.3 
110 Phenol, 2,6-dimethoxy- 61.859 10.7 61.827 6.0 
111 1,2,3-Trimethoxybenzene 65.477 3.5 65.448 0.4 
112 Benzene, 1,2,3-trimethoxy-5-methyl- 67.877 1.9 67.843 0.3 
113 1H-2-Benzopyran-1-one, 3,4-dihydro-8-hydroxy-3-methyl-* * * 69.368 0.1 
114 2,4-Hexadienedioic acid, 3,4-diethyl-, dimethyl ester, (Z,Z)- 70.695 0.4 70.681 0.1 
115 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester 72.799 0.2 72.805 0.2 
 
67 
 
116 Phenol, 2,6-dimethoxy-4-(2-propenyl)- 73.125 0.1 * * * 
RT = retention time; OC: occurrence of the compound; Blue box = occurrence in both types of vinegar; Green box – exclusive occurrence in eucalyptus vinegar; 
Orange box = exclusive occurrence in bamboo vinegar; (*) Negative occurrence 
 
 
68 
 
Araújo et al. (2017) also reported a varied chemical composition (also reported furfural and phenol as 1607 
the most significant component in Eucalyptus urograndis (GG100 clone). They observed furfural and phenol as 1608 
the most critical components in Mimosa tenuiflora vinegar. Ribeiro et al. (2012) commented that furfural is 1609 
formed during carbonization through the cleavage of cellulose and pentosans molecules in the lignocellulosic 1610 
raw materials. It has a solid inhibitory effectivity on several kinds of microorganisms. Another difference to 1611 
highlight about the chemical composition of the two types of vinegar assessed here (Table 3) is the more 1612 
expressive presence of organic acids in the bamboo vinegar compared to the product from eucalyptus. In the 1613 
bamboo vinegar, acetic (12.5%), propionic (5.8%), and butanoic (1.8%) are present in higher quantities. In 1614 
contrast, in the second type of vinegar, the contents of propionic and butanoic acids are significantly lower, 0.7 1615 
and 0.6%, respectively, while acetic acid is not present. However, the acid acetic takes part in the chemical 1616 
composition of eucalyptus carbonization vinegar. 1617 
As standardized by Pimenta et al. (2018), adding ammonium hydroxide in the samples of eucalyptus 1618 
vinegar before the liquid-liquid extraction with organic solvents is enough to neutralize the acetic acid present in 1619 
them. In this work, the total contents of acetic acid in the eucalyptus and bamboo vinegar after adding 1620 
ammonium hydroxide were 1.01 and 1.92% (weight/weight), and the water contents were 97.17 and 98.03%, 1621 
respectively. The organic fractions were 0.96 and 0.91% for the respective types of vinegar. The addition of 1622 
hydroxide ammonium before GC/MS analyses cited above was employed to suppress the peak of the acetic acid 1623 
that tended to be so strong in the chromatograms that the other peaks underwent a decrease enough to make it 1624 
challenging to attain their percentage areas. Since the method was standardized specifically to the eucalyptus 1625 
vinegar, in the case of bamboo vinegar, adding the base most likely was not sufficient to suppress the acetic acid 1626 
peak due to its higher concentration in the product. Organic acids play an essential role against pathogenic 1627 
microorganisms, and their application dates back over 100 years with several sorts of assessments (Tamblyn and 1628 
Conner 1997; Gómez-García et al. 2019; Peh et al. 2020; Rossi et al. 2021). 1629 
The main advantages of the presence of organic acids in potential antimicrobial natural products 1630 
concern their efficiency of these compounds in the inhibition of microorganisms' growth and also their non-1631 
toxicity to humans (Zhitnitsky et al. 2017). Acetic and propionic acids had a proven antimicrobial effect on E. 1632 
coli and S. aureus (Raftari et al. 2009). The employment of acetic acid efficiently controlled the action of 1633 
biofilm-forming bacteria isolated from patients with burn injuries (Halstead et al. 2015). Other studies report the 1634 
efficiency of acetic acid as an antimicrobial agent in several applications, including as a food preservative and 1635 
control of bovine mastitis (Fraise et al. 2013; Wali and Abed 2019; Pangprasit et al. 2020). Likewise, propionic 1636 
acid also plays a vital role in microbial control as an individual agent and modulator (Haque et al. 2009; Wang et 1637 
al. 2014; El-Adawy et al. 2018). However, in the case of different types of vinegar derived from the 1638 
carbonization of other lignocellulosic raw materials, their biological effectcomes not only due to the presence of 1639 
organic acids but, above all, a synergistic presence of organic acids, phenolic compounds, and furfural (Setiawati 1640 
et al. 2019; Suresh et al. 2019; Suresh et al. 2020). 1641 
The higher antimicrobial activity of the bamboo vinegar evaluated in this work is most likely associated 1642 
with its higher content of different organic acids. The combined effect of organic acids can enhance the 1643 
inhibitory action of some microorganisms (Wu et al. 2017). For instance, Peh et al. (2020) demonstrated that the 1644 
combination of different organic acids brought about higher values of MIC, which was not found when the acids 1645 
were assessed individually. That finding reinforces the statement that the higher performance of bamboo vinegar 1646 
as an inhibition agent can be associated with a synergistic effect of a mixture of organic acids. However, even 1647 
with a lower content of organic acids, eucalyptus vinegar also had a significant antimicrobial impact since other 1648 
compounds in its composition have this effect. 1649 
In addition to the organic acid content, both types of vinegar assessed here are rich in phenolic 1650 
compounds in their chemical constitution. The presence of phenolic compounds is one characteristic of 1651 
explaining the antimicrobial properties of pyroligneous acids from different sources (Li et al. 2019). These 1652 
compounds are antimicrobial agents proven by several researchers in the literature (Tyagi et al. 2015; Jang et al. 1653 
2018; Nascimento et al. 2021). Some studies have demonstrated the high potential of pyroligneous acids as 1654 
 
69 
 
microbial inhibitors by expressing their ability to control the development of bacterial biofilms (Ariffin et al. 1655 
2017; Macé et al. 2017; Nassima et al. 2019). However, as displayed in Table 3, the phenolic content of the 1656 
products tested in this research varied. This may be another possible explanation for the variability of 1657 
antimicrobial action demonstrated between the products assessed here and within the same type of vinegar 1658 
against different microorganisms. The literature reports that the other phenolic compounds and their varied 1659 
structural arrangements have various antimicrobial capacities (Maddox et al. 2010). This is reinforced by 1660 
Bouarab-Chibane et al. (2019), who found that the same phenolic compound can act differently against different 1661 
microorganisms. These authors also emphasize the fact that the antimicrobial effect of a product may not be 1662 
linked solely to the presence of a class of phenolic compounds, as demonstrated by their research. 1663 
Other compounds present in EPs also play an essential role as bioactive molecules against bacteria and 1664 
viruses, such as butyrolactone, capable of inhibiting Erwinia carotovora (Cazar et al. 2005); maltol, used both as 1665 
a flavoring agent and as an effective antimicrobial adjuvant (Saud et al. 2019; Naqvi et al. 2021; Ziklo et al. 1666 
2021); and 1,2,4- trimethoxybenzene, a bioactive molecule that has the selective ability to inhibit the activation 1667 
of the NLRP3 inflammasome and thus reduce autoimmune encephalomyelitis (Pan et al. 2021). Pyridine 1668 
compounds with several applications, including antimicrobial and antiviral (Marinescu and Popa 2022). All cited 1669 
compounds are present to a greater or lesser extent in the products assessed in this work. With this, it is 1670 
emphasized that the antimicrobial action performed by eucalyptus and bamboo vinegar is due to a set of 1671 
substances and that it would be difficult to assign the role of a single class in such an effect. With what has been 1672 
exposed so far, the great potential that pyroligneous acids have for developingalternative antimicrobial agents 1673 
has been increasingly proven, aiming to attenuate the pressure exerted by the emergence of microbial resistance. 1674 
Suresh et al. (2019) commented that this potential is due to their chemical constitution being expressly varied, 1675 
which can hinder the development of resistance mechanisms of microorganisms against it. 1676 
Scanning electron microscopy (SEM) analyses 1677 
At this stage, only carbonization vinegar obtained from bamboo was used, due to its greater efficiency and the 1678 
high costs of microscopic analyses. Furthermore, the micrographs of Staphylococcus aureus, Streptococuus 1679 
agalactiae, and Candida albicans were the best attained and are presented hereafter. For these three 1680 
microorganisms, the changes caused by the products were similar. To avoid redundancy, only the micrographs of 1681 
them before and after being subjected to the action of bamboo vinegar are presented in this section. However, it 1682 
is essential to highlight that despite being similar, the changes in the cell walls of the microorganisms treated 1683 
with bamboo vinegar were achieved in lower concentrations than those resulting from the action of eucalyptus 1684 
vinegar. As displayed in Figures 4, 5 and 6, there were significant changes in the cell walls of the 1685 
microorganisms after exposure to the action of bamboo vinegar compared to the non-exposed cells. The 1686 
magnification employed in the micrographs varied according to the characteristics of each microorganism. 1687 
 
Figure 4. SEM micrographs of Staphylococcus aureus before (A) and after 24-h exposure (B) to bamboo 
vinegar. Magnification: 30.000 X 
 
70 
 
 
Figure 5. SEM micrographs of Streptococcus agalactiae before (A) and after 24-h exposure (B) to bamboo 
vinegar. Magnification: 13,400 X 
 
Figure 6. SEM micrographs of Candida albicans before (A), after 24 h (B), and 48 h (C) of exposure to bamboo 
vinegar. Magnification: 5,000 and 7,410 X (arrows in the micrographs indicate cell budding) 
Figures 4, 5 and 6, parts 4A, 5A, and 6A, display the control treatments on which the microorganisms grew 1688 
without the influence of the pyroligneous acid. In the 4A and 5A parts, the S. aureus and S. agalactiae have their 1689 
cell wall surfaces regular, turgid, and with the typical spherical shape of the coccus (Jankowsky et al. 2018). The 1690 
C. albicans cells (Figure 6A) are unscathed and have an oval shape, a characteristic of this microorganism. See 1691 
in the arrows; the cell budding is represented by the circular structure in the cell edges, which is another species 1692 
characteristic (Grela et al. 2019). However, after exposure to the bamboo vinegar, all microorganisms’ cell walls 1693 
underwent morphological alterations compared to the respective controls. The microorganism more extensively 1694 
affected was S. agalactiae (Figure 4B), with practically all cells damaged. For the other strains, some cells 1695 
remained unaltered (Figures 4B, 5B, and 6C), especially C. albicans, 24 h after exposure (Figure 6B). This 1696 
observation corroborates the data from the previous sections, where it was determined that this microorganism 1697 
was more resistant to the effect of both types of vinegar compared to the other strains. Most likely, the 1698 
explanation for the higher susceptibility of the different microorganisms is related to the structural characteristics 1699 
of their cell walls and their internal organization. C. albicans is a yeast, a eukaryotic microorganism, while the 1700 
others are prokaryotic bacteria (Ibrahim et al. 2013). This fact may make it easier for C. albicans to resist the 1701 
effect of the pyroligneous acids. As cited in the literature, one of the essential effects of PA and organic acids is 1702 
to promote damage to the genetic material inside the microbial cell due to acid stress in low pHs (Jeong et al., 1703 
2008; Lund et al. 2020). 1704 
 
71 
 
In Figure 6C, treated C. albicans cells (Figure 6C), it can be seen that the cells in budding, that is, in cell 1705 
multiplication, there was more damage than the other cells of this species because these cells are more 1706 
vulnerable during the multiplication process (Deng et al. 2021). In contrast to these damaged cells, both for S. 1707 
aureus and for C. albicans, some cells remained with their structure relatively preserved, without such severe 1708 
deformations (Figures 4B, 5B, and 6C). The study by Cherrington et al. (1991) reported the same behavior, 1709 
where the action of organic acids was able to inhibit microorganisms such as E. coli and Salmonella spp., 1710 
without disturbing its membrane, which suggests that there are other forms of PA action on microorganisms but 1711 
the single disorganization of the cell wall. Organic acids in the chemical profile of bamboo and eucalyptus 1712 
vinegar can acidify the cytoplasm and lead to cell lysis (Wang et al. 2014). These authors proved that propanoic 1713 
acid, one of the PA constituents, lowered the intracellular pH of S. aureus, which is lethal for the microorganism. 1714 
Maltol, another chemical constituent of bamboo vinegar, was able to cause severe changes in C. albicans cells, 1715 
observed in the form of pores and extravasated cells (Ziklo et al. 2021), which are also observed in this research. 1716 
In addition to the substances already mentioned, carbonization vinegar has various phenolic compounds in its 1717 
chemical constitution. The antimicrobial action caused by this group of compounds has been established in the 1718 
literature (Jang et al., 2018; Bouarab-Chibane et al. 2019; Nassima et al. 2019; Nascimento et al. 2021). 1719 
According to a review by Bouarab-Chibane et al. (2019), these compounds can have different types of action on 1720 
the microorganism, including modifying the natural structure of the membrane and the microbial cell wall and, 1721 
consequently, the permeability of the membrane and the rigidity of the wall. These modifications may be one of 1722 
the responses to the morphological changes shown in the SEM images. In addition, these compounds are capable 1723 
of causing punctual ruptures in these structures, resulting in the impairment of their functions, which results in 1724 
extravasation of cellular content, causing irreparable damage to the microorganism (Piekarska-Radzik and 1725 
Klewicka 2021). Therefore, this is another factor that may explain the results shown in this research. 1726 
CONCLUSIONS 1727 
 1728 
In the context discussed above, both types of vinegar obtained from eucalyptus and bamboo grown in 1729 
Northeastern Brazil have a real potential to be employed as a basis for the production of antimicrobial agents 1730 
because of their chemical composition and the proven inhibitory effect on pathogenic microorganisms. Both 1731 
products, for the most part, had the same chemical compounds in their chemical composition. However, even 1732 
with the similarity, there were significant differences that are believed to have resulted in the demonstrated 1733 
inhibition differences between them. Bamboo vinegar had a higher content of organic acids and greater 1734 
inhibitory efficiency when compared to eucalyptus. Besides eucalyptus wood from sustainable forests, bamboo 1735 
biomass has been increasingly employed to produce charcoal on a large scale, so the harnessing of liquid 1736 
products from its carbonization constitutes an important alternative to add value to the charcoal-making chain. 1737 
Still, the increasing production of bamboo vinegar in industrial conditions is creating a basis to establish formal 1738 
ways to include it in new products for agricultural use or animal health. 1739 
AUTHORS’ CONTRIBUTIONS 1740 
G.S.P. Gama accomplished the carbonization runs; prepared and refined the pyroligneous acids; conducted all of 1741 
the experiments; collected and tabulated theexperimental data; interpreted the experimental data; drafted the 1742 
manuscript; and prepared the final version of the manuscript in Portuguese. A.S. Pimenta was responsible for the 1743 
general coordination of research activities and fundraising; allocated inputs and reagents; interpreted 1744 
experimental data; interpreted statistical data; translated the manuscript from Portuguese to English; and 1745 
reviewed the English of the manuscript until getting the submission version. F.M.C. Feijó coordinated the 1746 
microbiological assays, allocated inputs and reagents, interpreted experimental data, and reviewed the draft and 1747 
final versions of the manuscript. C.S. dos Santos trained the staff in microbiological assays, allocated inputs and 1748 
reagents, and supervised the microbiological assays. E.C. de Souza interpreted experimental data; ran the 1749 
statistical analyses; adjusted and selected the best regression models; and carried out charting. T.V.C. Monteiro 1750 
performed the gas chromatography and mass spectrometry (GC/MS) analyses, acquired the chromatograms and 1751 
mass spectra and tabulated the experimental data. M. Fasciotti coordinated GC/MS analysis; interpreted 1752 
 
72 
 
chromatograms and mass spectra; elaborated the table with GC data; interpreted the experimental data; and 1753 
reviewed the final version of the manuscript. T.K.B. de Azevedo assisted in fundraising; supported the allocation 1754 
of inputs and reagents; assisted in collecting experimental data; and reviewed the final version of the manuscript. 1755 
R.R. de Melo assisted in fundraising, supported in collecting experimental data, interpreted the experimental 1756 
data, and reviewed the final version of the manuscript. A.F. Dias Júnior assisted in fundraising and collecting 1757 
experimental data, interpreted the experimental data, and reviewed the final version of the manuscript. 1758 
 ETHICAL STATEMENT 1759 
This article does not contain any studies with either human participants or animals performed by any authors. 1760 
ACKNOWLEDGEMENTS 1761 
 1762 
The present research work was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – 1763 
Brazil (CAPES) – Finance code 001 and National Council for Scientific and Technological Development 1764 
(CNPq), a foundation linked to the Ministry of Science and Technology (MCT), and FAPERN (Fundação de 1765 
Apoio à Pesquisa do Estado do Rio Grande do Norte). 1766 
 1767 
COMPETING INTERESTS AND FUNDING 1768 
 1769 
The authors inform that no competing or conflicting interests are associated with the funders of the present 1770 
research work. All authors agree with the submission and publishing of the article. 1771 
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Considerações Finais 
_____________________________ 
6. CONSIDERAÇÕES FINAIS 
__________________________________________________________________________ 
 
Constata-se que os extratos pirolenhosos de Eucalyptus urograndis e Bambusa 2035 
vulgaris apresentaram ação antibiótica e antifúngica satisfatórias, inibindo o crescimento de 2036 
E. coli, P. aeruginosa, S. enteritidis, S. aureus, S. agalactiae e C. albicans. Ambos os EPs se 2037 
mostraram como fontes promissoras ao desenvolvimento de alternativas antimicrobianas 2038 
naturais. O pH em que o EP é utilizado influencia no seu potencial antimicrobiano, no 2039 
entanto, o EP extraído de E. urograndis desempenhou papel antimicrobiano mesmo em pH 2040 
neutro, requerendo concentrações mais elevadas conforme a neutralização avançava, para 2041 
resultar em inibição. Sendo assim, pode-se afirmar que a acidez deste produto influencia a 2042 
sua ação antimicrobiana, no entanto, não é a única responsável por tal ação. Vale ressaltar 2043 
a necessidade de testes in vivo para melhor estruturação de utilização dos EPs. Estudos 2044 
 
81 
 
posteriores são necessários para um maior conhecimento a respeito da aplicação deste 2045 
produto como antimicrobiano e as manipulações necessárias para melhorar suas condições. 2046 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
82 
 
 
 
 
 
 
 
 
 
 
 Anexos 
_____________________________ 
7. ANEXOS 
__________________________________________________________________________ 
 
ANEXO I 
Link de acesso às normas das revistas 
Capítulo 1: Revista Árvore 
http://revistaarvore.org.br/2857-
2/#:~:text=As%20principais%20diretrizes%20s%C3%A3o
%3A,de%20rodap%C3%A9%20n%C3%A3o%20s%C3%
A3o%20aceitas. 
http://revistaarvore.org.br/2857-2/#:~:text=As%20principais%20diretrizes%20s%C3%A3o%3A,de%20rodap%C3%A9%20n%C3%A3o%20s%C3%A3o%20aceitas
http://revistaarvore.org.br/2857-2/#:~:text=As%20principais%20diretrizes%20s%C3%A3o%3A,de%20rodap%C3%A9%20n%C3%A3o%20s%C3%A3o%20aceitas
http://revistaarvore.org.br/2857-2/#:~:text=As%20principais%20diretrizes%20s%C3%A3o%3A,de%20rodap%C3%A9%20n%C3%A3o%20s%C3%A3o%20aceitas
http://revistaarvore.org.br/2857-2/#:~:text=As%20principais%20diretrizes%20s%C3%A3o%3A,de%20rodap%C3%A9%20n%C3%A3o%20s%C3%A3o%20aceitas
 
83 
 
Capítulo 2: World Journal of 
Microbiology and Biotechnology 
https://www.springer.com/journal/11274/submission-
guidelines#Instructions%20for%20Authors_Format. 
 
https://www.springer.com/journal/11274/submission-guidelines#Instructions%20for%20Authors_Format
https://www.springer.com/journal/11274/submission-guidelines#Instructions%20for%20Authors_Format