Cost-effective urine recycling enabled by a synthetic osteoyeast platform for production of hydroxyapatite

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Article https://doi.org/10.1038/s41467-025-59416-8
Cost-effective urine recycling enabled by a
synthetic osteoyeast platform for
production of hydroxyapatite
Isaak E. Müller1,11, Alex Y. W. Lin2,11, Yusuke Otani1,3,11, Xinyi Zhang 4,11,
Zong-Yen Wu1,3, David Kisailus5, Nigel J. Mouncey 1,3, Jeremy S. Guest 4,6 ,
Behzad Rad 2 , Peter Ercius 2 & Yasuo Yoshikuni 1,3,7,8,9,10
Recycling human urine offers a sustainable solution to environmental chal-
lenges posed by conventional wastewater treatment. While it is possible to
recover nutrients like nitrogen and phosphorus from urine, the low economic
value of these products limits large-scale adoption. Here, we show that engi-
neered yeast can convert urine into hydroxyapatite (HAp), a high-value bio-
materialwidely used inbone anddental applications. Inspiredby thebiological
mechanisms of bone-forming cells, we develop a synthetic yeast platform
osteoyeast, which uses enzymes to break downurea and increase the pHof the
surrounding environment. This triggers the yeast vacuoles to accumulate
calcium and phosphate as amorphous calcium phosphate, which is then
secreted in vesicles and crystallized into HAp. We achieve HAp production at
titers exceeding 1 g/L directly from urine. Techno-economic analysis demon-
strates that this process offers clear economic and environmental advantages,
making it a compelling strategy for high-value resource recovery from
human waste.
Urine recycling is gaining traction as an approach for sustainable
wastewater management1–5. Although urine composes only 1% of total
wastewater, it contains roughly 70–90% of nitrogen (N) and 50–65% of
phosphorus (P) in waste streams6,7, leading to environmental issues
such as eutrophication. Because urine is relatively easy to separate
from other waste streams, researchers are exploring decentralized
urine diversion (UD) processes for their economic and environmental
benefits over traditional centralized processes4,5. In UD processes,
urine is concentrated to produce N, P, and potassium (K)-based
fertilizers for local agriculture. If fully utilized, urine could replace 21%,
12%, and 20% of global N-, P-, and K-fertilizer demands, respectively7–9.
Additionally, city-wide UD processes could reduce greenhouse gas
emissions, energy demands, freshwater usage, and eutrophication
potential by 20–60% compared to conventional centralized
processes10. These processes may prove crucial for the sustainable
growth of our economy.
Two main scenarios for UD processes are often considered: urine
concentration (UC) and struvite and ammonium sulfate (SAS)
Received: 2 December 2024
Accepted: 22 April 2025
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1The US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. 2The Molecular Foundry, Lawrence
Berkeley National Laboratory, Berkeley, CA, USA. 3Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley,
CA,USA. 4TheGraingerCollege of Engineering, Department ofCivil and Environmental Engineering, University of IllinoisUrbana-Champaign, Urbana, IL, USA.
5Department of Materials Science and Engineering, University of California at, Irvine, CA, USA. 6US Department of Energy Center for Bioenergy and
Bioproducts Innovation (CABBI), University of Illinois Urbana-Champaign, Urbana, IL, USA. 7Biological Systems and Engineering Division, Lawrence Berkeley
National Laboratory, Berkeley, CA, USA. 8US Department of Energy Center for Bioenergy and Bioproducts Innovation (CABBI), Lawrence Berkeley National
Laboratory, Berkeley, CA, USA. 9Global Institution for Collaborative Research and Education, Hokkaido University, Hokkaido, Japan. 10Institute of Global
InnovationResearch, TokyoUniversity of Agriculture andTechnology, Tokyo, Japan. 11These authors contributedequally: Isaak E.Müller, Alex Y.W. Lin, Yusuke
Otani, Xinyi Zhang. e-mail: jsguest@illinois.edu; brad@lbl.gov; percius@lbl.gov; yyoshikuni@lbl.gov
Nature Communications | (2025) 16:4216 1
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processes10. The UC process uses reverse osmosis (RO) to concentrate
urine and produce urea- and struvite (NH4MgPO4·6H2O)-based fertili-
zers. Due to the rapid decomposition of urea to ammonia by environ-
mental microbes, the SAS process involves adding Mg salts for struvite
production and using ion exchange to recover ammonium salts. While
producing chemical fertilizers from urine is environmentally beneficial,
their relatively low market prices (USD 300-400/ton) limit economic
incentives. The success of UD processes hinges on fully exploiting the
potential of urine to produce asmany value-added products as possible.
To enhance the economic attractiveness of UD processes, we sought to
produce other high-value commodity chemicals from urine.
Among the chemicals in urine, urea stands out due to its abun-
dance and its potential to induce biomineralization, making hydro-
xyapatite (HAp) an ideal target11. HAp, a calcium phosphate mineral
with the chemical formula Ca5(PO4)3OH, is a major component of
biocomposites such as bone and teeth in vertebrates12 and impact-
resistant shells in some marine invertebrates13,14. Due to its bio-
compatibility, HAp is extensively used in orthopedic, oral care, and
plastic surgery applications, aswell as in the restoration andprotection
of archaeologicalmaterials. HAp’s high capacity to absorbfluoride and
heavy metals makes it valuable for water purification and industrial
downstreamprocessing15,16. Themarket for HAp is projected to exceed
USD 3.5 billion by 2030, with a high sales price (over USD 80per kg)
enhancing the economic attractiveness of UD processes. HAp com-
posites, known for their lightweight, high mechanical strength,
toughness, anddurability, have the potential to serve as renewable and
biodegradable alternatives to various commodity materials like plas-
tics and building materials17–23. Reducing the cost of HAp production
could also lower the carbon and energy footprints of these processes,
facilitating the global expansion of UD processes.
In this study, we develop a synthetic yeast platformosteoyeast for
HAp production directly from urine, inspired by the biological
mechanisms of osteoblasts (Fig. 1a)24. Using correlative optical and
electron microscopy, we show that osteoyeast mimics osteoblast-like
mineralization behavior by accumulating and secreting amorphous
calcium phosphate (ACP), which crystallizes into HAp.4.0 International License,
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	Cost-effective urine recycling enabled by a synthetic osteoyeast platform for production of hydroxyapatite
	Results
	Design principle
	Accumulation of calcium in vacuoles
	HAp synthesis
	Secretion of ACP EVs
	Transformation of ACP EVs into HAp
	HAp synthesis from urine
	TEA of HAp synthesis
	Discussion
	Methods
	Strains, plasmids, and reagents
	Yeast transformation
	Cultivation conditions for HAp production
	X-Ray diffraction analysis
	Dry state transmission electron microscopy
	Live cell fluorescence microscopy
	Correlative optical and electron microscopy
	HAp production in urine
	TEA of HAp synthesis
	Reporting summary
	Data availability
	References
	Acknowledgements
	Author contributions
	Competing interests
	Additional informationThe engineered
yeast efficiently synthesizes HAp from urine, achieving titer exceeding
1 g/L. A techno-economic analysis demonstrates that this bioprocess
offers significant cost and sustainability advantages over conventional
urine diversion strategies. These findings establish osteoyeast as a
promising platform for high-value biomanufacturing and resource
recovery from human waste.
Results
Design principle
We leveraged the underlying biological mechanisms of HAp synthesis
by osteoblasts25–30 as the foundation for developing a design principle
to engineer our osteoyeast strains (Fig. 1). In osteoblasts, HAp synth-
esis occurs through a series of several distinct steps. First, dense cal-
cium phosphate particles are formed in the mitochondria and
subsequently interact with lysosomes. Next, osteoblasts transport,
accumulate, and store calcium and phosphate in their lysosomes,
acidic organelles critical for maintaining organismal homeostasis28.
These lysosomes contain polyphosphate29, which, along with their low
pH, likely stabilizes the ACP phase30. Once filled with ACP, lysosomes
are secreted into the extracellular environment as matrix vesicles
(MVs), where polyphosphate is gradually replaced with monopho-
sphate. These MVs interact with structural proteins such as collagen,
deform over time to release ACP, and ultimately facilitate the forma-
tion of platelet-like HAp crystals within collagen fibril gaps, using the
fibrils as templates for mineralization.
To engineer osteoyeast, Saccharomyces boulardii was selected as
the chassis due to its greater tolerance to pH variation compared to S.
cerevisiae31. The yeast vacuole, a lysosome-like acidic organelle
responsible for pH homeostasis, ion storage, and metal tolerance, was
chosen as the primary engineering target (Fig. 1b)32. The vacuole con-
tainspolyphosphate synthesizedby the vacuolar transporter chaperone
(VTC) complex33, while the H+ antiporter Vcx1 facilitates calcium trans-
port and intracellular pH regulation34–36. We hypothesized that
increasing cytosolic pH through the production of hydroxide ions,
generated via urea decomposition by ureolytic enzymes such as urea
amidolyase, would activate Vcx1 to pump H+ out of the vacuole to help
neutralize cytosolic pH. Consequently, calcium ions are accumulated in
the vacuoles as ACP (Fig. 1b, Step 1). Although the precise mechanisms
remain unclear, various fungal species are known to produce extra-
cellular vesicles (EVs) through diverse pathways. These include vesicle-
containing vacuoles37, Golgi apparatus-mediated secretory pathways38,
and the endosomal sorting complex required for transport (ESCRT)
Fig. 1 | Design principle (schematic) for our synthetic osteoyeast platform.
a Hydroxyapatite (HAp) synthesis catalyzed by osteoblasts. Dense calcium phos-
phate particles are formed inmitochondria, and they interact with lysosomes (Step
1). Osteoblasts start to transport, accumulate, and store phosphate and calcium
within their lysosomes (Step 2). As the lysosomes are filledwith amorphous calcium
phosphate (ACP), they are secreted into the extracellular milieu as matrix vesicles
(MVs) (Step 3). TheseMVs interact with proteins such as collagen and are gradually
deformed, releasingACP (Step4). Lastly, platelet-likeHAp is formedwithin the gaps
of collagen fibrils, which act as templates (Step 5). b The design principle used for
engineering the osteoyeast platform. The ureolytic enzyme is overexpressed,
which triggers the activity of an antiporter (Vcx1) to exchange cytosolic Ca2+ with
intra-vacuolar H+. Ca2+ is accumulated and stored in the form of ACP (Step 1). The
ACP in the vacuoles is translocated to extracellular vesicles (EVs) and secreted into
the media (Step 2). The EVs merge, and ACP inside the EVs transforms into HAp
(Step 3). VTC vacuolar transporter chaperone.
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machinery39,40. These pathways present an opportunity to engineer the
secretion of ACP via EVs (Fig. 1b, Step 2). Once secreted, polyphosphate
in EVs degrades to monophosphate catalyzed by Ppn1/2 and is not
replenished by VTC due to the absence of ATP41,42. The abiotic conver-
sion of ACP to HAp occurs under suitable environmental conditions
(Fig. 1b, Step 3). This systematic approach underpins our strategy to
develop a yeast-based platform for HAp synthesis.
Accumulation of calcium in vacuoles
We first engineered the S. boulardii strain (SB760) to constitutively
express genes for a urea amidolyase (dur12) (strain SB818), and both
dur12 and a urea transporter (dur3) (strains SB822-SB823) (Table 1).
Testing the urease activity of SB760, 818, and 822 (Supplementary
Fig. 1) revealed that only SB822 showed increased urease activity,
indicating that both dur12 and dur3 are required for urea decomposi-
tion. To confirm SB822’s ability to accumulate calcium in vacuoles, we
used brightfield and wide-field fluorescence microscopy during urea
decomposition. We visualized the vacuole membrane by expressing a
gene coding for V-type proton ATPase subunit A (Vph1) fused with an
mCherry fluorescent protein, creating strains SB824-SB825 (Table 1)43.
We also used calcein-acetoxymethylester (calcein-AM) to monitor
intracellular calcium accumulation, as membrane-permeable calcein-
AM is hydrolyzed by intracellular esterases to impermeable calcein,
which fluoresces upon binding to calcium ions44. SB824 and SB825
were inoculated in modified SD media (no ammonium sulfate, 50mM
Ca2+, and 20 g/L urea) and grown for 18.5 h at 37 °C. SB825 increased
the culture pH to 6.06, while SB824 did not (pH 3.97). After adding
calcein-AM to each culture, fluorescence microscopy showed that
calcein accumulated in the vacuoles of both strains, with stronger
signals in SB825, indicatinghigher calciumaccumulation (Fig. 2). These
results indicated successful engineering of vacuoles to transport,
accumulate, and store Ca2+. Interestingly, crystal-like substances
appeared only in SB825 cultures, suggesting its involvement in their
formation.
Table. 1 | S. boulardii strains used in this study
Strain name Parent strain Integration plasmid used Modification
SB760 – – wild type S. boulardii
SB818 SB760 fIM54 TEF1pr-DUR1/2-AOX1ter at the TRP1 site
SB822 SB818 fIM52 TEF1pr-DUR1/2-AOX1ter at the TRP1 site, TDH3pr-DUR3-AOX1ter at the HIS3 site
SB823 SB760 fIM58 TEF1pr-DUR1/2-AOX1ter and TDH3pr-DUR3-AOX1ter at the TRP1 site
SB824 SB760 fIM81 VPH1pr-VPH1-mCherry-AOX1ter at the HIS3 site
SB825 SB823 fIM81 TEF1pr-DUR1/2-AOX1ter and TDH3pr-DUR3-AOX1ter at the TRP1 site,VPH1pr-VPH1-mCherry-AOX1ter at
the HIS3 site
DUR1/2 theurea amidolyasegene,DUR3 theurea transporter gene,VPH1-mCherryV-typeprotonATPase subunit Agene fusedwith anmCherryfluorescentproteingene.Species andgenenamesare
italicized.
Fig. 2 | Engineered yeast showing increased accumulation of calcium in
vacuoles.Theoptical images of yeast strains SB824 (a–d) andSB825 (e–h) grown in
the YNB media with 20g/L urea, 50mM Ca2+, and 10 µM calcein-AM are shown.
Three yeast cells located at the center of the panels (box in h) are magnified and
shown (i–l). Bright field (a, e, i), channel for VPH1-mCherry (b, f, j), channel for Ca2+-
bound calcein (c, g, k), andmerged (d, h, i) images are shown. The calcein channel
contrast was set to the same minimum and maximum for each strain for compar-
ison. The scale bar represents 5μm in (a–h). A portion of panels (e–h) was mag-
nified 4.5× and presented in panels (i–l). The experiment was performed with at
least ten biological replicates, all yielding similar results. Representative data
are shown.
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HAp synthesis
To further characterize the vacuolar content and extracellular crystal-
like substances, we analyzed the SB822 culture using transmission
electron microscopy (TEM). After cultivating SB822 inmodified YNB
media for less than 24 h, white precipitants were visible and removed
(Fig. 3a). This material was washed with deionized water, dried, and
analyzed using high-angle annular dark-field scanning TEM (HAADF-
STEM) imaging (Fig. 3b). This analysis revealed that the precipitants
were mixtures of cells and inorganic substances in different phases.
The intracellular particles and platelet-like extracellular crystals likely
represented Ca2+ accumulated in the vacuoles and crystal-like sub-
stances, respectively (Fig. 3b, zones A and B). Elemental analysis using
STEM-energy-dispersive X-ray (EDX) spectroscopy of zones A and B
(indicated in Fig. 3b) showed that both substances were composed
mainly of calcium and phosphorus (Fig. 3c–f and Supplementary
Fig. 2). Selected-area electron diffraction analysis further confirmed
that the intracellular particles were filled with ACP (Fig. 3g) and the
extracellular platelet-like crystals were crystalline HAp (Fig. 3h).
Notably, polyphosphate and low pH in vacuoles might also play an
important role in maintaining the amorphous form of calcium phos-
phate. The crystal components were further separated by gradient
centrifugation. X-ray diffraction (XRD) analysis showed that the HAp
produced by the engineered strain was very similar to that of bone
(Fig. 3i)45. Together with the results from fluorescence microscopy,
these findings indicated that we had successfully engineered yeast
vacuoles to transport, accumulate, and store calcium in the form of
ACP, creating a yeast strain capable of mediating bone-like HAp
synthesis, which we named osteoyeast.
Secretion of ACP EVs
To directly correlate the phenomena observed using fluorescence
microscopy and TEM, we developed a correlative imaging method to
monitor the cellular processes underlying HAp synthesis (Fig. 4a). This
method uses reference/finder TEM grids to locate the same cells. First,
we used fluorescencemicroscopy tomonitor the cellular processes for
intracellular ACP formation and extracellular HAp synthesis. Next, we
dried the samples on the grids and analyzed themusingTEM.Although
the samples moved slightly during drying, the relative locations of
each cell and HAp crystal corresponded well enough to allow for the
correlation of the two different imaging modes. In the first step, the
SB825 strain was grown in modified YNB media on the TEM grid. We
took pictures every 45 s using fluorescence microscopy and created a
movie describing the cellular processes (Supplementary Movie 1).
Figure 4b–e show snapshots of the important events during HAp
synthesis. As expected, the SB825 strain first accumulated calcein
within the vacuoles. During this time, we noticed that small particles
appeared around the yeast cells at 2.083h (Fig. 4b, arrows). Between
3.760 and 3.823 h (Supplementary Movie 1 and Fig. 4c, d), many HAp-
like particles were formed. Notably, we observed the fusion of two EVs
at 3.785 h (Fig. 4c), and this larger EV was transformed into HAp-like
particles at 3.798 h (Fig. 4d). Although the intensity was low, we also
found that these particles were fluorescent in the mCherry channel,
Fig. 3 | HAp synthesis mediated by the osteoyeast platform analyzed
using TEM. a Material produced by the osteoyeast strain SB822, collected using
10μm filter paper. b HAADF-STEM image of the collected material. STEM-EDX
elemental maps for Ca (c) and P (d) in zone A, and Ca (e) and P (f) in zone B.
g, h Electron diffraction patterns corresponding to zone A and B, respectively.
i XRD analysis of HAp synthesized by osteoyeast, showing a pattern comparable to
that of bone. The experiment was performedwith at least ten biological replicates,
all yielding similar results. Representative data are shown.
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Fig. 4 | HAp synthesis mediated by the osteoyeast platform using correlative
imaging analyses. a Scheme for correlative imaging analysis. b–e The time
course of HAp synthesis mediated by the osteoyeast platform. The arrows indi-
cate (b) emergence of extracellular vesicles, (c) fusion of two extracellular vesi-
cles to a single vesicle, (d) conversion to HAp, and (e) exudate from the ruptured
yeast cell. f–h Optical imaging of extracellular vesicles and HAP-like particles.
The EVs and HAP-like particles appear in the mCherry channel (f) and the calcein
channel (g) and are observable by bright-field imaging (h). i–n Analysis of a
ruptured yeast cell during HAp synthesis using fluorescence microscopy. j A
HAADF-STEM analysis of a ruptured yeast cell. k A STEM-EDX analysis of Ca from
the ruptured yeast cell. i–k The asterisks denote the same locations analyzed
using different microscopy methods. l Analysis of yeast cells during HAp
synthesis using fluorescencemicroscopy.m, n TEM analysis of (l) with increased
magnification. l–n The asterisks denote the same locations analyzed using dif-
ferent microscopy methods. o A HAADF-STEM analysis of the zone denoted with
the rectangle. p–r STEM-EDX analyses of Ca, P, and C. The experiment was per-
formed with at least three biological replicates, all yielding similar results.
Representative data are shown.
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suggesting that they were derived from the vacuoles (Fig. 4f–h).
Interestingly, the nascent HAp-like particles showed high calcein sig-
nals (Fig. 4c, d), but these signals gradually decreased over time
(Fig. 4e), possibly indicating the transformation of ACP into HAp.
At 4.748 h, one of the yeast cells underwent necrosis, and the cell
contents were exuded (Fig. 4e). The exudate was also coveredwith red
fluorescence, suggesting that vacuoles were translocated outside the
cells. We therefore investigated this exudate as a possible mechanism
for HAp synthesis (Fig. 4i–k). However, correlative TEM analysis sug-
gested that the exudate contained ACP rather than HAp (Fig. 4l–n).
Additionally, the proportion of yeast cells that underwent necrosiswas
low, ~1 cell per 200 cells, indicating that necrosis is unlikely theprimary
mechanism for ACP translocation. Further TEM analysis showed many
EVs ~20 to 150 nm in diameter, around the yeast cells (Fig. 4n). STEM-
EDX and electron diffraction revealed that these EVs were filled with
ACP (Fig. 4o–r). The presence of carbon around each ACP particle
suggests that the particles were encapsulated in lipid membranes
(Fig. 4r). The TEM image also showed that several EVs gathered closely
and may have been about to fuse into larger EVs. Our results suggest
that yeast cells can translocate vacuolar ACP into EVs, which are then
secreted. Although the mCherry signal was of low intensity, EVs also
carriedmCherry signals on their membranes (Fig. 4f–h), suggesting an
association with vacuoles. However, the dilution of red fluorescence
may mean that a fraction of the vacuoles might fuse with another
organelle or the plasma membrane during EV formation. After being
secreted, EVs couldmerge into larger EVs, and ACP in the EVs could be
transformed into HAp if a certain size is reached or if the media pH
becomes high enough to catalyze the transformation of ACP into HAp.
Transformation of ACP EVs into HAp
In cells, polyphosphate within vacuoles undergoes a dynamic cycle of
replenishment. The Ppn1/2 enzymes decomposepolyphosphate, while
the VTC complex synthesizes polyphosphate using ATP as a substrate.
Once extracellular vesicles (EVs) are secreted into the medium, ATP is
no longer available, leading to the degradation of polyphosphate into
monophosphate. This degradation plays a critical role in crystal
synthesis, as polyphosphate likely inhibits crystal formation. Notably,
significant calcein fluorescence signals were detected following HAp
formation between 3.785 and 3.798 h (Fig. 4c, d), indicating the pre-
sence of loosely bound calciumwithin the HAp crystals. Over time, the
calceinsignal diminished (Fig. 4e), suggesting that free calcium was
progressively incorporated into the HAp crystal structure or released
into the surrounding medium.
Furthermore, the transformation of ACP within EVs into HAp
particles occurred simultaneously (Fig. 4c, d). This transformation
appears to be triggered by pH changes during fermentation, which
occur as the culture pH rises over time due to urea degradation. This
process mirrors the co-precipitation method commonly used for HAp
production. To investigate this, a time-course experiment was con-
ducted to monitor the relationship between pH changes and HAp
formation during fermentation (Supplementary Fig. 3). Initially, the
culture pH was 4.4. After 15 h of fermentation, the pH increased to 5.8,
and no visible crystals were observed. Approximately 30min later,
crystalflakes began to form in the culture, coincidingwith a drop in pH
to 5.2. In this pH range, phosphate predominantly exists asH2PO4
−. The
observed pH drop is likely associated with HAp crystallization, driven
by the deprotonation of H2PO4
−. By 92 h, the pH had further
increased to 8.2.
TEM analysis of samples collected at different pH levels revealed
distinct structural changes. At pH 5.8, ACP vesicles, which likely
represent vacuoles, were observed (Supplementary Fig. 3a). At pH 5.2,
platelet-like HAp crystals began to form (Supplementary Fig. 3b), and
by pH 8.2, flower-like HAp structures were present in the culture
(Supplementary Fig. 3c). Interestingly, HAp formation occurred at a
relatively low pH, despite conventional co-precipitation methods
typically requiring higher pH values (10–11). In vertebrates, HAp for-
mation is templated by collagen at physiological pH. Similarly, it is
plausible that macromolecules secreted alongside ACP vesicles serve
as templates for HAp nucleation and crystallization, enabling the
process to occur under these conditions. To illustrate these findings,
we have summarized the comparison of wild-type and engineered S.
boulardii strains for HAp synthesis as schematic diagrams (Supple-
mentary Fig. 4).
HAp synthesis from urine
We tested the osteoyeast’s ability to produceHApdirectly fromhuman
urine (purchased from Innovative Research Inc.). The pH of the urine
was adjusted to 5.5 using acetate, a level optimal for yeast cultivation
but inhibitory to bacterial growth, thereby slowing urea decomposi-
tion. Various concentrations of CaCl2 (10–50mM) were added to the
urine, as the calcium concentration in urine varied between 2.5 and
5.0mMfrombatch to batch. Fermentationwas initiated by inoculating
the SB823 strain at afinalODof0.1. The cultureswere grown for 5days,
after which the remaining calcium concentrations were measured
(Fig. 5a). Based on these measurements, total calcium consumption
and the percentage of calcium consumed were calculated (Fig. 5b).
When osteoyeast was cultivated in urine supplemented with an addi-
tional 10mM calcium, it consumed over 10mM of calcium, corre-
sponding to 85% of the total calcium. However, when cultured in urine
supplementedwith 40mMcalcium,osteoyeast consumedonly 13mM,
and the consumption rate dropped to about 30%.
We observed that calcium consumption plateaued at 12, 13mM,
even when higher calcium concentrations were added. We hypothe-
sized that this limitation could bedue to the inoculumsize. To test this,
we increased the inoculum to a final OD of 0.15 and selected urine
supplemented with 30mM calcium for a time-course experiment
using SB823 and its control strain, SB760 (Fig. 5c). Calcium con-
sumption occurred at a rate of 12mM/day, with osteoyeast ceasing
calcium consumption two days after fermentation began. On day 10,
the cultures were terminated, and the precipitates were collected by
centrifugation. The precipitates were oven-dried, and HAp production
was estimated to be ~1.1 g/L compared to the control sample (Fig. 5d).
Although precise biomass measurements in SB823 cultures were not
possible, a growth comparison between SB760 and SB823 in mYNB
mediumwithout calcium suggests that the biomass yield of SB823 was
reduced to ~30% of that of SB760 (Supplementary Fig. 5). This obser-
vation supports the validity of our analysis. The sample was further
analyzed using optical microscopy, XRD, TEM, and electron diffrac-
tion, confirming HAp synthesis (Fig. 5e–i).
TEA of HAp synthesis
To investigate the financial viability of HAp synthesis from urine, we
designed, simulated, and conducted a techno-economic analysis (TEA)
of a city-scale HAp production system using the open-source process
simulators QSDsan46,47 and BioSTEAM48–50. Given the transient nature
of urea in fresh, undiluted humanurine and the volatility of ammonia51,
we assumed that HAp synthesis reactors would be distributed across a
densely populated city such as San Francisco and serve somewhere
between 10,000 and 80,000 people. Precipitates from the reactors
would be regularly collected, transported, and processed into HAp
products at a centralized facility. Osteoyeast would be cultivated
centrally and distributed to the reactors. The TEA system boundary
includes the HAp synthesis reactors, city-wide precipitate collection
and material distribution system, centralized osteoyeast production,
HApprocessing, and auxiliary facilities. A summary of end-to-endmass
balance, unit operation design, and equipment costing of the pro-
posed system is included in the Supplementary Materials. The mini-
mumproduct selling price (MPSP) of HAp for the system to break even
was chosen as the indicator of financial viability. Monte Carlo simula-
tions (n = 2000) were performed to incorporate the uncertainty of
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process design, system performance, and deployment contexts into
MPSP evaluation, and Spearman’s rank correlation coefficients were
characterized to determine the sensitivity of MPSP to each individual
assumption.
Despite the uncertainty, the TEA demonstrated this system offers
a potentially profitable approach for sustainable HAp production. At
the simulated scales, the system can recover P from urine and produce
65.4 tonne HAp per year (median value, the 5th–95th percentiles were
18.7–127 tonne/year, which will be presented in brackets hereinafter).
The median MPSP from Monte Carlo simulations was 18.8 (12.6–36.9)
USD/kg HAp (Fig. 6a), all within or below the market price ranges of
HAp at various grades (Supplementary Data 1–6)52–54. Operating
expenses (OPEX) over the 10-year project lifetime account for
68.3–87.6% of the system’s life cycle cost (Fig. 6b). The system’s
deployment context drives the onsite operating cost of these reactors
and the costs of logistics, contributing 59.2% to the total OPEX.
Osteoyeast production and precipitate post-processing also sig-
nificantly contribute to OPEX. Sensitivity analysis demonstrated that
contextual factors and technical performance of the HAp synthesis
process are themost impactful drivers for theMPSP (Fig. 6c). Covering
a larger population or increasing centralization can lower the unit cost
of HAp production.
The median unit cost of urine treatment with the HAp synthesis
system was estimated to be 54.5 USD/m³ urine. Assuming the recov-
ered HAp was sold at 50USD/kg (a moderate wholesale price of
industrial grade HAp), we estimated a median annual profit of 1.4
(0.2–2.4) million USD. Incorporating HAp synthesis into existing N
recovery technologies can make both N and P recovery from source-
separated urine financially viable. Compared to conventionalmethods
(e.g., solid-state, chemical precipitation, sol-gel, hydrothermal)55, HAp
Fig. 5 | Production of HAp from urine. a Remaining calcium concentration in the
cultures 5 days after the fermentation began. b Calculated calcium consumption
amount (bars) and percentage (plot) based on the samples in (a). c Calcium con-
sumption in the cultures. The experiment shown in (a) was repeated using the
higher inoculum size.d Dry weight of pellets collected from cultures grown for
10 days. e An optical image of the SB823 culture. f HAADF-STEM analysis of the
SB823 culture. g, h XRD spectra of the dried pellets from the SB760 and SB823
cultures. i Electron diffraction analysis of the product identified in (f). Each dot
(a, c, d) represents an individual measurement from a biological triplicate. Bars
(a, b, d) and lines (c) indicate the mean values, and error bars represent the stan-
dard deviation (a, c, d). Source data are provided as a Source Data file.
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synthesis through the osteoyeast platform requires fewer exogenous
chemical inputs by fully utilizing the existing phosphorus and urea in
fresh urine. Further, the osteoyeast-facilitated process features milder
reaction conditions and exhibits high HAp selectivity that is relatively
insensitive to variations in reaction conditions, making it suitable for
distributed applications at various scales.
Discussion
Urine recycling is an important concept for the development of a
sustainable economy. This process can produce a large amount of
fertilizers for agricultural applications and significantly reduce the
costs and environmental footprints associated with wastewater treat-
ment. However, the utility of this processhasbeen limiteddue to a lack
of economic incentives. Tomake the process more attractive, we have
demonstrated the development of the osteoyeast platform, utilizing
urine as a feedstock for the production of high-value chemical
hydroxyapatite (HAp).
The TEA suggests that the cost of HAp production using the
osteoyeast platform is highly attractive. Assuming anHAp selling price
of 50USD/kg, the annual profit from HAp synthesis was estimated to
Fig. 6 | Techno-economic analysis of a HAp synthesis system that recovers
phosphorus from urine at distributed locations. a Minimum product selling
price (MPSP) of the recovered HAp.Whiskers, boxes, and themiddle line represent
5th/95th, 25th/75th, and 50th percentiles, respectively, from Monte Carlo simula-
tion samples (n = 2000). Each sample represents a unique stochastic realization of
the TEA model (i.e., technical replicates under varying input parameters). “×”
indicates the mean MPSP of all samples. The shaded gray regions indicate the
market price ranges for HAp of various grades (e.g., food, industrial, cosmetic,
medical; 90-99.5% purity; detailed in Supplementary Data 4). Horizontal gray lines
indicate price values from literature73,74. b Breakdown of the system’s life cycle cost
into operating expenses (OPEX) and capital expenditures (CAPEX). Samples were
sorted with ascending contributions of a single category for better visualization.
c The relative sensitivity of HAp MPSP to different parameters, indicated by the
magnitudes of the Spearman’s rank correlation coefficients (ρ) between MPSP and
individual parameters. The null hypotheses of no monotonic relationship between
MPSP and individual parameters (i.e., ρ=0) were tested using two-sided t-
approximations (df = 1998). Exact ρ and p values are provided in Supplementary
Data 5 and 6. Source data are provided as a Source Data file.
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range between $19.1 and $138/m3 of urine, depending on the system
performance and deployment context. In contrast, nitrogen recovery
as ammonium sulfate liquid fertilizer through the UD process was
estimated to cost around $12–$33/m3 of urine56. Thus, the profit from
HAp production can make the UD process financially attractive.
Although this margin may not be guaranteed as the scale increases,
reducing the cost of HAp synthesis could significantly expand its
applications, including air and water purification, soil treatment16,
flame resistance and thermal protection57, plastic replacement, and
construction materials.
Given the low MPSP and the high market prices of nano-HAp52–54,
additional processes could also be employed to improve the HAp
product quality (e.g., purity, size, and morphology) and potentially
further increase the profit margin of this system despite the additional
costs. To fully realize this potential, future research and development
efforts should focus on advancing purification techniques, optimizing
scale-up processes, and exploring regionally-specific market entrance
opportunities to ensure broader adoption and commercial success.
Our TEA suggests that total phosphorus (TP) concentration and HAp
yield are two of the major cost drivers. TP can be easily increased by
adding cheap phosphate to the culture, andwill naturally increasewith
diet shifts from animal to vegetal protein sources8. In contrast,
improvingHAp titer and yieldwill likely require furthermodification of
the osteoyeast platform.
To increase HAp production, several geneticmodifications can be
explored. Urine contains sufficient urea to synthesize 60–90mM of
HAp, whereas our current production is limited to ~4mM. Optimizing
the reaction may involve increasing the Ca and P fluxes into both the
cytosol and vacuoles, synthesizing polyphosphate in vacuoles, and
breaking down polyphosphate to monophosphate in extracellular
vesicles (EVs). Additionally, we can explore accelerating the secretion
of ACP EVs. For instance, it is understood thatdiverse pathways suchas
the vesicle-containing vacuoles, Golgi apparatus secretory pathways,
and ESCRTmachinery are involved in EV secretion38,39,58,59. Modulating
these pathways could change the number of EVs secreted or their size
and morphology. Furthermore, the cell wall is an obstacle to EV
secretion. Deleting genes for cell wall biosynthesis or engineering
enzymes in EVs that modify cell walls could also improve EV secretion.
In addition to serving as a platform for HAp synthesis via the UD
process, the osteoyeast platform holds significant potential for a wide
range of applications. While we acknowledge the serendipitous nature
of this discovery, a relatively simple modification—expression of
ureolytic enzymes—successfully activated molecular mechanisms
strikingly similar to thosemediating HAp synthesis in osteoblasts. This
observation suggests that the molecular machinery underlying HAp
synthesis in both systems may have evolved from shared ancestral
mechanisms. These ancestral pathways likely provided stress resis-
tance against unexpected cytosolic pH fluctuations or facilitated the
detoxification of calciumandothermetal ions. Over evolutionary time,
these mechanisms appear to have been co-opted for the synthesis of
lightweight, durable, and impact-resistant bionanocomposites, such as
bone, teeth, and crustacean shells. Studying osteoblasts or crustacean
cells involved in HAp synthesis remains a challenge due to their bio-
logical complexity. However, the osteoyeast platform introduces an
alternative paradigm in biomaterial research, offering a simplified and
alternative model system for investigating the synthesis of HAp-based
bionanocomposites. Additionally, it provides a versatile platform for
producing bionanocomposites with commercial potential, thereby
opening opportunities for both fundamental studies and applied
research in biomaterials science.
In conclusion, the osteoyeast platform offers a promising
approach to transforming urine into a valuable resource for hydro-
xyapatite (HAp) production, delivering significant economic and
environmental benefits. By mimicking molecular mechanisms strik-
ingly similar to those used by osteoblasts for HAp synthesis, this
platform underscores the divergent and adaptive evolution of these
pathways. It also provides a unique opportunity to study and engineer
the molecular mechanisms underlying HAp synthesis across diverse
eukaryotic species involved in the formation of bionanocomposites.
Further optimization of the genetic and biochemical pathways in the
osteoyeast platformcould improve its efficiency and broaden its
applications, contributing to a more sustainable and economically
viable bioeconomy.
Methods
Strains, plasmids, and reagents
Escherichia coli Top10 (Invitrogen) was used as the cloning host. The S.
boulardii strain carrying auxotrophic mutations (trp1, his3, ura3) was
provided by the Jin lab60, and all S. boulardii strains used in this study
are listed in Table 1. Unless otherwise specified in the main text, all
chemical reagents were purchased from MilliporeSigma. For genomic
integration, the following constructs were generated: DUR3 (fIM52)
encoding urea amidolyase, DUR1/2 (fIM54) encoding the urea trans-
porter, both DUR3 and DUR1/2 (fIM58), and VPH1 fused to red fluor-
escent protein (fIM84). Plasmid maps are shown in Supplementary
Fig. 6, and annotated sequences are available in Supplementary
Data 7–10. The primers used in the creation of these plasmids can be
found in Supplementary Table 1.
Yeast transformation
To integrate the DNA fragment into the S. boulardii genome, yeast
transformations were performed using the lithium acetate method61.
Briefly, overnight yeast cultures were diluted 1:50 in YPD medium (1%
yeast extract, 2% peptone, and 2% glucose) and grown until the optical
density at 600nm (OD600) reached ≥0.4. A 50mL culture was then
harvested by centrifugation (3000× g, 5min), washed twice with 25mL
of sterile water, and resuspended in 1mL of water. A 100 µL aliquot of
the cell suspension was pelleted in a microcentrifuge tube and resus-
pended in a transformationmixture containing 240 µLof 50%PEG3350,
36 µL of 1M LiAc, 10 µL of 10mg/mL single-stranded DNA, and 74 µL of
linearizedDNAandwater. The yeast cellswere heat-shocked at 42 °C for
45min, then plated onto yeast synthetic defined (SD) agar medium
(Sunrise Science) supplementedwith CSM-trp, CSM-his, or CSM-trp-his.
Plates were incubated at 37 °C until colonies appeared.
Cultivation conditions for HAp production
Engineered yeast cells were grown overnight in YPD medium, washed
with PBS buffer, and inoculated into modified yeast nitrogen base
(mYNB) medium consisting of 1.71 g/L Yeast Nitrogen Base without
ammonium sulfate (#1500, Sunrise Science), appropriate amino acid
supplements, 20 g/L glucose, and 50mM CaCl2. Urea (20 g/L) and/or
additional CaCl2 (50mM) were optionally added at the start or later
during cultivation. Cultures were grown at 37 °C for up to 96 h (as
shown in Fig. 3). Crystals and cells were collected by filtration through
2.7μm filter paper, washed with water, and air-dried at room tem-
perature. In some cases, cell or cell-crystal suspensions were analyzed
immediately without filtration.
X-Ray diffraction analysis
The cultures were spin down at 3000 × g for 10min. Pellets were dried
at 80 °C for 48 h prior to analysis. The driedmaterial was analyzed on a
Rigaku MiniFlex 6 XRD.
Dry state transmission electron microscopy
Dry pellets containing yeast cells and inorganic materials were dis-
persed in distilled water and deposited onto lacey carbon 300 mesh
copper grids. TEM imaging and selected-area electron diffraction
(SAED)wereperformedat theNational Center for ElectronMicroscopy
on an FEI ThemIS TEM equipped with an X-FEG gun and a Ceta2
complementarymetal oxide semiconductor (CMOS) camera operating
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at 300 kV. High-angle annular dark field (HAADF) images were
acquired in scanning transmission electron microscopy (STEM) mode
at 300 kV with a convergence semi-angle of 11.3mrad. To determine
the compositions of the inorganic materials, energy dispersive X-ray
spectroscopy (EDS) was performed using a Bruker SuperX windowless
EDS detector, which has a solid angle of 0.7 steradian enabling high
count rateswithminimal dead time for fast STEM-EDSmapping. STEM-
EDS elemental mapping was performed at 300 kV with a 5 to 10min
acquisition time.
Live cell fluorescence microscopy
For long time-lapse fluorescence and bright field microscopy, yeast
cells were imaged on sterile glass-bottom dishes. Glass-bottom dishes
(35mm with 14mm #1.5 glass, Cellvis) were treated with 2mg/mL of
ConcanavalinA from JackBean (SigmaAldrich) in 1x PBS and allowed to
incubate for 30min.We then rinsed the glass-bottom 1x PBS to remove
unbound protein. Yeast cells grown overnight were then diluted in
YNB media supplemented with 20 g/L urea with or without 50mM
CaCl2 as described above and allowed to bind to the surface.
Bright field and fluorescence imaging were performed on an
inverted Zeiss Elyra 7 microscope using a Plan-Apo 63x/1.46 NA oil
immersion objective (Zeiss) and a Sapphire 488 nm (0.5W), 561 nm
(0.5W) and a Lasos 642 nm (550mW) laser, an MBS 405/488/561/641
and EF LBF 405/488/561/641 filter cube followed by a LP 560 or a BP
570-620+LP655beamsplitter.Calcein andmCherryfluorophoreswere
excited with the 488 nm and 561 nm laser lines, respectively. Bright-
field images were taken using a transmission filter cube. The fluores-
cence image was split on a Duolink adapter using the appropriate
beamsplitter and imaged on 2 pco.edge 4.2 high-speed scientific
CMOS cameras. Images were then analyzed using Zeiss Zen Black,
ImageJ, or FIJI software.
Correlative optical and electron microscopy
A 3mm Au TEM finder grid (Ted Pella) was plasma cleaned in Ar for
20 s then affixed to a glass-bottom dish using ultra-thin (0.0015 in)
polyimide tape. The entire dishwith the TEM grid was submergedwith
a 2mg/mL solution of Concanavalin A for 30min at 37 °C. We then
washed the dishwith 1x PBS and transferred 1 µL of overnight yeast cell
cultureonto the petri dish. Thedishwith the cell culturewas incubated
for another 30min at 37 °C before the cellmedia was aspirated off and
replacedwithmYNB (crystal growth)media (see cultivation conditions
for HAp production section above). Regions of interest near the
alphabet letter markers on the TEM finder grid were selected for live
cell imaging. The images are collected in a fashion similar to the one
outlined in the previous section, using a Plan-Neofluar 40x/1.3 DIC
WD=0.21 M27 objective to image a wider field of view. At the end of
the fluorescent imaging analysis, themedia was aspirated and the dish
and 1 µL of PBS were gently deposited away from the TEM grids to
avoid any excessive turbulent flowwhile rinsing away the media. After
being rinsed three times, the polyimide tapewas gently removed using
sharp tweezers, and the TEM grids were allowed to dry in air. After the
TEM grids were fully dry, they were loaded into a FEI ThemIS TEM, and
the regions of interest identified during the optical microscopy
experiments were imaged using the protocols described in the Dry
State Transmission Electron Microscopy section.
HAp production in urine
We tested and optimized the culture conditions of strain SB823 for the
production of HAp in human urine (purchased from Innovative
Research Inc.), which was adjusted to pH 5.5 using acetate (as shown in
Fig. 5). We added varying concentrations of CaCl2 (10–50mM) and
inoculated the strain to a final OD of 0.1 in a total culture volume of
15mL. HAp production was carried out for 5 days, after which we
measured the calcium concentration in the supernatant using calcium
assay kit (Abcam) and estimated calcium consumption.
After optimizing the conditions,we tested the ability of SB823 and
its control strain SB760 to produce HAp. Both strains were inoculated
into 15mL of human urine supplemented with 30mM CaCl2, adjusted
to pH 5.5 using acetate. We collected 50 µL of samples daily and
measured the calcium concentration. After 10 days of cultivation, the
samples were collected by centrifugation at 3000× g for 15min. The
resulting precipitatewaswashedwith deionizedwater and centrifuged
again at 3000 × g for 15min. The pellets were then dried at 80 °C for
48 h prior to dry-weight measurement.
TEA of HAp synthesis
We assumed each location had two fed-batch reactors in parallel for
continuous HAp synthesis.Similarly, we assumed two fed-batch fer-
menters in parallel for osteoyeast cultivation at the central facility.
These reactors are equippedwith a cleaning-in-place system, agitators,
recirculation pumps, and heat exchangers for automatic operation.
HAp reactors and equipment were sized based on the urine flow rate
and the design batch time62. Osteoyeast fermenter and equipment
were sized based on the system-wide demand for yeast biomass, and
the cultivation was assumed to resemble a typical industrial produc-
tion process of S. boulardii63. Blowers are included and sized based on
aeration duty for osteoyeast cultivation. Fresh osteoyeast cells were
separated from the liquid mixture using centrifuge without further
drying. Precipitates collected from the HAp reactors were assumed to
be dried with a recessed plate filter press and then incinerated. Total
chamber volume of the filter press was estimated based on the
moisture content and flowrate of the precipitates64. Fuel input for
incineration was estimated based on the higher heating value and dry
solid loading rate of the dried precipitates64. Purchase costs (Cp) of the
reactors, the centrifuge, the filter press, the incinerator, and all
equipment were estimated as power functions of their sizing factor (S)
as shown in Eq. 1. Baseline parameter values (i.e., a, b, and c) of these
power functions as well as their ranges of uncertainty were calibrated
with supplier price data (Supplementary Table 2 and 3).
Cp =a � Sb + c ð1Þ
We considered three major inputs (i.e., source-separated fresh urine
(Supplementary Table 4), CaCl2 powder, and osteoyeast inoculum)
and two main products (i.e., precipitates and supernatants) for the
HAp synthesis reactors. The mass balance of the HAp reactors can be
describedby Eqs. 2–6,whereMi,j indicates the averagemassflowrate of
component i in the input or output stream j at a deployed location:
MCaCl2, in
=MP,urine × 5:9721533 ð2Þ
MP,urine =CP,urine � Qurine � p=n ð3Þ
where 5.9721533 was derived from the stoichiometry of the HAp pre-
cipitation process, Cp,urine indicates the average TP concentration of
fresh urine, Qurine indicates the average urination rate, and p and n
represent the total population served and the number of deployed
locations across the city, respectively.
MHAp,precipitate =MP,urine � f HAp=0:184987 ð4Þ
where fHAp is the specified percentage of the theoretical maximum
yield, and 0.184987 is the phosphorus content of HAp.
Mbiomass,precipitate =Myeast, inoculum +MCOD,urine � ybiomass ð5Þ
Myeast, inoculum =Qurine � Cinoculum ð6Þ
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where Cinoculum indicates the design inoculum concentration in the
HAp reactors, ybiomass indicates the overall yeast biomass yield (g bio-
mass/g urine COD) due to yeast growth. ybiomass was calibrated against
a yeast-to-HAp mass ratio in the precipitate of 0.34 in the baseline
scenario (i.e., fHAp = 66%andCinoculum = 0.5 g/L), which is representative
of the experimental result (Fig. 5d).
Sulfuric acid was dosed to lower the pH of fresh urine to inhibit
other microbial activities, urea degradation, and the formation of
other precipitates during the HAp synthesis process. We assumed a
one-time dosage of 60meq H2SO4/L fresh urine for a conservative
estimation of the corresponding operational cost65.
The mixed influent of source-separated fresh urine, osteoyeast
inoculum, and CaCl2 was assumed to have cooled down to 30 °Cwhen
it reaches the HAp reactor. A heat exchanger is included for each HAp
reactor to maintain a reaction temperature of 37 °C. The heat utility
required for continuous operation of the HAp reactors was estimated
using a previously defined algorithm for similar fed-batch
fermenters62, where the heating duty is calculated as the difference
in total enthalpy flows between reactor outputs and reactor inputs
(accounting for differences in temperature and in composition). No
subsequent cooling is needed for the HAp reactors.
For the osteoyeast cultivation reactors, the feedstocks include
organic carbon (in forms of molasses and/or glucose), water, macro-
nutrients (in forms of ammonium sulfate and phosphoric acid), and
micronutrients (including minerals and vitamins), besides seed
osteoyeast. The output fermentation broth contains significant
amounts of osteoyeast cells, ethanol, and water. The mass balance is
described by the Eqs. 7–9:
Myeast,broth =n �Myeast, inoculum=f viable ð7Þ
Where fviable indicates the average fraction of viable cells63.
Msugar, f eed =Myeast, broth=yyeast ð8Þ
Qbroth =Msugar, f eed=Csugar ð9Þ
where the feedstock flowrate Msugar,feed is determined by the required
production rate of osteoyeast cells Myeast,broth, and the specified fer-
mentation yield yyeast (kg yeast/kg sugar), and the average volumetric
flowrate of the fermentation broth Qbroth (m
3/hr) is dependent on the
design sugar concentration Csugar (kg/m3). The aeration duty of an
osteoyeast fermenter is calculated using Eq. 10.
Qair =qduty � Vbroth ð10Þ
where qduty indicates the amount of air supply required per unit
volume of fermentation broth (m3·(hr·m3 broth)−1), and Vbroth is the
volumeof broth in the fermenter at its full capacity.Vbroth is dependent
on the fermenter volume, which is determined by the design retention
time, the average feedstock flowrate, and the number of fed-batch
reactors in parallel62. Key parameter values for the osteoyeast
fermenters can be found in Supplementary Table 5. All feedstocks
were assumed to be at 20 °C when fed to the fermenters. Heat
exchangers are installed and operated to maintain a fermentation
temperature of 35 °C. Heat utility was estimated using the same
algorithm as that for the HAp reactors62.
Precipitates from the HAp reactors are collected and then pro-
cessed at the central facility, first dried mechanically and then incin-
erated with additional fuel. The recessed plate filter press is sized
based on the total flow and moisture content of the precipitates col-
lected, following an established algorithm64. The incinerator is sized to
accommodate the required burn rate, which is determined by the
average output mass flow rate of the dryer. The final product is
assumed to be a powder composed of HAp and ash.
Mash,product =Mbiomass,precipitate � f inert ð11Þ
where finert represents the inert fraction of biomass. finert was fixed at
0.128, a value higher than the ash content of yeast cells to account for
other impurities66–68. Detailed design and TEA results can be found in
the Supplementary Fig. 7 and Supplementary Data 1–6.
To realistically evaluate the costs associated with material dis-
tribution and precipitate collection, we assumed the locations for HAp
reactor deployments were randomly distributed within a rectangular
area that spanned ~8.3 miles longitudinally and 7.0 miles latitudinally,
which resembles the size of San Francisco. The location for the central
facility was also randomly sampled in this area. Then a capacitated
vehicle routing (CVR) problem was formulated, given service time
required per location and vehicle capacity as constraints. We used
Google OR-Tools to solve the CVR problem69, and the optimized
routes for distribution and collection were input to the calculation of
traveling distances and time for costing.
For TEA, we assumed a 10-year project lifetime and a 5% discount
rate. CAPEX includes installed costs of all unit operations andequipment.
All installed costs were converted into 2023 USD using the Chemical
Engineering Plant Cost Index70. OPEX includes operation and main-
tenance labor costs (e.g., fermenteroperators, vehicledrivers), electricity
and fuel input, material costs (e.g., feedstock for yeast cultivation, CaCl2
for HAp synthesis), and others (e.g., central facility rent, vehicle rental
costs). All code for design, costing, and simulation of this system are
made available in the EXPOsan Python repository71. TEA was performed
usingthe TEA module in the QSDsan Python package46,72. Parameters
varied inMonteCarlo simulations are detailed in Supplementary Table 2.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
Data supporting the findings of thiswork are availablewithin the paper
and its Supplementary Information files. A reporting summary for this
Article is available as a Supplementary Informationfile. Source data are
provided with this paper.
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Acknowledgements
We would like to thank Professor Ro Cusick at University of Illinois
Urbana-Champaign for valuable comments to our project. Thisworkwas
supported by laboratory-directed research and development (LDRD).
The work conducted by the U.S. Department of Energy (DOE) Joint
Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science
User Facility, is supported by the Office of Science of the U.S. Depart-
ment of EnergyoperatedunderContractNo. DE-AC02-05CH11231.Work
at theMolecular Foundrywas supported by theOffice of Science,Office
of Basic Energy Sciences, of the U.S. Department of Energy under
Contract No. DE-AC02-05CH11231. This research was developed with
funding from the Defense Advanced Research Projects Agency (DARPA)
as part of the Bio-inspired Restoration of Aged Concrete Edifices
(BRACE) program. D.K. would like to acknowledge funding from AFOSR
(FA9550-23-1-0209). Any opinions, findings, conclusions, or recom-
mendations expressed in this publication are those of the author(s) and
do not necessarily reflect the views of DOE or the US Government. The
views, opinions, and/or findings expressed are those of the author and
should not be interpreted as representing the official views or policies of
the Department of Defense or the US Government. We thank Anita
Wahler for professional editing of this manuscript. We acknowledge
Eduardo de Ugarte for his artistic contribution to Fig. 1.
Author contributions
Y.Y., P.E., B.R., and I.E.M. conceived of the research project. I.E.M.
developed the osteoyeast strain. I.E.M., Z.Y.W., and A.Y.W.L. performed
imaging analyses. Y.O. performed HAp production from urine. X.Z.
performed design, simulation, and TEA of the HAp production system.
D.K. and N.J.M. provided valuable input throughout the course of this
work. Y.Y., I.E.M., A.Y.W.L., Y.O., and X.Z. drafted the manuscript and
generated figures and tables with the support of B.R., P.E., and J.S.G. All
authors reviewed and edited the manuscript and approved the final
version.
Competing interests
I.E.M., Y.Y., P.E., and A.Y.W.L. have filed a PCT and US patent application
(PCT/US2023/018715) for the osteoyeast platform. The remaining
authors declare no competing interests.
Additional information
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