Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Orkin, Jeff, and Deb Roy. “Semi-Automated Dialogue Act Classification for Situated Social Agents in Games.” Agents for Games and Simulations II. Ed. Frank Dignum. Berlin, Heidelberg: Springer Berlin Heidelberg, 2011. 148-162. As Published http://dx.doi.org/10.1007/s00253-010-2699-8 Publisher Springer-Verlag Version Original manuscript Accessed Wed May 25 18:24:16 EDT 2016 Citable Link http://hdl.handle.net/1721.1/66951 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike 3.0 Detailed Terms http://creativecommons.org/licenses/by-nc-sa/3.0/ Applied Microbiology and Biotechnology Manuscript for review Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha r Fo Journal: Manuscript ID: Manuscript Category: Complete List of Authors: AMB-10-19908.R1 Original Paper Pe Date Submitted by the Author: Applied Microbiology and Biotechnology n/a er Yang, Young-Hun; Konkuk University, Microbial Engineering Brigham, Christopher; Massachusetts Institute of Technology, Biology Budde, Charles; Massachusetts Institute of Technology, Chemical Engineering Boccazzi, Paolo; Massachusetts Institute of Technology, Biology Willis, Laura; Massachusetts Institute of Technology, Biomaterials Science and Engineering Hassan, Mohd Ali; Universiti Putra Malaysia, Biotechnology and Biomolecular Science Yusof, Zainal; SIRIM Berhad, Research and Technology Division Rha, ChoKyun; Massachusetts Institute of Technology, Biomaterials Science and Engineering Sinskey, Anthony; Massachusetts Institute of Technology, Biology ew vi Re Keyword: polyhydroxyalkanoate, Ralstonia eutropha, organic acid, mixture model Page 1 of 38 1 2 Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha 3 4 5 Yung-Hun Yang1, 2), Christopher J. Brigham1), Charles F. Budde3), Paolo Boccazzi1), 6 Laura B. Willis1,4), Mohd. Ali Hassan5), Zainal Abidin Mohd. Yusof 6), ChoKyun Rha4) 7 and Anthony J. Sinskey1,7,8)* 8 10 11 13 15 16 18 ev 17 rR 14 Department of Biology, 3)Department of Chemical Engineering, 4)Biomaterials Science and Engineering Laboratory, 7)Division of Health Sciences Technology, 8) Engineering Systems Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA 2) Current address: Department of Microbial Engineering, Konkuk University, Seoul, Republic of Korea 5) Department of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 6) Research & Technology Division, SIRIM Berhad, P.O. Box 7035, 40911 Shah Alam, Malaysia ee 12 1) rP 9 Fo 19 iew 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 20 21 22 23 24 * Author for correspondence (E-mail: asinskey@mit.edu, Tel#: 1-617-253-6721, Fax#: 1-617-253-8550) 25 1 Applied Microbiology and Biotechnology 1 Abstract 2 We employed systematic mixture analysis to determine optimal levels of acetate, 3 propionate, and butyrate for cell growth and PHA production by Ralstonia eutropha 4 H16. Butyrate was the preferred acid for robust cell growth and high PHA production. 5 The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of 6 propionate initially present in the growth medium. The proportion of acetate 7 dramatically affected the final pH of the growth medium. A model was constructed 8 using our data that predicts the effects of these acids, individually and in combination, 9 on cell dry weight (CDW), PHA content (% CDW), PHA production, 3HV in the 10 polymer, and final culture pH. Cell growth and PHA production improved 11 approximately 1.5-fold over initial conditions when the proportion of butyrate was 12 increased. Optimization of the phosphate buffer content in medium containing higher 13 amounts of butyrate improved cell growth and PHA production more than 4-fold. The 14 validated organic acid mixture analysis model can be used to optimize R. eutropha 15 culture conditions, in order to meet targets for PHA production and/or polymer HV 16 content. By modifying the growth medium made from treated industrial waste, such as 17 palm oil mill effluent (POME), more PHA can be produced. iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18 2 Page 2 of 38 Page 3 of 38 1 Keywords: polyhydroxyalkanoate, Ralstonia eutropha, organic acid, mixture model 2 iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 3 Applied Microbiology and Biotechnology 1 Introduction 2 Bioplastics are among the biomass-based products currently being developed to replace 3 petroleum-based products (Frazzetto 2003; Young 2003). A large number of bioplastics 4 have been discovered, with over 150 varieties (Williams et al. 1999), encompassing a 5 wide range of thermoplastic properties (Cornibert and Marchessault 1972). PHAs are 6 naturally occurring biopolymers produced by a diverse group of bacteria (Madison and 7 Huisman 1999; Sudesh et al. 2000) that possess characteristics similar to those of 8 chemically-synthesized polymers (Steinbüchel and Füchtenbusch 1998). 9 In general, the microbial biosynthesis of PHA results from the limitation of a nutrient, 10 such as nitrogen, sulfur, oxygen, or phosphate (Anderson and Dawes 1990) in the 11 presence of abundant carbon. These polymers are thought to serve in nature as storage 12 molecules for carbon and reducing equivalents, and in some bacterial strains, PHA can 13 account for more than 80% of CDW (Khanna and Srivastava 2005). The molecular 14 weight and monomer composition of polymer can be controlled by utilizing specific 15 PHA synthase enzymes and specific carbon substrates (Anderson and Dawes 1990; Sim 16 et al. 1997). PHAs exhibit different thermal and mechanical properties depending on the 17 types of monomers incorporated (Doi et al. 1995; Jendrossek and Handrick 2002). 18 These polymers can be rapidly hydrolyzed in soil, sea water, and lake water by iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 Page 4 of 38 Page 5 of 38 1 microorganisms (Mergaert et al. 1992; Khanna and Srivastava 2005). Many 2 environmental factors can influence the polymer properties, which can, in turn, affect 3 the rate at which the polymer degrades (Khanna and Srivastava 2005). 4 PHAs, which have better biocompatibility than petroleum-based plastics, have been 5 used to develop medical devices, artificial organs, and therapeutic composites (Chen 6 and Wu 2005; Misra et al. 2006; Wu et al. 2009a). Despite these advantages, production 7 costs of bioplastics remain higher than those of conventional plastics and will need to be 8 lowered for bioplastics to compete in the marketplace (Choi and Lee 2000). The overall 9 cost of polymer production could be decreased by the efficient use of inexpensive 10 carbon feedstocks (Nikel et al. 2006). Several studies have investigated the production 11 of bioplastics from industrial waste streams (Ahn et al. 2000; Füchtenbusch et al. 2000; 12 Marangoni et al. 2002; Chua et al. 2003; Liu et al. 2008), which have potential as 13 inexpensive carbon sources. The fermentation of these substrates yields favorable 14 amounts of cell mass and bioplastic. However, few systematic or statistical approaches 15 have been taken to enhance the production of the desired polymer products (Nikel et al. 16 2005). 17 To develop a cost-effective strategy to produce bioplastics from oil palm waste products 18 (Hassan et al. 2002), the possibility of using anaerobically treated, clarified POME was iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 5 Applied Microbiology and Biotechnology 1 examined. POME is an aqueous stream generated in large quantities during the milling 2 of oil palm fruits. The oil palm tree, Elaeis guineensis, is the most productive oilseed 3 plant under cultivation and is an important agricultural crop in Africa and Southeast 4 Asia (Basiron 2007; Wu et al. 2009b). In Malaysia, approximately 46 million tons of 5 POME are produced annually (Econ.mpob.gov.my/economy/Overview_2008_latest 6 130109. htm). 7 Malaysia, 2.5 tons of POME are generated. 8 POME is a colloidal suspension containing 95-96.5% water, 0.6-0.7% oil, and 4-5% 9 total solids that has high chemical oxygen demand (COD) and biological oxygen 10 demand (BOD), making it a severe pollutant that must be treated (Hassan et al. 2002; 11 Ahmad et al. 2003). POME can be fermented anaerobically by wild-type bacteria to 12 produce the volatile organic acids acetate, propionate, and butyrate in the mass ratio of 13 3:1:1, respectively (Yee et al. 2003). The feasibility of utilizing this treated POME as a 14 carbon source for the production of PHAs by Ralstonia eutropha has been demonstrated 15 (Hassan et al. 2002). In order to confirm and extend these preliminary findings, we have 16 used a mixture analysis model to optimize the concentrations of volatile organic acids in 17 minimal medium. We have determined the effects that altering the ratio of these organic 18 acids has on microbial cell growth, PHA production, and monomer composition of the Fo It is estimated that for every ton of crude palm oil produced in iew ev rR ee rP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 6 Page 6 of 38 Page 7 of 38 1 polymer. We report here the steps we have taken toward the goals of enhancing 2 bioplastic productivity and lowering production costs. 3 4 Materials and Methods 5 Bacterial strains and growth media 6 R. eutropha H16 (Wilde 1962) was precultured in 3 mL of dextrose free Tryptic Soy 7 Broth (TSB) for 24 h at 30°C. The cells were harvested and washed twice with sterile 8 water, then used to inoculate 5 mL of PHB minimal medium, prepared as previously 9 described (York et al. 2003) with a final NH4Cl concentration of 0.1 g/L. Cell growth 10 was monitored by measuring optical density (OD) at 600 nm, starting from an initial 11 OD600 of 0.05. For media containing high phosphate buffer (10X), we used 67 mM of 12 Na2HPO4, 62.5 mM of NaH2PO4, 26 mM of K2SO4, and 10 mM of NaOH. Cells for 13 high phosphate media growth experiments were grown initially in 5 mL PHB minimal 14 medium in a test tube for 48 h on a roller drum or shaker. For larger volume mixed acid 15 experiments with high phosphate media, 1 mL of cells from an overnight culture grown 16 in TSB was used to inoculate 50 mL of high phosphate minimal medium in 250 mL 17 flasks and then grown for 48 h at 30°C. 18 cells from an overnight culture grown in TSB was used to inoculate 50 mL of TSB in a iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology For 1 L cultures with mixed acids, 1 mL of 7 Applied Microbiology and Biotechnology 1 250 mL flask, which was then incubated for 24 h at 30°C. These cells were harvested, 2 washed with sterile water twice, used to inoculate 1 L of high phosphate minimal 3 medium, and then grown for 72 h at 30°C before harvesting. The cells were harvested, 4 washed twice with cold water, and lyophilized for 48 h. 5 acid concentrations, all values refer to the % (w/v) of the organic acid sodium salt added 6 to the media. 7 gentamicin. ee 9 All media used in this study were supplemented with 10 µg/mL rP 8 Note that when describing Fo Design of experiments and mixture analysis rR 10 To develop a strategy for optimizing cell growth and PHA production, a mixture 11 analysis model of three acids in the minimal medium was developed and populated 12 using a standard mixture analysis methodology (Nowruzi et al. 2008) and the Minitab 13 V14 program (http://www.minitab.com). For the design of mixture analysis experiments 14 to populate the model, we used a simplex lattice method augmented by the design of 15 axial points (0.17:0.17:0.67, 0.17:0.67:0.17, and 0.67:0.17:0.17 for acetate: propionate: 16 butyrate). The degree of lattice for this mixture analysis is three; therefore, the 17 experimental design contains the set of all 13 combinations, where each value of 18 organic acid is 0.00, 0.33, 0.67 and 1.00 (Table 1). The experimental data for response iew ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 8 Page 8 of 38 Page 9 of 38 1 trace plots are also shown in Table 1. All experiments were performed using 50 mL 2 cultures with 0.3% total organic acid content. 3 regression using the model fitting method was applied with full quadratic component 4 terms initially included. In the data analysis, the coefficients with p-value below 0.1 5 were used as parameters (Online Resource 1). 6 To plot mixture contours, a mixture rP Fo 7 Analytical methods 8 PHA quantity and composition were determined by gas chromatography using a slight 9 modification of a method described previously (Brandl et al. 1988). Approximately 10 10 mg of freeze-dried cells from each experiment were weighed and placed in Teflon- 11 stoppered glass vials. For methanolysis, 1 mL CHCl3 and 1 mL CH3OH/H2SO4 (85:15 12 v/v) were added to the samples, which were then incubated at 105°C for 2 h, cooled to 13 room temperature, and incubated on ice for 10 min. After adding 0.5 mL of cold water, 14 the samples were vortexed for 1 min, then centrifuged at 2000 × g. The organic phase 15 was extracted by pipette, and moved to clean borosilicate glass tubes containing Na2SO4. 16 These samples were then filtered and injected into a gas chromatograph (Agilent, CA, 17 USA) equipped with a DB-Wax capillary column (Agilent, 30 m x 0.32 mm x 0.5 µm). 18 The volatile acids were monitored by high-performance liquid chromatography (HPLC) iew ev rR ee 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 9 Applied Microbiology and Biotechnology 1 using an Aminex HPX-87H column (Bio-Rad, CA, USA) at 50°C with a diode array 2 detector at 210 nm. The mobile phase was 5 mM sulfuric acid, with a flow rate of 0.6 3 mL/min. 4 5 Results 6 Optimizations of mixed acid concentration, buffer concentration, and C/N ratio. 7 Initial experiments were performed to determine the experimental range of organic acid 8 concentrations to be explored for the mixture analysis. Specifically, we examined the 9 growth of R. eutropha H16 in minimal media containing individual organic acids or 10 combinations of mixed acids as sole carbon sources, various buffer concentrations, and 11 various carbon to nitrogen (C/N) ratios. We tested utilization of the individual acids, 12 and found R. eutropha exhibited maximal growth in defined minimal medium 13 containing 0.5% acetate, 0.4% propionate, or 0.5% butyrate as the sole carbon source 14 (Fig. 1). When R. eutropha H16 was grown on the 3:1:1 (acetate: propionate: butyrate) 15 maximum growth was observed with a 0.3% total acid concentration (Fig. 2a). 16 The growth of R. eutropha in the standard minimal medium was accompanied by an 17 increase in the final pH to values as high as 9.5. Therefore, experiments were performed iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 10 Page 10 of 38 Page 11 of 38 1 to study cell growth on the mixed acid carbon source using different concentrations of 2 phosphate buffer added to control pH. A range of phosphate buffer concentrations from 3 10 mM to 500 mM was examined, and media with up to 130 mM phosphate buffer 4 exhibited an increase in final culture optical density that was proportional to the amount 5 of buffer added (Fig. 2b). The buffer concentration of 130 mM phosphate (i.e., high 6 phosphate media, compared to our initial media) was therefore employed in all further 7 mixed acid culture experiments. Various C/N ratios were examined by adding different 8 levels of mixed acids to the standard minimal medium. 9 for the most robust cell growth using mixed acids as the carbon source (Fig. 2c), while rR ee rP Fo We found that 40 C/N allowed 10 the % PHA of CDW was highest at 80 C/N (Fig. 2d). 11 highest PHA content, the low cell densities of these cultures made them difficult to 12 analyze. 13 The initial pH of the culture medium also affected cell growth, especially with 14 propionate and acetate as carbon sources. With increases in the initial pH of the growth 15 medium, R. eutropha showed a lower final OD600 on acetate as the main carbon source. 16 However, when cells were grown on propionate, a different trend emerged: 17 optical density increased as the initial pH was increased up to 7.3, then went down with 18 further increases in pH (Fig. 3a). The increase in pH of the cultures was greater when Although 80 C/N provided the ev We therefore used 40 C/N in all further mixed acid experiments. iew 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 11 the final Applied Microbiology and Biotechnology 1 acetate was used as the carbon source than when the other organic acids were used (Fig. 2 3b). The reason why acetate consumption increased the pH of the growth medium was 3 not clear, and no significant production of by-product was detected by HPLC analysis 4 (data not shown). A similar pH increase was observed in R. eutropha cultures in another 5 study (Wang and Yu 2001), but no explanation for this phenomenon was given. Fo 6 rP 7 Modelling the effects on Cell Growth, pH, Amount of PHA, and 3HV Content 8 To populate the mixture analysis model of the effect of the three acids on cell growth, 9 PHA production, and 3HV content, R. eutropha was grown with 13 culture 10 compositions of mixed acids, as described in Materials and Methods (Table 1). Each 11 culture condition was grown for 48 h and tested in duplicate. For each culture, we 12 measured the cell growth, medium pH, amount of PHA per 50 mL culture, PHA content 13 in the cells (% of CDW), residual CDW (CDW minus PHA), and the 3HV content of 14 PHA. 15 Contour plots of each variable, generated by the modelling software, were used to 16 predict growth and PHA production over the range of compositions tested (Fig. 4). To 17 examine the accuracy of these models, three additional mixture compositions, which iew ev rR ee 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 12 Page 12 of 38 Page 13 of 38 1 had not been included in the first set of 13 conditions, were selected and tested. The 2 three conditions used for the model validation tests were (acetate: propionate: butyrate): 3 0.1:0.1:0.8, 0.1:0.4:0.5, and 0.6:0.3:0.1. The results of these cultures validated the 4 predictive power of the model for CDW, PHA content, total amount of PHA per volume 5 of culture, and 3HV%. The results indicated that the model contour plots accurately 6 reflected each of the empirical trends (Fig. 5), and that the model could be used to 7 reliably predict the results of growth on various acid mixtures. From the results outlined 8 in Fig. 4, an increase in the initial concentration of butyrate augmented cell growth and 9 PHA production. Interestingly, the optimal acid composition for total PHA production 10 predicted by the model is not pure butyrate, but rather 0.2:0.0:0.8. The reason that a 11 small fraction of acetate in the medium is beneficial is not currently understood. 12 Butyrate concentration correlated positively with cell weight and amount of PHA, while 13 propionate concentration correlated positively with 3HV content in the final PHA. 14 We determined that the effect of butyrate on cell growth and PHA production was not 15 due simply to an increase in total carbon available in the media. When we normalized 16 carbon levels to 124 mM of total carbon and examined differences in growth between 17 conditions of 0.3% (3:1:6), 0.34% (3.6:1:1) and 0.33% (3:1.5:1), the culture with the 18 highest concentration of butyrate reached the highest OD600 (Online Resource 2). iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 13 Applied Microbiology and Biotechnology 1 The amount of 3HV in the final PHA was proportional to the amount of propionate 2 added to the culture, as previously reported (Bhubalan et al. 2008). With a constant 3 amount of propionate in the media, and different acetate:butyrate ratios, combinations 4 with higher acetate led to PHAs containing more 3HV. For example, polymer from the 5 0.67:0.33:0 (acetate: propionate: butyrate) cultures contained 7.7% 3HV, while the 6 polymer from the 0:0.33:0.67 (acetate: propionate: butyrate) culture contained 4.6% 7 3HV. 8 (Table 1). 9 experiments, can be applied to adjust conditions to optimize cell growth and polymer 10 production. The model can also be used to maximize polymer production with a given 11 monomer composition (i.e. culture conditions can be identified that will maximize 12 polymer accumulation with a minimum 3HV content). rP Fo However, cultures containing higher acetate yielded less PHA and total biomass These results, which are based on model predictions and empirical iew ev rR ee 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 13 14 PHA Production 15 We have found that increasing the ratio of butyrate to the other organic acids increased 16 total biomass and PHA content of R. eutropha, consistent with previous reports 17 (Chakraborty et al. 2009). Based on our model predictions and experiments, the 14 Page 14 of 38 Page 15 of 38 1 reported acid composition from treated POME (3:1:1 acetate: propionate: butyrate) is 2 not an optimal ratio for maximizing cell growth and PHA production (Hassan et al. 3 2002). Our model shows that supplementing the original 3:1:1 mixture to change the 4 ratios of the volatile acids could yield several alternative media compositions that are 5 more favourable for cell growth and production of PHAs (Online Resource 3). To test 6 some of the predictions listed in Online Resource 3, we compared 3:1:1 (acetate: 7 propionate: butyrate, Fig. 6a) to 3:1:6 (acetate: propionate: butyrate, Fig. 6b) conditions 8 using larger (1 L) culture volumes. 9 monitored by HPLC. The concentration of each volatile acid was ee rP Fo CDW and PHA content increased continuously until 41 h (Fig. rR 10 6a and b). When the results in Fig. 6a and 6b are compared, it can be seen that the 11 maximum PHA obtained from 3:1:6 (41 h, CDW 1.33 g/L, PHA 0.86 g/L) is 12 significantly higher than that obtained from 3:1:1 (41 h, CDW 1.10 g/L, PHA 0.64 g/L). 13 In addition, measuring the consumption of volatile acids showed that butyrate was 14 depleted first, regardless of its initial concentration in the medium (Fig. 6a and b). This 15 observation was confirmed by using a culture medium containing a uniform percentage 16 of each volatile acid (1:1:1 acetate: propionate: butyrate) (Online Resource 4). The rapid 17 depletion of butyrate is consistent with a report which suggested that R. eutropha can 18 direct 67% of butyrate into the TCA cycle, whereas only 33% of acetate can enter the iew ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 15 Applied Microbiology and Biotechnology 1 TCA cycle (Chakraborty et al. 2009). Our experiments clearly show that butyrate 2 increased PHA accumulation more effectively than acetate. This may be related to the 3 observation that butyrate can be incorporated into PHA without decomposition of the 4 molecule (Doi et al. 1988). 5 Fo 6 Discussion 7 Many efforts have been undertaken to seek renewable sources of carbon for the 8 production of PHA (Tan et al. 1997; Loo et al. 2005; Nikel et al. 2005). Industrial and 9 agricultural effluents are promising because these aqueous streams contain various 10 sugars, organic acids, and other organic compounds that can be recovered and used as 11 substrates for fermentation. Despite the advantages of turning waste streams into carbon 12 feedstocks, several problems remain to be solved regarding the use of industrial wastes 13 as carbon sources for PHA production. First and foremost, the carbon composition of an 14 untreated waste stream is unlikely to be optimal for biological polymer production. 15 Untreated waste streams may contain harmful components, which could inhibit bacterial 16 growth and, in turn, limit PHA production. Furthermore, the composition of these 17 streams may not be easy to control and reproduce (Zaldivar et al. 2001). One strategy 18 that can therefore be employed is to treat a waste stream in order to generate a more iew ev rR ee rP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 Page 16 of 38 Page 17 of 38 1 desirable feedstock, as has been demonstrated by using anaerobic fermentation of 2 POME to produce organic acids (Hassan et al. 1996). 3 In this study, we applied systematic optimization modelling of mixed acid ratios to 4 establish the effect of each volatile fatty acid and to maximize cell growth and PHA 5 production. We first reformulated our minimal medium, raising the phosphate buffer 6 concentration so as to increase buffering capacity and changing the C/N ratio. We then 7 populated a model using data from cultures with various mixed acid compositions, and 8 confirmed the predicted results with additional mixed acid cultures. With the validated 9 model we can predict growth and PHA production of R. eutropha on any combination rR ee rP Fo 10 of acetate, propionate, and butyrate. 11 medium resulted in differences in cell growth: depending on the type and concentration 12 of each organic acid, different final pH values were observed, suggesting that 13 fermentation proceeds by different routes and different metabolites are produced. These 14 results suggest several strategies to make treated POME a better substrate for cell 15 growth and PHA production. One way to stimulate biomass production, starting with a 16 3:1:1 (acetate: propionate: butyrate) mixture, is to increase the amount of butyrate, 17 which is consumed by R. eutropha faster than the other volatile acids tested in this work. 18 More butyrate would increase CDW as well as PHA content. To increase the proportion We also observed that the initial pH of the growth iew ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 17 Applied Microbiology and Biotechnology 1 of 3HV in the resulting PHA, cells can be grown with more propionate and higher 2 initial pH. Previous work has shown that introducing 3HV into a PHB chain reduces the 3 melting temperature and increases the toughness of the resulting plastic, making it a 4 more useful material (Anderson and Dawes, 1990). Copolymers of 3HB and 3HV 5 synthesized by R. eutropha are also known to have lower molecular weights, compared 6 to the PHB homopolymer (Kunioka and Doi 1990; Scandola et al. 1992). The mixed 7 acid model allows one to formulate a medium for production of PHA with a desired 8 3HV content. 9 Furthermore, the validated mixture optimization model can also guide the development 10 of alternative procedures for upstream treatment of POME, in order to improve the 11 proportions of certain acids in the final composition. For example, the composition of 12 organic acids generated from POME has been shown to be affected by controlling the 13 pH of the treatment conditions (Hassan et al. 1996). 14 In this work, a model designed to improve PHA production from mixed organic acids 15 by R. eutropha H16 was constructed and validated. 16 represent a significant step towards the production of low-cost bioplastic using organic 17 waste streams as a carbon feedstock. 18 18 iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The results discussed here Page 18 of 38 Page 19 of 38 1 Acknowledgements 2 The authors thank Mr. John Quimby and Ms. Karen Pepper for their contributions to 3 this manuscript. This work was supported by a grant from the government of Malaysia 4 and the Malaysian Office of Science, Technology and Innovation (MOSTI). The result 5 of this grant, the Malaysia-MIT Biotechnology Partnership Program (MMBPP), is a 6 collaboration between MIT, SIRIM Berhad, Universiti Putra Malaysia, and Universiti 7 Sains Malaysia. The authors would like to thank the members of this program for their 8 collegial collaborations. iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 19 Applied Microbiology and Biotechnology 1 References 2 Ahmad AL, Ismail S, Bhatia S (2003) Water recycling from palm oil mill effluent 3 (POME) using membrane technology. Desalination 157:87-95 4 Ahn WS, Park SJ, Lee SY (2000) Production of Poly(3-hydroxybutyrate) by fed-batch 5 culture of recombinant Escherichia coli with a highly concentrated whey solution. 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Macromolecules 28:4822-4828 9 Doi Y, Tamaki A, Kunioka M, Soga K (1988) Production of copolyesters of 3- 10 hydroxybutyrate and 3-hydroxyvalerate by Alcaligenes eutrophus from butyric and 11 pentanoic acids Appl Microbiol Biotechnol 28:330-334 12 Frazzetto G (2003) White biotechnology - The application of biotechnology to industrial 13 production holds many promises for sustainable development, but many products still 14 have to pass the test of economic viability. EMBO Rep 4:835-837 15 Füchtenbusch 16 polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from 17 an oil remaining from biotechnological rhamnose production. Appl Microbiol 18 Biotechnol 53:167-172 19 Hassan MA, Nawata O, Shirai Y, Rahman NAA, Yee PL, Bin Ariff A, Ismail M, Karim 20 A (2002) A proposal for zero emission from palm oil industry incorporating the 21 production of polyhydroxyalkanoates from palm oil mill effluent. J Chem Eng Jpn 35:9- 22 14 23 Hassan MA, Shirai Y, Kusubayashi N, Karim MIA, Nakanishi K, Hashimoto K (1996) 24 Effect of organic acid profiles during anaerobic treatment of palm oil mill effluent on ASM, Takabatake H, Satoh H, Mino T (2003) Production of B, Wullbrandt D, ev rR ee rP Fo Steinbüchel A (2000) Production of iew 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 21 Applied Microbiology and Biotechnology 1 the production of polyhydroxyalkanoates by Rhodobacter sphaeroides. J Ferment 2 Bioeng 82:151-156 3 Jendrossek D, Handrick R (2002) Microbial degradation of polyhydroxyalkanoates. 4 Annu Rev Microbiol 56:403-432 5 Khanna S, Srivastava AK (2005) A simple structured mathematical model for 6 biopolymer (PHB) production. 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Process Biochem 38:137-141 21 Mergaert J, Anderson C, Wouters A, Swings J, Kersters K (1992) Biodegradation of 22 polyhydroxyalkanoates. FEMS Microbiol Rev 9:317-321 23 Misra SK, Valappil SP, Roy I, Boccaccini AR (2006) Polyhydroxyalkanoate 24 (PHA)/inorganic Huisman phase GW (1999) composites for 22 Metabolic engineering iew LL, ev rR ee 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 22 of 38 tissue engineering of poly(3- applications. Page 23 of 38 1 Biomacromolecules 7:2249-2258 2 Nikel PI, de Almeida A, Melillo EC, Galvagno MA, Pettinari MJ (2006) New 3 recombinant Escherichia coli strain tailored for the production of poly(3- 4 hydroxybutyrate) from agroindustrial by-products. 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Trends Biotechnol 16:419-427 20 Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of 21 polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 25:1503-1555 22 Tan IKP, Kumar KS, Theanmalar M, Gan SN, Gordon B (1997) Saponified palm kernel 23 oil and its major free fatty acids as carbon substrates for the production of 24 polyhydroxyalkanoates in Pseudomonas putida PGA1. Appl Microbiol Biotechnol rate of bacterial rR ee rP Fo poly(3-hydroxybutyrate-co-3-hydroxyvalerate). iew ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 23 Applied Microbiology and Biotechnology 1 47:207-211 2 Wang J, Yu J (2001) Kinetic analysis on formation of poly(3-hydroxybutyrate) from 3 acetic acid by Ralstonia eutropha under chemically defined conditions J Ind Microbiol 4 Biot 26:121-126 5 Wilde E (1962) Untersuchungen über Wachstum und Speicherstoffsynthese von 6 Hydrogenomonas. Arch Mikrobiol 43:109-& 7 Williams SF, Martin DP, Horowitz DM, Peoples OP (1999) PHA applications: 8 addressing the price performance issue: I. Tissue engineering. Int J Biol Macromol 9 25:111-121 rP Fo 10 Wu Q, Wang Y, Chen GQ (2009a) Medical Application of Microbial Biopolyesters 11 Polyhydroxyalkanoates. Artif Cell Blood Sub Biot 37:1-12 12 Wu TY, Mohammad AW, Jahim JM, Anuar N (2009b) A holistic approach to managing 13 palm oil mill effluent (POME): Biotechnological advances in the sustainable reuse of 14 POME. Biotechnol Adv 27:40-52 15 Yee PL, Hassan MA, Shirai Y, Wakisaka M, Karim MIA (2003) Continuous production 16 of organic acids from palm oil mill effluent with sludge recycle by the freezing-thawing 17 method. J Chem Eng Jpn 36:707-710 18 York GM, Lupberger J, Tian J, Lawrence AG, Stubbe J, Sinskey AJ (2003) Ralstonia 19 eutropha 20 hydroxybutyrate] depolymerase genes. J Bacteriol 185:3788-3794 21 Young AL (2003) Biotechnology for food, energy, and industrial products - New 22 opportunities for bio-based products. Environ Sci Pollut R 10:273-276 23 Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from lignocellulose: a 24 challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol H16 encodes two and iew ev rR ee 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 24 of 38 possibly 24 three intracellular Poly[D-(-)-3- Page 25 of 38 1 56:17-34 2 3 iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 25 Applied Microbiology and Biotechnology 1 Table. 1 2 Experimental design points chosen through a simplex lattice methodology for the 3 mixture analysis model and their experimental results. CDW ID # Acetate PHA content Propionate Butyrate 3HV% (mg/ 50mL) (wt%) 1 1.00 0.00 0.00 38.6±1.3 66.5±0.5 0.0±0.0 2 0.67 0.33 0.00 46.9±0.1 71.1±1.9 7.7±1.7 3 0.67 0.00 0.33 57.5±0.5 76.8±0.5 0.0±0.0 4 0.33 0.67 0.00 51.7 ±2.7 72.9±1.4 17.1±3.2 5 0.33 0.33 0.33 61.3±2.2 77.6±0.8 8.5±1.3 6 0.33 0.00 0.67 71.7±1.5 83.0±3.3 0.0±0.0 7 0.00 1.00 0.00 43.0±3.9 70.0±3.9 35.1±2.7 8 0.00 0.67 0.33 60.2±1.0 76.8±2.2 15.4±2.8 9 0.00 0.33 0.67 66.0±1.9 83.7±0.2 4.6±0.7 10 0.00 0.00 1.00 66.1±4.9 82.2±0.3 0.0±0.0 11 0.67 0.17 0.17 49.1±0.6 77.7±3.7 4.7±0.8 12 0.17 0.67 0.17 53.2±1.5 74.6±0.9 18.4±3.6 13 0.17 0.17 0.67 70.0±0.1 78.1±2.6 2.0±0.3 iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 26 of 38 4 5 6 26 Page 27 of 38 1 Figure legends 2 3 Fig. 1 Growth of R. eutropha in minimal medium with acetate, propionate, or 4 butyrate as the sole carbon source. Cultures were inoculated to an initial 5 OD600 of 0.05 and the reported values were measured after 48 h of growth. 6 7 Fig. 2 Optimizations of total mixed acid concentration (A), buffer concentration Fo 8 (B), C/N ratio (C) and PHA production (D). 9 after 48 h of growth. Measurements were made rP For (A), media was buffered with 70 mM phosphate 10 buffer. In all experiments a ratio of 3:1:1 (acetate:propionate:butyrate) was 11 used. 12 Fig. 3 For (B-D), 0.5% total mixed acids were used. rR 13 ee Effect of initial pH on cell growth. OD600 (A) and final pH (B) were 14 measured after 48 h of growth as functions of initial pH. 15 contained 0.5% (w/v) acetate (filled circles), 0.4% (w/v) propionate (open 16 circles), or 0.5% (w/v) butyrate (closed triangles). iew 17 18 Cultures ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology Fig. 4 Optimization of mixed acid composition. The effects on CDW (A), final 19 pH (B), amount of PHA produced (C), PHA content (wt%) (D), 3HV content 20 (wt%) (E) and residual CDW (F) of different mixed acid compositions. 21 22 23 24 Fig. 5 Confirmation of mixed acid model by comparison of experimental values (solid bars) and predicted values (white bars) at 0.1:0.1:0.8 (acetate: 27 Applied Microbiology and Biotechnology 1 propionate: butyrate), 0.1:0.4:0.5 and, 0.6:0.3:0.1 using high phosphate 2 buffer. 3 4 Fig. 6 Production of PHAs from 3:1:1 (A) and 3:1:6 (B) organic acids (acetate: 5 propionate: butyrate) in high phosphate buffer media. Acetate (●), 6 propionate (○), butyrate (▼), CDW (△) and PHA (■) concentrations were 7 monitored. Depletion of NH4+ occurred within 12 h. iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 28 Page 28 of 38 Page 29 of 38 1 2 3 Fig. 1 iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 29 Applied Microbiology and Biotechnology 1 2 3 ev rR ee rP Fo Fig. 2 4 iew 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 6 7 8 9 10 11 30 Page 30 of 38 Page 31 of 38 1 2 Fig. 3 iew ev rR ee rP 3 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 31 Applied Microbiology and Biotechnology 1 ev rR ee rP Fo 2 iew 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 4 Fig. 4 32 Page 32 of 38 Page 33 of 38 1 2 Fig. 5 iew ev rR ee rP Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 33 Applied Microbiology and Biotechnology 1 2 iew ev rR ee rP 3 Fig. 6 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 34 Page 34 of 38 Page 35 of 38 1 2 Online Resource 1 3 Parameters for mixture contour in these experiments CDW Coef P coef P coef P A1) 39.8 - 8.93 - 26.2 - 66.17 - 0.03 - P2) 47.0 - 8.03 - 34.6 - 75.63 - 37.86 - 3) B 70.7 - 8.29 - 58.1 - 83.68 - -0.02 - AP 32.3 0.000 0.79 0.032 27.2 0.005 - - -18.24 0.000 AB 37.3 0.000 0.55 0.098 44.4 0.001 28.70 0.003 - - BP 25.5 0.001 0.79 0.029 26.7 0.005 - - -32.17 0.001 AP(A-P) -35.8 0.003 - - -33.9 0.024 - - 12.52 0.001 AB(A-B) -23.6 0.010 - - -32.2 0.025 - - - - BP(B-P) - - AAPB - APPB - - - - - - - - - -12.26 0.043 - - - - 66.48 0.020 -348.5 0.002 - APBB - - - AP(A-P)sq - - - AB(A-B)sq - - - - BP(B-P)sq - - - - - -391.1 0.009 - - 247.09 0.000 - - - - - -78.71 0.007 - - - - - - - - - - - - - - - - - 160 0.107 iew 8 P ev 7 3 HV % Coef rR 6 PHA % P5) ee 5 PHAs Coef4) rP 4 pH Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology R-sq 99.88% 98.05% 99.67% 92.63% 95.20% R-sq(pred) 98..04% 94.97% 96.42% 80.59% 84.73% 1) A: Acetate 2) P: Propionate 3) B: Butyrate 4) Coef.: coefficient 5) P: Statistical probability 9 1 Applied Microbiology and Biotechnology 1 2 3 rR ee rP Fo 4 Online Resource 2 Comparison of growth between compositions with same number of 5 carbons (3:1:6, 3.6:1:1 and 3:1.5:1(acetate: propionate: butyrate)) and 6 control (3:1:1(acetate: propionate: butyrate)) for 48hr at 30°C iew ev 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Page 36 of 38 Page 37 of 38 1 2 Online Resource 3 Predicted amount of cell mass, PHA and wt % of 3HV. 3 Ratio 1) CDW (mg) 2) PHAs (mg) % CDW (%) 3 HV wt% 3:1:1 52.3 38.9 75.0 6.5 3:1:4 65.4 53.7 81.5 2.4 3:1:6 68.7 57.4 82.8 1.3 1:1:1 58.6 45.6 78.3 9.9 4 1) 5 2) 6 mL mimimal media in 250 mL flasks at 30°C for 48 hrs. 8 iew ev rR ee rP 7 Acetate: Propionate: Butyrate CDW predicted when cultures were grown using the given carbon source ratio in 50 Fo 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Applied Microbiology and Biotechnology 3 Applied Microbiology and Biotechnology 2 3 ev rR 1 ee rP Fo 4 Online Resource 4 Consumption of volatile acids in 1:1:1 (acetate: propionate: 5 butyrate) composition. Acetate (●), propionate (○), and butyrate (▼) were measured iew 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 6 4 Page 38 of 38