Biogas-based polyhydroxyalkanoates production by Methylocystis hirsuta: A step further in anaerobic digestion biorefineries

1 Biogas-based polyhydroxyalkanoates production by Methylocystis hirsuta: a step further in anaerobic digestion biorefineries Juan C. López, Esther Arnáiz, Laura Merchán, Raquel Lebrero, Raúl Muñoz,* Department of Chemical Engineering and Environmental Technology, School of Industrial Engineerings, University of Valladolid, C/Dr. Mergelina s/n, 47011 Valladolid, Spain. *Corresponding author: [email protected], Tel. +34 983186424, Fax: +34 983423013. Abstract The potential of biogas (with and without H2S) and volatile fatty acids (VFAs) to support microbial growth and accumulation of polyhydroxyalkanoates (PHAs) in type II methanotrophs was evaluated batchwise under aerobic conditions. Methylocystis hirsuta was able to grow on artificial biogas (70 % CH4, 29.5 % CO2, 0.5 % H2S) and accumulate PHA up to 45 ± 1 % (wt %) under N-limited conditions. The presence of CO2 and H2S did not significantly influence the growth and PHA synthesis in M. hirsuta compared to control tests provided with pure CH4 at similar concentrations. Likewise, *Revised Manuscript (clean for typesetting) Click here to view linked References 2 the addition of VFAs to the cultivation broth at initial concentrations of 100–200 mg L-1 did not hamper the growth of this strain on artificial biogas. Indeed, the addition of 10 % extra carbon in the form of individual VFAs resulted in an increase in the maximum PHA yield and final PHA content up to 0.45–0.63 gPHA gSubstrate-1 and 48–54 % (wt %), respectively, at the expense of a higher energy demand. Valeric acid supplementation supported the highest 3-hydroxyvalerate content (13.5 %) within the biocomposite. In this context, this study demonstrated for the first time that 3- hydroxyvalerate synthesis by M. hirsuta did not depend on CH4 assimilation. Keywords Biorefinery, methane, methanotroph, polyhydroxybutyrate, polyhydroxyvalerate, volatile fatty acid. 3 1 Introduction Methane (CH4), which accounts for 10–16% of the global warming impact worldwide, represents nowadays the second most important greenhouse gas. In nature, CH4 is mainly emitted from the anaerobic decomposition of organic matter in wetlands and oceans. However, more than 60% of CH4 emissions worldwide are anthropogenic [1-3]. Waste and wastewater treatment plants (WWTPs) represent one of the most significant emission sources of CH4 (20000 ktons CO2-eq in 2014 in the EU-28), which is often released in the form of a biogas typically composed of 50–70% CH4, 30–50% CO2 and 0–0.5% H2S (v/v) [4,5]. Anaerobic digesters in such facilities process different types of organic feedstock, liquid and solid waste, while producing i) sludge that can be used as an agricultural fertilizer and ii) biogas to be employed for electricity and/or heat production. In this regard, the European Biogas Association (EBA) report claimed that by the end of 2013 more than 14000 anaerobic digesters were in service in Europe with at least 7400 MW of electricity generation capacity [6]. However, despite the potential of biogas as a renewable energy source for heat and electricity generation, the high investment costs needed for on-site energy recovery or the high costs associated to biomethane production (1.08 € Nm-3 in the EU market compared to 0.30–0.67 € Nm-3 for natural gas) promote biogas flaring or venting to the atmosphere in low-medium size facilities [7,8]. In addition, the huge reserves of shale gas worldwide, along with its affordable extraction costs, do not forecast a scenario of increased natural gas prices (where biogas could advantageously compete). In this context, the development of costeffective technologies for the bioconversion of biogas into high-added value products could eventually mitigate biogas emissions from waste/wastewater treatment facilities along with the implementation of anaerobic digestion as a platform for organic pollution control. 4 Polyhydroxyalkanoates (PHAs), such as poly-3-hydroxybutyrate (PHB), poly-3- hydroxyvalerate (PHV) and their copolymer (PHBV), are polyesters biologically produced under unbalanced nutrient conditions (e.g. N limitation). PHAs have the potential to substitute conventional plastics such as polyethylene or polypropylene due to their biocompatibility, biodegradability and their versatile thermal and mechanical properties. The market price of PHAs ranges from 4 to 20 € kgPHA-1, which greatly depends on the monomer composition of the biocomposite, the carbon source, the microbial strain used and the product purity [9]. Despite its rapid decrease in the past 5 years, the market price of PHAs is still higher than that of fossil-based polyesters due to the high costs of biopolymer downstreaming and carbon source acquisition, the later accounting for 30–40% of the final PHA price [8,9]. In this regard, CH4 has recently emerged as a low-cost and environmentally friendly feedstock for PHA production [10,11]. To the best of the authors’ knowledge, most studies reported to date on methanotrophic PHA production have been mainly focused on the use of pure CH4 or natural gas as substrate [12-14]. Controversy still exists in literature about the technical and microbiological feasibility of biogas (containing the toxic and acid gases CO2 and H2S) as a feedstock for PHA production [12,15]. Moreover, the direct addition to the methanotrophic cultivation broth of volatile fatty acids (VFAs), which are readily available during anaerobic digestion, could increase PHA yields and tailor the composition of the biocomposite during biogas bioconversion. However, the few studies reported to date restrict the use of VFAs to their corresponding salts (i.e. sodium valerate, sodium propionate or sodium 3-hydroxybutyrate), which overcome the pH- associated effects of VFAs but hinder their applicability within this biorefinery concept. In this context, neither the potential of biogas nor the influence of VFA supplementation on PHA accumulation by methanotrophs have been yet systematically addressed [16- 5 19]. A successful bioconversion of biogas into VFA-tailored biopolymers would represent the cornerstone of a new generation of biogas biorefineries supporting a lowcost and environmentally friendly conversion of residual organic matter into multiple high-added value products. This study aimed at evaluating the feasibility of artificial biogas as a feedstock to support the growth of the type II methanotroph Methylocystis hirsuta coupled to the synthesis of PHAs. Additionally, the potential of acetic, butyric, propionic and valeric acids to support M. hirsuta growth and modify the composition of the biogas-based PHA biocomposite was here evaluated for the first time. 2 Materials and methods 2.1 Strain, chemicals and culture conditions The methanotrophic strain Methylocystis hirsuta was acquired from DSMZ culture collection (DSM No. 18500, Leibniz Institut, Germany). This type II methanotroph was selected based on i) its ability to produce PHB from CH4 through the serine pathway and ii) the fact that the highest PHA contents up to date have been recorded for this strain [13]. Synthetic biogas (70 % CH4, 29.5 % CO2, 0.5 % H2S), CH4 (≥ 99.5 %), He (≥ 99.5 %), O2 (≥ 99.5 %) and CO2 (≥ 99.9 %) were purchased from Abelló Linde S.A. (Barcelona, Spain). Poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid] (molar ratio 88/12, ≥ 99.99 %), valeric acid (≥ 99 %) and butyric acid (≥ 99 %) were obtained from Sigma-Aldrich® (Sigma-Aldrich, St. Louis, USA). Acetic acid (≥ 99 %) was purchased from Cofarcas S.A. (Burgos, Spain). Additional reagents and chemicals were purchased from Panreac® (Barcelona, Spain) with a purity of at least 99 %. 6 Balanced growth cultures were cultivated in Whittenbury nitrate mineral salt (NMS) medium (pH of 6.8) [20]. NMS medium supplemented with agar at 1.5 % (w/v) was used to test culture purity along the experiment. In contrast, unbalanced growth cultures devoted to accumulate PHAs were incubated in a nitrate-free Whittenbury mineral salt medium (NFMS). 2.2 Experimental procedures Inocula M. hirsuta inocula were prepared in 125-mL serum bottles capped with butyl-rubber stoppers and crimp-sealed under a CH4:O2 headspace (35:65 % v/v) and sterile conditions (Figure 1). The serum bottles contained 50 mL of NMS inoculated at 10 % (v/v) and were incubated in an orbital shaker at 250 rpm and 25 ºC for 7 days, which entailed five CH4:O2 headspace renewals. The final optical density of the cultures at 600 nm (OD600) was 4.0 ± 0.4 (total suspended solid concentration – TSS – of 1690 ± 169 mg L-1). Unless otherwise specified, this inoculum was used for Test Series 1 – 4. <Fig. 1> Test Series 1: Influence of artificial biogas on M. hirsuta growth The ability of M. hirsuta to grow on artificial biogas (with and without H2S) was assessed in triplicate in 2.15-L serum bottles capped with butyl-rubber stoppers and aluminium crimp seals under three different O2-supplemented headspace atmospheres (v/v): H2S-free biogas (CH4:O2:CO2:He at 31.5:55.0:13.27:0.23 %), biogas (CH4:O2:CO2:H2S at 31.5:55.0:13.27:0.23 %) and control (CH4:O2:He at 31.5:55.0:13.5 %). The headspace mixtures were prepared in 25 L-Tedlar bags (Sigma-Aldrich®, St. Louis, USA) using the appropriate volumes of each gas component from the cylinders and further pumped into the corresponding bottles in order to completely flush the air 7 atmosphere out. The cultures, which contained 400 mL of NMS inoculated at 3 % (v/v) (initial OD600 of 0.13 ± 0.01, corresponding to 55 ± 2 mgTSS L-1), were magnetically stirred at 300 rpm (Multipoint 15 Variomag, Thermo Fisher Scientific, Bartlesville, USA) and 25.0 ± 0.5 ºC in a temperature-controlled room. Abiotic controls for the three headspace mixtures were also prepared as above described to rule out any potential CH4 removal due to adsorption or photolysis. Test Series 2: Influence of artificial biogas on PHA synthesis by M. hirsuta M. hirsuta was initially grown as above described in 2.15-L serum bottles containing 400 mL of NMS inoculated at 3 % (v/v) under a CH4:O2:CO2:H2S atmosphere (31.5:55.0:13.27:0.23 %) for 9–12 days (to completely deplete CH4 from the headspace). The methanotrophic biomass was harvested by centrifugation (10000 rpm, 8 min) and resuspended in NFMS. Then, the ability of biogas-grown M. hirsuta to accumulate PHAs was assessed in triplicate in 2.15-L serum bottles capped with butylrubber stoppers and aluminium crimp seals under three different O2-supplemented headspace atmospheres (v/v): H2S-free biogas (CH4:O2:CO2:He at 31.5:55.0:13.27:0.23 %), biogas (CH4:O2:CO2:H2S at 31.5:55.0:13.27:0.23 %) and control (CH4:O2:He at 31.5:55.0:13.5 %). The bottles were incubated under magnetic agitation at 300 rpm and 25.0 ± 0.5 ºC in a temperature-controlled room. Test Series 3: Influence of the type and concentration of VFA on biogas-based M. hirsuta growth M. hirsuta was grown in VFA-supplemented 125 mL serum bottles capped with butylrubber stoppers and crimp-sealed under sterile conditions and an artificial biogas headspace (CH4:O2:CO2:H2S at 31.5:55.0:13.27:0.23 %). The bottles, which initially contained 50 mL of NMS inoculated at 4 % (v/v) (initial OD600 of 0.15 ± 0.01, 8 corresponding to 65 ± 2 mgTSS L-1), were incubated at 300 rpm and 25.1 ± 0.3 ºC in a temperature-controlled room. The influence of the concentration of acetic, butyric, propionic and valeric acid (named C1–C5, where C1 represented the lowest and C5 the highest VFA concentration) on M. hirsuta growth was evaluated in duplicate in separate batch assays. The initial concentrations of acetic acid were 92 ± 10, 197 ± 5, 243 ± 2, 324 ± 5 and 482 ± 4 mg L-1, of butyric acid 68 ± 1, 139 ± 3, 182 ± 7, 273 ± 2 and 345 ± 3 mg L-1, of propionic acid 64 ± 2, 123 ± 2, 214 ± 26, 258 ± 0 and 320 ± 3 mg L-1 and of valeric acid 57 ± 5, 114 ± 1, 177 ± 3, 238 ± 4 and 292 ± 0 mg L-1. These concentrations represented 16, 31, 47, 63 and 78 % of the C initially supplied as CH4. Abiotic controls under a CH4:O2:CO2:H2S atmosphere (31.5:55.0:13.27:0.23 %) and an initial C2 concentration for each VFA were prepared to rule out any potential CH4 or VFA removal due to adsorption or photolysis. Individual biogas-deprived controls at an initial C2 concentration for each VFA were included in this batch assay to evaluate the ability of the strain to remove the VFA in the absence of biogas. Cosubstrate-deprived controls under a CH4:O2:CO2:H2S atmosphere (31.5:55.0:13.27:0.23 %) were also conducted. Test Series 4: Influence of the type of VFA on biogas-based PHA synthesis by M. hirsuta M. hirsuta was initially grown as above described in 2.15-L serum bottles containing 400 mL of NMS inoculated at 3 % (v/v) under a CH4:O2:CO2:H2S atmosphere (31.5:55.0:13.27:0.23 %) for 9–12 days (to completely deplete CH4 from the headspace). The methanotrophic biomass was again harvested by centrifugation (10000 rpm, 8 min) and resuspended in NFMS supplemented with either acetic, butyric, propionic and valeric acids at concentrations of 181 ± 16, 123 ± 2, 139 ± 1 and 130 ± 6 mg L-1, respectively (corresponding to 10 % of the C initially supplied as CH4). The bottles were finally crimp-sealed, filled with a fresh CH4:O2:CO2:H2S atmosphere 9 (31.5:55.0:13.27:0.23 %) and incubated at 300 rpm and 25.0 ± 0.5 ºC. Abiotic controls under a CH4:O2:CO2:H2S atmosphere (31.5:55.0:13.27:0.23 %) and/or an initial C2 concentration for each VFA were prepared to rule out any potential CH4 or VFA removal due to adsorption or photolysis. Individual biogas-deprived controls at an initial C2 concentration for each VFA were included in this batch assay to evaluate the ability of the strain to produce PHAs in the presence of VFA without artificial biogas. Cosubstrate-deprived controls under a CH4:O2:CO2:H2S atmosphere (31.5:55.0:13.27:0.23 %) were also carried out to assess the influence of VFAs on the content and composition of the PHA synthesized. The headspace concentration of CH4, CO2, O2 and H2S was periodically measured by GC-TCD in all test series. Liquid samples (3 mL) were periodically drawn to monitor the concentration of VFAs, PHAs, TSS and OD600 in all test series. Liquid samples (1 mL) were also randomly withdrawn to measure the SO42- concentration in the liquid phase by HPLC-IC in Test Series 1 and 2. The pH of the cultivation broth was measured at the beginning and at the end of each test series. Cultivation broth samples (100 μL) were systematically drawn from all test series to test strain purity in agar plates incubated under CH4:O2 atmosphere (35:65 % v/v) in 2 L-Tedlar bags. 2.3 Analytical methods CH4, O2, CO2 and H2S gas concentrations were determined according to López et al. in a Bruker 430 GC-TCD (Bruker, Palo Alto, USA) equipped with a CP-Molsieve 5A (15 m × 0.53 mm × 15 mm) and a CP-PoraBOND Q (25 m × 0.53 mm × 10 mm) columns [21]. The determination of OD600 and TSS concentration was performed as described elsewhere [22]. SO42- concentration in the liquid phase was determined by HPLC-IC according to López et al. [21]. Cultivation broth samples of 1 mL were filtered (0.22 μm) and acidified with 20 μL H2SO4 (96-97 % (w/v)) prior to VFAs analysis in an 16 the maximum PHA content being 2.8 % (> 91 % mol 3HB fraction) (Table 1). Surprisingly, the highest content of PHAs among the control tests without biogas was obtained with valeric acid (up to 9.0 ± 1.7 % PHA), which entailed a high 3HV fraction of 83 mol %. These results suggested that the synthesis of PHV in M. hirsuta was not strictly linked to the assimilation of CH4. In contrast, Myung et al. [17] found that Methylocystis parvus OBBP was not able to accumulate PHAs using valerate as the sole carbon and energy source, which highlights the higher metabolic versatility of M. hirsuta. It must be noted that no biomass formation was observed during M. hirsuta cultivation on VFAs as the sole carbon source during the accumulation phase, where the carbon belonging to VFAs was mainly deviated towards the production of CO2 (Table S4). The use of artificial biogas as the sole substrate during the accumulation phase supported similar maximum YPHA and PHA contents to those found during Test Series 2 (0.41 ± 0.02 gPHA gSubstrate-1 and 43 ± 2 %, respectively), with 3HB as the main monomer within the biocomposite (Table 1, Table S4). <Table 1> The addition of propionic, acetic or butyric acid as co-substrates during biogas biodegradation by M. hirsuta enhanced PHA accumulation, which increased from 43.1 ± 1.8 % up to 47.9 ± 0.7, 52.3 ± 0.7 and 52.2 ± 2.1 %, respectively. This represented a 10–20 % increase in PHA accumulation over the basal content obtained only with artificial biogas and matched the increase in the maximum YPHA (by 10–30 %) achieved (Table S4). To the best of the authors’ knowledge, there are no previous studies evaluating the supplementation of acetate as co-substrate during CH4-based PHA accumulation by methanotrophic bacteria, which could presumably act as direct precursor for the synthesis of 3-hydroxybutyryl-CoA and thus, of 3HB units (Figure 4). The final PHA content here reported for the simultaneous cultivation of M. hirsuta in 17 propionic and biogas were higher than those found by Myung et al. using pure CH4 and propionate at 100 mg L-1 (32 ± 4 %) [17]. Surprisingly, the 3HV fraction obtained by these authors with M. parvus OBBP (25 mol %) significantly differed from the one obtained in M. hirsuta in the present study (2 mol %), which suggests that propionic acid bioconversion in methanotrophic bacteria is species-dependent [17]. The PHA contents and HB:HV ratios here obtained were comparable to those obtained in M. parvus OBBP when butyrate was supplemented together with CH4 (55 ± 3 %, 100:0 ratio) [18]. In our particular study, the highest PHA content was found when valeric acid was used as co-substrate during biogas-based M. hirsuta cultivation, which resulted in a final PHA content of 53.8 ± 0.8 % (corresponding to a maximum YPHA = 0.63 ± 0.05 gPHA gSubstrate-1) and a 3HB:3HV ratio of 75:25 (Table 1, Table S4). Maximum PHA contents of 54 ± 4 %, with 3HB:3HV ratios of 75:25 and YPHA of 0.67 gPHA gSubstrate-1 have been reported in Methylocystis species when valerate was added together with CH4 [16,18,19]. Likewise, previous studies have consistently demonstrated that fatty acids assimilation by methanotrophic bacteria is an energy intensive process, which increases fe when co-substrates such as valerate are supplemented [17,32]. In this regard, Bahr et al. demonstrated that the lower the carbon oxidation-reduction state (CORS) of a pollutant, the higher the energy requirements (and therefore the oxygen demands) during the cultivation of a methanotrophic bacterial consortium [33]. In our particular study, a gradual increase in fe was observed as the CORS of the VFA decreased. Thus, fe increased from 0.52 ± 0.02 under acetic acid (CORS = 0) cultivation to 0.94 ± 0.05 under valeric acid (CORS = -6) co-addition, the later likely explaining the reduced biomass growth observed (Table S4). These fe values were higher than those previously obtained during PHA accumulation under valerate 18 co-supplementation, which could be attributed to the lower pHs here encountered [16,17]. <Fig. 4> 4 Conclusions Artificial biogas (with and without H2S) supported a similar growth and PHA accumulation (under nitrogen limitation) to pure CH4 in M. hirsuta cultures, which confirmed for the first time the feasibility of biogas-based biorefineries devoted to the production of these high-added value product. M. hirsuta was able to use acetic, butyric, propionic and valeric acids as the sole carbon and energy source. This study also demonstrated the potential of the individual supplementation of these VFAs to modify the composition of the biocomposite, valeric acid supporting up to 25 % HV fraction within the whole biopolymer. Further research is still needed to elucidate the effect of i) including other trace gases within the biogas (i. e. volatile sulfur compounds such as COS or CS2), and ii) the multiple supplementation of VFAs on biogas-based M. hirsuta growth and PHA synthesis. Acknowledgments This research was supported by the Spanish Ministry of Economy and Competitiveness, the European Union through the FEDER Funding Program (CTM2015-70442-R and RED NOVEDAR projects and BES-2013-063922 contract) and the Regional Government of Castilla y León (UIC71). J. Prieto and E. 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