Simultaneous methane abatement and PHB production by Methylocystis hirsuta in a novel gas-recycling bubble column bioreactor

1 Simultaneous methane abatement and PHB production by Methylocystis hirsuta in a 1 novel gas-recycling bubble column bioreactor 2 3 Teresa García-Péreza,b, Juan C. Lópeza, Fabiana Passosc, Raquel Lebreroa, Sergio Revahb, 4 Raúl Muñoza,* 5 6 a Department of Chemical Engineering and Environmental Technology, School of 7 Industrial Engineerings, University of Valladolid, Dr. Mergelina, s/n, 47011, Valladolid 8 Spain. 9 b Departamento de Procesos y Tecnología. Universidad Autónoma Metropolitana Unidad 10 Cuajimalpa, Avenida Vasco de Quiroga 4871. Col. Santa Fé Cuajimalpa. Delegación 11 Cuajimalpa de Morelos, Ciudad de México, México. 12 c Department of Sanitary and Environmental Engineering, Universidade Federal de Minas 13 Gerais (UFMG), Av. Antonio Carlos 6627, 31270-901, Belo Horizonte, Brazil. 14 15 *Corresponding author: [email protected] 16 17 *Revised Manuscript (clean for typesetting) Click here to view linked References 2 Abstract 18 The limited gas-liquid mass transfer represents the main challenge in the operation of cost-19 effective bioreactors devoted to the treatment of poorly soluble gas pollutants such as 20 methane (CH4). This study evaluates the influence of internal gas-recycling strategies on 21 the enhancement of CH4 abatement in a bubble column bioreactor inoculated with the 22 methanotroph Methylocystis hirsuta. Maximum CH4 removal efficiencies of 72.9 ± 0.5 % 23 (corresponding to elimination capacities of 35.2 ± 0.4 g m-3 h-1) were recorded under 24 process operation at an empty bed residence time of 30 min and 0.50 m3gas m-3reactor min -1 of 25 internal gas-recycling rate. The accumulation of poly-3-hydroxybutyrate (PHB) in M. 26 hirsuta was evaluated batchwise under limitations of potassium, manganese, nitrogen, and 27 nitrogen with excess of iron. Nitrogen starvation resulted in the highest PHB content (28 ± 28 1 %). Likewise, the implementation of sequential N starvation cycles in a continuous 29 bubble column reactor operated at a gas residence time of 30 min and an internal gas-30 recycling rate of 0.50 m3gas m-3reactor min-1 supported a PHB content of up to 34.6 ± 2.5 %, 31 with a volumetric PHB productivity of 1.4 ± 0.4 kg m-3 d-1 and elimination capacities of 32 16.2 ± 9.5 g m-3 h-1. 33 34 Keywords: Biological gas treatment; greenhouse gas; methanotroph; 35 polyhydroxyalkanoate; suspended growth bioreactor. 36 3 1. Introduction 37 Methane (CH4) emissions account for 20-30 % of the global warming effect worldwide 38 based on the 25-times higher ability of this greenhouse gas (GHG) to absorb Earth’s 39 radiation compared to CO2 [1, 2]. This GHG is mainly released to the atmosphere from 40 cattle farming, waste management and mining at low concentrations (< 20 % v/v), which 41 limits its potential energy valorization. In this context, the absence of specific regulations 42 targeting CH4 emissions, along with the lack of viable technical alternatives to produce 43 energy from dilute CH4 emissions, promote the uncontrolled release of CH4 to the 44 atmosphere without prior treatment. Therefore, the development of cost-efficient and 45 environmentally-friendly technologies for the abatement of CH4 is mandatory to achieve an 46 effective climate change mitigation [3]. 47 48 Biotechnologies, such as biofiltration, have consistently shown comparable removal 49 efficiencies and robustness to those of physical-chemical technologies during the treatment 50 of malodours and volatile organic pollutants [4]. Nonetheless, biofilters still present severe 51 operational drawbacks limiting their long-term treatment performance and consequently 52 their widespread implementation for air pollution control. These limitations include the 53 poor mass transfer of poorly water-soluble compounds from the gas phase to the biofilm, 54 and the occurrence of packed bed clogging and channeling as a result of biomass 55 overgrowth [5, 6]. In this context, suspended growth bubble column bioreactors (BCBs) 56 allow for an easy biomass control and harvesting, while they overcome mass transfer 57 limitations due to the recent commercial availability of ultrafine bubble diffusers with 58 micropores < 0.5 µm. In addition, the performance of BCBs can be further boosted via 59 internal gas-recycling, which allows the decoupling of the actual gas residence time and 60 4 turbulence in the microbial broth from the overall empty bed residence time (EBRT). 61 However, the potential of internal gas-recycling in BCBs has been poorly explored for off-62 gas treatment [7-9]. 63 64 Methanotrophs are microorganisms capable of metabolizing CH4 as their sole carbon and 65 energy source by using the enzyme methane monooxygenase (MMO) [10, 11]. 66 Methanotrophic bacteria are typically classified into two different types based on their 67 metabolic and physiological differences: I (which belong to γ-Proteobacteria class) and II 68 (α-Proteobacteria class). Interestingly, type II methanotrophs (e.g. Methylocystis, 69 Methylosinus and Methylocella genera) are able to co-produce polyhydroxyalkanoates 70 (PHAs) under nutrient-limited conditions via the so-called serine pathway [12]. In this 71 regard, CH4 represents a low-cost substrate for the production of these high added-value 72 products (market price of 4-20 € kg-1), whose competitiveness is up to date jeopardized by 73 the high cost of the carbon source employed. Commercial PHAs are nowadays produced 74 through fermentation of glucose or agricultural sugar substrates, which account for 30-40% 75 of the total production costs [13-15]. To date, CH4-based biopolymer production has been 76 focused on the synthesis of poly-3-hydroxybutyrate (PHB), which presents similar 77 mechanical and thermal characteristics to those of conventional plastics and is 78 biodegradable, thus enabling its rapid decomposition in the environment [16,17]. Recent 79 attention has been paid to the optimization of PHB accumulation from a microbiological 80 point of view by identifying the key limiting macro/micronutrients that boost PHB 81 synthesis in methanotrophs. However, to the best of the authors’ knowledge, the influence 82 of micronutrients such as Mn, Fe, and K on methanotrophic PHB synthesis has been 83 scarcely studied [18, 19]. Moreover, few studies have evaluated the simultaneous 84 5 abatement of dilute CH4 emissions and co-production of PHB in gas-phase bioreactors 85 under continuous operation [20]. 86 87 This study aimed at optimizing the continuous abatement of diluted CH4 emissions (4 % 88 v/v) coupled to PHB accumulation at high productivities in a novel internal gas-recycling 89 BCB using Methylocystis hirsuta as a model type II methanotroph. The influence of the 90 EBRT and internal gas-recycling rates on the CH4 removal were first investigated in a lab-91 scale BCB. In addition, the role of different nutrient-limiting conditions (N, K, Mn, and N 92 with excess of Fe) on PHB accumulation in M. hirsuta was also assessed. Finally, the 93 potential of the internal gas-recycling BCB for simultaneous CH4 abatement and PHB co-94 production was evaluated under the optimum EBRT, internal gas-recycling rate and 95 nutrient-limiting conditions previously identified. 96 97 2. Material and methods 98 2.1. Mineral salt medium, chemicals and inoculum 99 The mineral salt medium (MSM) used for M. hirsuta cultivation was modified from 100 Mokhtari-Hosseni et al. [21]. The MSM was composed of (g L-1): 2.25 NaNO3, 0.1 101 MgSO4·7H2O, 0.02 CaCl2·2H2O, 0.68 KH2PO4, 6.14 Na2HPO4·12H2O, 1.3 × 10-3 102 FeSO4·7H2O, 3.5× 10-3 MnCl2·4H2O, 1.5× 10-3 ZnSO4·7H2O, 0.04 × 10-3 Na2MoO4·2H2O, 103 0.04× 10-3 CuSO4·5H2O, 0.32 × 10-3 CoCl2, and 0.2 × 10-3 H3BO3. Unless otherwise 104 specified, all reagents and chemicals were purchased from Panreac® (Barcelona, Spain) 105 with a purity of at least 99 %. CH4 (≥ 99.5 %) and O2 (≥ 99 %) were purchased from Abelló 106 Linde S.A. (Barcelona, Spain). Poly [(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric 107 6 acid] (molar ratio 88/12, ≥ 99.99 %) was obtained from Sigma-Aldrich® (Sigma-Aldrich, 108 St. Louis, MO, USA). 109 110 M. hirsuta (DSMZ 18500) inocula were initially prepared in sterile 120 mL gas-tight serum 111 bottles containing 40 mL of sterile MSM inoculated at 1 % (v/v). These cultures were 112 incubated at 25 °C and 250 rpm for 48 h under a 33:67 % (v/v) CH4:O2 headspace. The 113 cultivation broths were finally transferred to sterile 1.25 L gas tight serum bottles made-up 114 with sterile MSM to a final liquid volume of 200 mL, and incubated at 25º C and 600 rpm 115 to a final optical density of the cultures at 600 nm (OD600) of 1.1 (corresponding to a total 116 suspended solid concentration – TSS – of 295 ± 16 mg L-1). 117 118 2.2. Influence of the EBRT and internal gas-recycling rate in the BCB on CH4 119 biodegradation 120 A lab-scale PVC bubble column bioreactor (0.08 m internal diameter × 0.6 m height) with a 121 working volume of 2.5 L was used in the present study (Fig. 1). The polluted air emission, 122 which contained CH4 at 4 % (v/v), was sparged at the bottom of the bioreactor using three 123 0.5 µm-pore stainless steel diffusers. This synthetic emission was composed of a pure CH4 124 stream supplied via a mass flow controller (AalborgTM, USA) and pressurized air. A 1-L 125 jacketed condenser cooled with water at 20 ºC was implemented within the internal gas-126 recycling line. The temperature in the reactor was maintained at 25 °C. The reactor was 127 inoculated at 194 ± 4 mg L-1 and initially operated for 13 days (to reach steady-state) at 60 128 min of EBRT without internal gas-recycling during the start-up phase. The influence of the 129 EBRT (120, 60, 30 and 15 min) and internal gas-recycling ratio (QR/Q = 0, 2, 3, 6, 10 and 130 7 15, where QR is the recycling gas flow rate and Q the gas flow rate fed to the overall 131 system) was investigated in order to optimize the CH4 abatement performance (Table 1). To 132 ensure an optimum balance of nutrients and a stable pH (7.3 ± 0.2) within the bioreactor, 133 500 mL of cultivation broth were drawn every 48 h, centrifuged (10000 rpm, 7 min) and the 134 biomass pellet (resuspended in fresh 500 mL MSM) was returned to the BCB. 135 136 The inlet and outlet CH4, O2 and CO2 gas concentrations were daily monitored by GC-TCD. 137 OD600, pH, TSS and total nitrogen (TN) concentrations in the cultivation broth were 138 determined every 48 h. The elimination capacity (EC), removal efficiency (RE), CO2 139 production rate (PCO2), PHB content, PHB productivity and the maximum rate of CH4 140 consumption (fitting the data to the Gompertz model) were calculated according to Zuñiga 141 et al. [2]. 142 143 2.3. Influence of micro/macro nutrient limitation on PHB accumulation 144 The influence of N (at low and high Fe2+ concentrations), K and Mn limitations on PHB 145 accumulation and CH4 biodegradation in M. hirsuta cultures were evaluated batchwise. The 146 batch assays involved a growth phase of 15 days in MSM followed by a PHB accumulation 147 phase of 10 days under nutrient limiting conditions according to Table 2. The assays were 148 carried out in duplicate in 2 L gas-tight serum bottles containing 400 mL of MSM 149 inoculated with an initial biomass concentration of 128 ± 17 mg L-1. The glass bottles were 150 sealed with butyl septa and aluminum crimp seals, and CH4 was then added to the 151 headspace both in the growth and accumulation stages at an initial concentration of 193 ± 7 152 g m-3 (32.5 ± 1.1 % v/v) in a pure O2 atmosphere. The biomass was centrifuged at the end 153 of the growth phase and resuspended in the corresponding nutrient-limited MSM prior to 154 8 the accumulation phase. Control tests with the original MSM were conducted as above 155 described. The CH4, O2 and CO2 composition of the headspace, and the biomass (measured 156 through OD660) in the cultivation broth were periodically monitored throughout the 25 days 157 of experiment while PHB concentrations were monitored throughout the limitation tests. 158 159 2.4. Continuous CH4 abatement and PHB co-production in the internal gas-recycling 160 BCB under optimum operational conditions 161 The performance of the internal gas-recycling BCB was assessed under continuous mode 162 using the optimum operational conditions identified in sections 2.2 and 2.3 (EBRT = 30 163 min, internal gas-recycling rate = 0.50 m3gas m-3reactor min-1 and nitrogen limitation as stress 164 to induce PHB production) at an inlet load (IL) of 49.8 ± 11.8 g CH4 m-3 h-1. The BCB was 165 inoculated with M. hirsuta at an initial biomass concentration of 152 ± 1 mg L-1 and 166 initially operated under nutrient-sufficient conditions and continuous CH4 supply in order 167 to reach a biomass concentration of 4.4 ± 0.6 g TSS L-1. Then, nine sequential nitrogen 168 feast-famine cycles (1 day in excess of nitrogen and 2 days under nitrogen limitation) were 169 applied to evaluate the continuous co-production of PHB during CH4 abatement. N-170 supplemented or N-free MSM were supplied at a dilution rate (D) of 0.1 d-1 during the feast 171 and famine periods, respectively. N concentration in the N-supplemented MSM was 172 adjusted to 61 ± 8 mg N L-1 during the feast periods to ensure a complete depletion within 173 the following 24 h. The inlet and outlet CH4, O2 and CO2 gas concentrations were daily 174 monitored in the BCB. Likewise, 20 mL liquid samples were daily withdrawn to determine 175 the OD600, pH, TSS concentration and PHB content. 176 177 9 2.5. Analytical methods 178 CH4, O2, and CO2 gas concentrations were measured in a Bruker 430 GC-TCD (Palo Alto, 179 USA) equipped with a CP-Molsieve 5A column (15 m × 0.53 µm × 15 µm) and a CP-180 PoraBOND Q column (25 m × 0.53 µm × 10 µm). The oven, injector, and detector 181 temperatures were maintained at 45 °C, 150 °C and 200 °C, respectively. Helium was used 182 as the gas carrier at 13.7 mL min-1. TSS concentration was determined according to 183 standards methods [22]. Culture absorbance was measured at 600 nm using a Shimadzu 184 UV-2550 UV/Vis spectrophotometer (Shimadzu, Japan). TN concentration was quantified 185 following sample filtration (0.45 µm) in a TOC-VCSH analyzer (Shimadzu, Japan) coupled 186 with a chemiluminescence detection TN module (TNM-1) (Shimadzu, Japan). PHB 187 accumulation was quantified in a GC-MS (Agilent Technologies: GC System 7820A MSD 188 5977E, Santa Clara, USA) equipped with a DB-wax column (30 m × 250 µm × 0.25 µm) 189 according to López et al. [20]. 190 3. Results and discussion 191 3.1. Influence of the EBRT and internal gas-recycling rate in the BCB on CH4 192 biodegradation 193 Process operation at an EBRT of 120 min in the absence of internal gas-recycling allowed 194 elimination capacities of 4.7 ± 0.48 g m-3 h-1, corresponding to REs of 38 ± 4 %, while 195 values ranging from 6.6 ± 0.3 to 9.8 ± 0.1 g m-3 h-1 were obtained at QR/Q of 2, 3, 6, 10, 15 196 (corresponding to internal gas-recycling rates of 0.02, 0.03, 0.05, 0.08 and 0.13 m3 m-3 min- 197 1). Similarly, higher gas-recycling rates resulted in concomitant increases in EC, RE and 198 PCO2 (Fig. 2). Thus, the REs increased from 38 ± 4 to 54 ± 2, 60 ± 1, 69 ± 2, 73 ± 1, and 199 16 References 338 [1] G. Koneswaran, D. Nierenberg, Global farm animal production and global warming: 339 impacting and mitigating climate change, Environ. Health Perspect. 116 (2008) 578-340 582. 341 [2] C. Zúñiga, M., Morales, S. Le Borgne, and S. 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Schematic of the experimental set up. (1) BCB, (2) sampling port, (3) rotameter, (4) 439 mass flow controller, (5) cooling water, (6) condenser, (7) internal gas-recycling peristaltic 440 pump and (8) liquid sampling port. 441 442 Fig. 2. Influence of the internal gas-recycling rate on the EC ( ), RE ( ) and CO2 443 production rate ( ) at EBRTs of 60 min (A) and 120 min (B) in the BCB. 444 445 Fig. 3. Influence of the EBRT on the EC ( ), RE ( ) and CO2 production rate ( ) 446 at a constant internal gas-recycling ratio of 15 in the BCB. 447 448 449 Fig. 4. Influence of nutrient limitation on PHB accumulation (A) and final biomass 450 concentration (B) in the stationary phase of the batch assays. 451 452 Fig. 5. Time course of the PHB cell content (A), biomass concentration (B), CO2 453 production rate (C) and eliminations capacities (D) in the BCB during the continuous 454 abatement of CH4 coupled to PHB production. Vertical arrows indicate nitrogen addition 455 during each N feast-famine cycle. Dashes lines indicate the start of limitation cycles. 456 457 458 459 21 460 Table 1. Experimental conditions evaluated during the optimization of CH4 abatement in the internal gasrecycling BCB. Condition EBRT (min) Inlet load (g m-3 h-1) QR/Q Recycling rate (m3gas m-3reactor min-1) Virtual residence time (min) Mineralization ratio (PCO2/EC) 1 120 12 0 0.000 120 2.5 ± 0.2 2 2 0.017 40 2.1 ± 0.2 3 3 0.025 30 1.8 ± 0.4 4 6 0.050 17 1.7 ± 0.2 5 10 0.083 11 1.9 ± 0.3 6 15 0.125 8 2.0 ± 0.1 7 60 24 0 0.000 60 1.4 ± 0.2 8 2 0.033 20 1.7 ± 0.5 9 3 0.050 15 1.7 ± 0.2 10 6 0.100 9 1.6 ± 0.4 11 10 0.167 5 1.7 ± 0.1 12 15 0.250 4 1.7 ± 0.4 13 30 48 10 0.333 3 1.8 ± 0.2 14 15 0.500 2 1.9 ± 0.2 15 15 96 15 1.00 0.94 2.0 ± 0.1 22 Table 2. Micro and macro-nutrients limiting conditions evaluated during batch cultivation of M. hirsuta. Conditions Nutrient limitation Nutrient in excess Fe Concentration (µM) Control - - 4.6 1 K - 4.6 2 Mn - 4.6 3 N - 4.6 4 N Fe 60 461 Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image