Microsoft Word - 26jonassen.docx CCHHEEMMIICCAALL EENNGGIINNEEEERRIINNGG TTRRAANNSSAACCTTIIOONNSS VOL. 40, 2014 A publication of The Italian Association of Chemical Engineering www.aidic.it/cet Guest Editor: Renato Del Rosso Copyright © 2014, AIDIC Servizi S.r.l., ISBN 978-88-95608-31-0; ISSN 2283-9216 Effect of Pollutant Concentration During Isolation on the CH4 Biodegradation Kinetics, Population Structure and PHB Accumulation Juan C. López, Guillermo Quijano, Rebeca Pérez, Raúl Muñoz* Department of Chemical Engineering and Environmental Technology, Escuela de Ingenierías Industriales, Sede Dr. Mergelina, University of Valladolid, Dr. Mergelina s/n, 47011, Valladolid, Spain mutora@iq.uva.es Methane-oxidizing bacteria were enriched in three stirred tank reactors continuously supplied with CH4- laden air at 20, 2 and 0.2 gCH4 m -3 to assess the effect of pollutant concentration on the biodegradation kinetics, population structure and polyhydroxyalkanoate (PHA) production under sequential nitrogen limitations. CH4 concentration influenced the population structure of the enriched cultures, which were mainly constituted by type I and, in a lesser extent, by type II methanotrophs. Microorganisms enriched at 2 gCH4 m -3 presented the highest maximum specific degradation rate (qmax) and those enriched at 20 and 0.2 gCH4 m -3 exhibited the lowest half-saturation constant (KS), which ruled out a potential correlation between CH4 concentration and kinetic parameters. Maximum polyhydroxybutyrate (PHB) contents of 1.0, 12.6 and 1% (w/w) were obtained at 20, 2 and 0.2 gCH4 m -3, respectively. Polyhydroxyvalerate (PHV) was also detected at PHB:PHV ratios of up to 12:1 and 4:1 in the cultures enriched at 20 and 0.2 gCH4 m -3, respectively. 1. Introduction Methane (CH4) contributes to approximately 20% of the worldwide greenhouse gas (GHG) emissions and increased its atmospheric concentration by 150% from the pre-industrial era (IPCC 2013). CH4 exhibits a global warming potential 25 times higher than that of CO2 and is mainly emitted from organic waste treatment activities such as landfilling, composting and wastewater treatment (122 million tCO2-eq in the EU-15), coal mining (6 million tCO2-eq in the EU-15) and livestock farming (120 million tCO2-eq in the EU- 15) (EEA 2013). Its concentration in anthropogenic emissions varies in the range of 0-0.2 g CH4 m -3 for compost piles or livestock farms, and of 20-100 g CH4 m -3 for old landfills (Nikiema et al. 2007). Considering the gradual application of restrictive legislations on CH4 emissions, cost-efficient and sustainable methods such as biotechnologies must be properly implemented for an active abatement of CH4 on-site (Copelli et al. 2012). However, conventional biotechnologies such as biofilters or biotrickling filters are prone to CH4 mass transfer limitation from the gas to the liquid phase (and consequently to the microorganisms) due to the low CH4 solubility in water (dimensionless Henry’s law constant of 30). Besides, there is a lack of knowledge on the kinetics and the microbiology involved, especially at the trace level CH4 concentrations (~mg m -3) encountered under real applications (López et al. 2013). In this context, microorganisms with high specific biodegradation rates (qmax) and affinity towards CH4 (low half- saturation constant, KS) are desirable to guarantee an efficient abatement and a reduction of the start-up period during the implementation of biotechnologies. On the other hand, the potential of methanotrophic bacteria to co-produce high-added value products such as biopolymers (i.e. PHB) could positively impact the economic sustainability of biological CH4 treatment, although this technological alternative has been poorly explored (Zúñiga et al. 2013). The present work evaluated the effect of CH4 concentration on their biodegradation kinetic parameters and population structure. Furthermore, the influence of the CH4 DOI: 10.3303/CET1440036 Please cite this article as: Lopez J.C., Quijano G., Perez R., Munoz R., 2014, Effect of pollutant concentration during isolation on the ch4 biodegradation kinetics, population structure and phb accumulation, Chemical Engineering Transactions, 40, 211-216 DOI: 10.3303/CET1440036 211 concentration and the CH4/biomass ratio on the ability to accumulate PHB under sequential nitrogen limitations was assessed. 2. Materials and methods 2.1 Experimental set-up Three 500-mL jacketed stirred tank (STRs) with mineral salt medium were initially seeded with a mixed inoculum in order to start the experiment with a high diversity of methanotrophs. The inoculum contained fresh aerobic activated sludge from a wastewater treatment plant, soil from an abandoned landfill cover and sludge from an aerobic lagoon stabilizing effluents from a full-scale anaerobic digester (working liquid volume of 400 mL). Diffusers located at the bottom of the reactors 1 (R1), 2 (R2) and 3 (R3) were used to continuously supply CH4 (diluted in air) via aeration (400 mL min -1) at 20 g m-3, 2 g m-3 and 0.2 g m-3, respectively. Figure 1 depicts the set-up and summarizes the key experimental conditions used during microbial enrichment. The reactors were operated under 8 sequential periods of N limitation (48 – 72 h per period) in order to promote the production of PHB in the methanotrophic cultures. The N-NO3 - concentration was restored to 249 ± 65, 48 ± 23, 17 ± 7 mg L-1 in R1, R2 and R3, respectively, after each limitation period. At the end of the 8th N limitation cycle, the influence of the CH4/biomass ratio on microbial PHB accumulation under N limiting conditions was assessed for a period of 18 days by diluting the biomass concentration in R1 and R2 to the levels of R3. Liquid samples were periodically drawn to measure culture absorbance (OD650) and the concentration of dissolved total organic carbon (TOC), total nitrogen (TN) and total suspended solids (TSS). Liquid samples were drawn on weeks 14 and 19 to determine the biodegradation kinetic parameters. At the end of each 3 days nitrogen limitation period, liquid samples were also drawn to quantify bacterial PHB content. CH4 and CO2 gas concentrations were monitored by GC-TCD at the inlet and outlet of the reactors. Figure 1: Schematic representation of the experimental set-up (1 = CH4 gas bottle, 2 = thermostatic water bath connection, 3 = pH and temperature acquisition system, 4 = air compressor connection). 2.2 Kinetics of CH4 biodegradation The determination of the maximum specific CH4 degradation rate qmax (g CH4 g -1 biomass h -1) and the Monod half-saturation constant KS (g m -3) in R1, R2 and R3 cultures was carried out in 120-mL bottles containing 20 mL of MSM and biomass at an initial concentration of 51.7 ± 14.7 gbiomass m -3, which ensured that the kinetic parameters were obtained under non-limiting CH4 mass transfer conditions. CH4 was supplied at initial headspace concentrations of 91.5 ± 3.9 g m-3, 17.9 ± 0.8 g m-3 and 4.7 ± 0.4 g m-3. The bottles were incubated at 25ºC under orbital agitation at 150 rpm for 25 h. The concentrations of CH4 and CO2 in the headspace of the bottles were periodically measured by GC-TCD. The Lineweaver-Burk correlation (Eq. 212 (1)) was used to determine the biodegradation kinetic parameters from the initial CH4 biodegradation rates (Walkiewicz et al., 2012): max4max 1 ][ 11 qCHq K q S +⋅= (1) where q represents the initial CH4 biodegradation rate (g CH4 m -3 liq h -1) and [CH4] the CH4 concentration in aqueous phase (g m-3liq) estimated using the dimensionless Henry’s law at 25ºC and 1 atm. 2.3 Molecular biology analysis To evaluate the richness and composition of the microbial community, biomass samples from the inoculum (A) and from R1, R2 and R3 were collected on week 4 (B, C and D, respectively), 14 (E, F and G, respectively) and 19 (H, I and J, respectively) and stored immediately at –20ºC. DNA extraction, PCR and qPCR amplifications, DGGE analysis, sequencing and DNA sequence analysis were carried out according to Lebrero et al. (2013). The sequences were deposited in GenBank Data Library under accession numbers KF957448 – KF957465. Similarity and Shannon-Wiener diversity (H) indexes were also determined according to Lebrero et al. (2013). 2.4 Measurement of PHB The quantitative determination of PHB was carried out according to Zúñiga et al. (2011) using chloroform as extraction solvent and a GC-MS for quantification. 3. Results and discussion 3.1 Structure of the enrichment communities The lowest Shannon-Wiener diversity index was found in the seed (H = 2.6). The samples retrieved from the STRs on week 4 onwards exhibited a higher microbial diversity as a result of the continuous CH4 supply, although no significant differences were found among communities growing at different CH4 concentrations (Table 1). In contrast, Estrada et al. (2012) determined for toluene that high concentrations of the pollutant supported lower H indexes, which was attributed to the high toxicity and water solubility of this aromatic compound. The analysis of the pair-wise similarity indexes revealed a low correspondence between the inoculum and the cultures in the three STRs. Hence, similarity coefficients of 8%, 18% and 34% were recorded between the seed and R1, R2 and R3 by week 4, respectively, thus confirming the rapid dynamics of the methanotrophic communities in the STRs. The highest similarity coefficients were obtained between the communities on weeks 14 and 19 (88% in R1, 80% in R2 and 75% in R3), which confirmed the stabilization of the methanotrophic populations from week 14 onward regardless of the CH4 concentration evaluated. Moreover, the comparison of communities in the STRs by week 19 revealed that cultures enriched at CH4 concentrations differing in one order of magnitude were more similar (similarities of 51% between R1 and R2, and 66% between R2 and R3) than those enriched at CH4 concentrations differing in two orders of magnitude (32% between R1 and R3). Thus, these results confirmed the significant influence of CH4 concentration during culture enrichment on the structure of the microbial populations. DGGE analysis revealed the presence of three different phyla in the STRs (Proteobacteria, Firmicutes, Actinobacteria), with a dominance of type I methanotrophs (Methylosarcina, Methylomicrobium, Methylosoma and Methylobacter genera) over the rest of microorganisms (Table 1). Type II methanotrophs (Methylocystis genus) were also present in the three STRs and gradually increased their abundance in R2 and, in a lesser extent, in R3 (samples F, G, I and J). The fact that type II methanotrophs exhibited a low abundance in the three STRs can be attributed to the enrichment of cultures at high Cu2+ concentrations. In this context, preliminary quantitative-PCR results revealed the higher expression of particulate methane monooxygenases (pMMO) compared to the type II-specific soluble methane monooxygenases (sMMO), which was predominantly detected in R2 and, in a lesser extent, in R3 (Figure 2). Methylotrophic bacteria belonging to the Methylobacillus and Hyphomicrobium genera were also significantly detected among the samples, which agreed well with previous findings in biofilters treating CH4 (Kim et al. 2013). Moreover, bacteria from the Dokdonella, Rhodanobacter, Turicibacter, Acidobacterium and Rhodococcus were initially detected in the STRs and gradually disappeared, likely due to their incapability to consume CH4 or CH4-derived metabolites. 213 Table 1: Microbiological analysis of the enrichment communities in the STRs Source of origin H index Significant similarities Dominant genera (closest relatives in Blast with similarity ≥ 94%) A 2.6 34% D, <19% rest Acidobacterium, Turicibacter, Methylocystis B 2.9 52% E, 67% C Methylomicrobium, Dokdonella, Rhodanobacter, Methylocystis, Methylosoma C 3.2 53% F, 37% D Methylomicrobium, Methylocystis, Dokdonella, Acidobacterium, Rhodococcus D 3.1 38% G, 23% B Methylobacter, Acidobacterium, Turicibacter, Methylocystis E 2.4 88% H, 66% F Methylosarcina, Methylomicrobium, Hyphomicrobium, Methylocystis F 3.1 80% I, 46% G Methylobacter, Methylosoma, Methylocystis, Methylomicrobium, Hyphomicrobium G 2.8 75% J, 27% E Methylocystis, Dokdonella, Hyphomicrobium, Methylomicrobium, Methylosoma H 2.9 47% B, 51% I Methylosarcina, Methylomicrobium, Methylobacillus, Hyphomicrobium I 2.7 60% C, 66% J Methylomicrobium, Methylocystis, Methylosoma, Hyphomicrobium J 2.7 29% D, 32% H Methylocystis, Methylosoma, Hyphomicrobium, Methylomicrobium Figure 2: sMMO (upper gel) and pMMO (lower gel) expression profiles of the bacterial communities present in the STRs. The size of the amplified fragments and the names of the samples are shown in the left and the upper parts of the gels, respectively. 3.2 Determination of kinetic parameters Table 2 summarizes the CH4 biodegradation kinetic parameters qmax and KS obtained at weeks 14 and 19. No significant differences were found in terms of maximum specific biodegradation rate between the enrichment cultures of R1 and R3 at week 14, while the community of R2 exhibited the highest qmax obtained during the whole experiment (4.8 × 10-4 ± 0.8 × 10-4 gCH4 gbiomass -1 h-1), possibly due to the high biodiversity for both type I and II methanotrophs revealed by the DGGE analysis. The qmax values here recorded were higher than those previously reported in literature, which typically ranged from 4.2 × 10-5 to 1.3 × 10-4 gCH4 gbiomass -1 h-1. These differences can be explained by the fact that in the present study biomass concentration was optimized in order to avoid CH4 mass transfer limitations, thus resulting in a more realistic parameter estimation. The qmax values for the communities of R1 and R2 significantly decreased at week 19 compared to those obtained at week 14 (1.1 × 10-4 ± 0.3 × 10-4 and 1.9 × 10-4 ± 0.5 gCH4 gbiomass -1 h-1, respectively), while qmax in R3 remained constant. These results suggested that culture aging negatively influenced the biodegradation capacity of the communities exposed to 20 and 2 gCH4 m -3. 214 Table 2: Influence of CH4 concentration during enrichment on the CH4 biodegradation kinetic parameters qmax and KS at weeks 14 and 19 Kinetic parameter R1 (week 14) R2 (week 14) R3 (week 14) R1 (week 19) R2 (week 19) R3 (week 19) qmax (gCH4 gbiomass -1 h-1) 2.7 ± 0.6 × 10-4 4.8 ± 0.8 × 10-4 1.6 ± 0.2 × 10-4 1.1 ± 0.3 × 10-4 1.9 ± 0.5 × 10-4 1.9 ± 0.0 × 10-4 KS (gCH4 L -1) 19.2 ± 3.2 × 10-5 20.8 ± 4.8 × 10-5 16 ± 6.4 × 10-5 8 ± 1.6 × 10-5 25.6 ± 1.28 × 10-5 8 ± 0.7 × 10-5 Moreover, the KS values of the bacterial communities obtained at week 14 showed no significant differences among the samples (Table 2). In contrast, KS values recorded at week 19 significantly decreased in the methanotrophic consortia of R1 and R3 (0.5 × 10-5 ± 0.0 M in both cases), which represents an enrichment of high affinity (low KS) microorganisms mediated by the long-term exposure to CH4. These findings were in agreement with the fact that type I (which often present the highest affinities for CH4) were dominant over type II in both R1 and R3 during almost the whole enrichment. In this regard, Whalen et al. (1990) reported KS values as low as 4 × 10 -5 gCH4 L -1 for type I-methanotroph like cultures, while Delhoménie et al. (2009) obtained higher KS values (108.8 × 10 -5 – 75.2 × 10-4 gCH4 L -1) for type II methanotrophs. 3.3 PHB accumulation Considering the feasibility of coupling CH4 abatement to PHA production under nutrient limiting conditions (Wendlandt et al. 2001; Zúñiga et al. 2011), the reactors were operated under 8 sequential N limitations in order to evaluate the influence of different CH4 concentrations and CH4/biomass ratios on PHB accumulation by methanotrophic consortia. The PHB cell contents in R1, R2 and R3 following the N limitation periods varied within the ranges 0.3-0.5%, 2.9-9.7% and 0.1-0.8%, respectively (Figure 3). The biopolymer content was low and neither correlated with CH4 concentration nor with the time course of the enrichment, possibly due to a lack of naturally CH4 accumulating methanotrophs or a low bioavailability of the C source. The highest PHB cell contents in R1 and R2 were achieved when the CH4/biomass ratio was increased at the end of the 8th N limitation, likely due to a higher C availability. 0 2 4 6 8 10 12 14 1 2 3 4 5 6 7 8 8 (3) 8 (6) 8 (10) 8 (18) % PH B Cycle Figure 3: Specific PHB content in the biomass enriched in R1 (▲), R2 (♦) and R3 (□) following N limitation. The differences in PHB cell content among the communities enriched agreed well with the different structure of the methanotrophic communities revealed by the DGGE analysis. In this regard, Pieja et al. (2011) confirmed that only type II methanotrophs exhibit the ability to produce PHB under nutrient limiting conditions. In the present study, the highest PHB contents were achieved in R2, which presented the highest abundance of type II methanotrophs (Methylocystis genera). GC-MS analyses also revealed the 215 production of PHV in the enrichment cultures. The highest PHV:PHB ratios were obtained in R1 (up to 12:1) and R3 (up to 4:1), which interestingly corresponded to the reactors where the lowest PHB contents were achieved. These results suggested that the low PHB cell contents detected in both reactors could be attributed to the preferential accumulation of PHV by the methanotrophic cultures. In this context, Zúñiga et al. (2013) found maximum PHV:PHB ratios of 7:11 for Methylobacterium organophilum in a STR fed with CH4 and citrate as a co-substrate. However, as fas as the authors know, this is the first work on PHA accumulation from CH4 removal performed in continuous mode without additional carbon sources. Thus, comparisons with previous reports must be carefully done. 4. Conclusions The pair-wise similarity indexes revealed that CH4 concentration during enrichment influenced the structure of the bacterial populations. The cultures were characterized by a rapid population dynamics and a high species evenness and richness. 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