Biodegradation https://doi.org/10.1007/s10532-018-9825-1
ORIGINAL PAPER
Anaerobic biodegradation of partially hydrolyzed polyacrylamide in long-term methanogenic enrichment cultures from production water of oil reservoirs Hao Hu . Jin-Feng Liu . Cai-Yun Li . Shi-Zhong Yang . Ji-Dong Gu . Bo-Zhong Mu
Received: 4 January 2017 / Accepted: 24 January 2018 Ó Springer Science+Business Media B.V., part of Springer Nature 2018
H. Hu J.-F. Liu (&) C.-Y. Li S.-Z. Yang B.-Z. Mu State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China e-mail:
[email protected]
incubated for over 328 days, and analyzed using both molecular microbiology and chemical characterization methods. Gel permeation chromatography, Highpressure liquid chromatography and Fourier-transformed infrared spectroscopy results indicated that, after 328 days of anaerobic incubation, some of the amide groups on HPAM were removed and released as ammonia/ammonium and carboxylic groups, while the carbon backbone of HPAM was converted to smaller polymeric fragments, including oligomers and various fatty acids. Based on these results, the biochemical process of anaerobic biodegradation of HPAM was proposed. The phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichments showed that Proteobacteria and Planctomycetes were the dominant bacteria in the culture with HPAM as the source of carbon and nitrogen, respectively. For archaea, Methanofollis was more abundant in the anaerobic enrichment. These results are helpful for understanding the process of HPAM biodegradation and provide significant insights to the fate of HPAM in subsurface environment and for possible bioremediation.
J.-D. Gu School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China
Keywords Partially hydrolyzed polyacrylamide Anaerobic biodegradation Microbial community Mechanism Oil reservoir production water
Abstract The increasing usage of partially hydrolyzed polyacrylamide (HPAM) in oilfields as a flooding agent to enhance oil recovery at so large quantities is an ecological hazard to the subsurface ecosystem due to persistence and inertness. Biodegradation of HPAM is a potentially promising strategy for dealing with this problem among many other methods available. To understand the responsible microorganisms and mechanism of HPAM biodegradation under anaerobic conditions, an enrichment culture from production waters of oil reservoirs were established with HPAM as the sole source of carbon and nitrogen
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10532-018-9825-1) contains supplementary material, which is available to authorized users.
B.-Z. Mu Shanghai Collaborative Innovation Center of Biomanufacturing Technology, Shanghai 200237, People’s Republic of China
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Introduction Partially hydrolyzed polyacrylamide (HPAM) with high-molecular-weight has been widely and increasingly used in oil industries for enhancing oil recovery (Liu et al. 2012). As a more resistant material to degradation under natural conditions (Deshmukh et al. 1985; Elmamouni et al. 2002), HPAM becomes a chemical hazard to the subsurface ecosystem as it accumulates over time (Kim and Rhee 2003; Zhao et al. 2008). On the other hand, although it is regarded as being low or non-toxic, HPAM may be slowly degraded into acrylamide monomers naturally, a toxic compounds to nervous system (Tyl and Friedman 2003). Thus, research on effective treatment of residual HPAM in the environment receives increasingly attention. Apart from the recently developed physico-chemical methods for degradation of HPAM, biodegradation has been recognized as a potentially effective, safe and environmentally friendly means to remove HPAM from the contaminated environments (Kay-Shoemake et al. 1998b; Nakamiya and Kinoshita 1995). Earlier investigations showed that HPAM could be degraded by microorganisms under aerobic or anaerobic conditions (Grula et al. 1994; Kay-Shoemake et al. 1998a). As a result, Bacillus cereus, Bacillus sp., Bacillus flexu, and Rhodococcus from production water of oil reservoirs (Bao et al. 2010; Sang et al. 2015; Wen et al. 2010), Enterobacter agglomerans and Azomonasm acrocytogenes from soil (Nakamiya and Kinoshita 1995) as well as members of the sulphate-reducing bacteria from oilfield (Ma et al. 2008), were all identified to be HPAM degraders. In HPAM transportation pipelines before injection into oil reservoirs, Pseudomonadaceae, Desulfobulbaceae, Oxalobacteraceae, Flavobacteriaceae and Alcaligenaceae were detected to be primarily responsible for in situ HPAM biodegradation into lower molecularweight polymers and oligomers (Li et al. 2015). With respect to the mechanism of HPAM biodegradation, HPAM could be used as the carbon source by cleavage of the carbon backbone and as the nitrogen source by hydrolyzing the amide groups into free ammonium in anaerobic digestion for dewatered sludge (Dai et al. 2014). Till now, the knowledge about the microbial community, characteristics and specific biochemical processes involved for HPAM biodegradation in anaerobic enrichment cultures are still very limited.
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In this study, an anaerobic enrichment culture derived from production waters of oil reservoirs was established with HPAM as the sole source of carbon and nitrogen, and incubated for over 328 days. The microbial community of this enrichment was investigated and the predominant microorganisms were characterized. Based on the analytical results of metabolic products, the characteristics of HPAM biodegradation were summarized and the biodegradation process was proposed.
Materials and methods Description of samples Production water samples were collected from oil reservoirs of Jiangsu, Shengli and Daqing oilfields in China and mixed for anaerobic enrichment culturing with amendment of HPAM in laboratory. The HPAM sample (Sinopharm Chemical Reagent Co. Ltd, Shanghai, China) with an average molecular weight of 3.0 9 106 Da, solid content of 85% (w/w) and hydrolysis degree of 20% was used in this research. Culturing techniques The culture medium used in experiment contained (mg/L): HPAM 1000, NaHCO3 405, CaCl2 50, K2HPO4 155, NaCl 500, MgSO4 100, FeCl2 20, trace elements solution 1.0 ml/L, vitamins solution 1.0 ml/ L (Wang et al. 2011), and NaNO3 1000 mg/L when HPAM was used as the sole carbon source (C-HPAM), or sucrose 1000 mg/L when HPAM was used as the sole nitrogen source (N-HPAM). The pH of culture media was adjusted to 7.0–7.2 with 0.1 M NaOH solution or 0.1 M HCl solution. Approximately 55 mL of the culture medium were transferred aseptically into each 120 mL sterile serum bottle and flushed with N2 (99.99%) before sealing with butyl rubber stoppers (Bellco Glass, Inc., Vineland, NJ) and aluminum crimp. Then, 5 mL of the mixed culture from the production waters were injected into each serum bottle followed by incubation at 37 ± 1 °C in dark. Each assay was prepared with the blank control (without HPAM) and samples in triplicate.
Biodegradation
Chemical analysis About 200 lL of the headspace gases in each serum bottle were taken with air-tight syringe and analyzed periodically on gas chromatograph (GC) equipped with a flame ionization detector (FID) for CH4 detection, a thermal conductivity detector (TCD) for H2 detection, and a furnace (GC112A, Shanghai Precision Scientific Instrument Co., Ltd., Shanghai, China) for conversion of CO2 into CH4 for detection (Wang et al. 2011). The concentration of ions and volatile fatty acids in the enrichment cultures were analyzed by ion chromatography. About 10 mL culture were centrifuged at 12,0009g at 4 °C for 20 min, and the supernatant was filtered by polycarbonate membrane filters (0.22-lmpore-size, Millipore, Bedford, MA, USA). Then, the residual HPAM was eliminated by a Dionex Onguard C18-RP separation column. The concentration of anions, cations and volatile fatty acids (VFAs) in these samples were quantified using DIONEX ICS1000 ion chromatography (IC, model Dionex 600, Sunnyvale, CA, US) equipped with DIS-5C and DIS5A suppressors. The content of the acrylamide in samples was analyzed by HPLC (Wufeng, China) equipped with an Elitehplc C18 (5 lm) column (250 9 4.6 mm). Ten mL of culture aliquot were mixed with methanol and then centrifuged at 12,0009g for 20 min to remove HPAM. The supernatant was filtered through polycarbonate membrane filters (0.22-lm-pore-size, Millipore, Bedford, MA, USA) and subjected to HPLC analysis with a mobile phase consisting of 50:50 water–methanol (v/v) at a flow rate of 0.5 mL/min and a UV-detector at 210 nm. The HPAM chemical structure was analyzed by FT-IR (Thermo Nicolet 6700, USA) and its molecular weight by GPC (Shimadzu LC-20AD, Japan). The enrichment culture was directly centrifuged at 12,0009g at 4 °C for 20 min and the supernatant was lyophilized at - 60 °C. Approximately, 100 mg of the dried sample were dissolved in deionized water and analyzed by GPC with the mobile phase water at a flow rate of 0.5 mL/min.
respectively, by centrifugation at 12,0009g, followed by total genomic DNA extraction using DNA Miniprep Kit (AxygenÒ Biosciences, Inc., CA, USA). 16S rRNA gene sequences were amplified using 8F/805R primer set for bacteria (Savage et al. 2010) and 349F/ 806R for archaea (Takai and Horikoshi 2000), respectively. PCR products were visualized on agarose gel (1.8%, w/v) and purified using a DNA purification kit (AxygenÒ Biosciences, Inc., CA, USA). After purification, DNA was cloned into Escherichia coli according to the pMD19Ò-T Simple vector protocol, and positive colonies (white ones) obtained after overnight cultivation were checked by colony PCR for correct insert. Sequencing was performed on an ABI 3730 automated sequencer. Valid sequences were obtained after the vectors trimming and chimera checking, and then grouped with the FastGroupII program (Yu et al. 2006) into operational taxonomic units (OTUs) at a cut-off value of 97%. The classifier tool of the Ribosomal Database Project II (Wang et al. 2007) was used to classify each 16S rDNA gene sequence. The nearest relatives of each OTU were identified using the NCBI BLAST network service at http://www.ncbi.nlm.nih.gov/blast/ (Altschul et al. 1990; McGinnis and Madden 2004). Phylogenetic trees were constructed based on the Kimura two-parameter model (Kimura 1980) and neighbor-joining algorithm (Saitou and Nei 1987) for nucleotide sequences using the MEGA6.0 software. Bootstrap analysis with 1000 replicates was applied to assign confidence levels to the nodes in the trees. Partial 16S rRNA gene sequences were deposited into GenBank database under accession numbers of KU563115-KU563145, KU563088-KU563114, KU563065-KU563087 KU563027-KU563049, KU563050-KU563064, KU563017-KU563022 and KU563023-KU563026. Statistical analysis Statistical analysis of means was conducted using the Student’s t test (p value \ 0.05) for the differences in fatty acids and ions for different treatments.
Microbial analysis Microbial cells in the inoculum and enrichment cultures (200 mL of each) were collected,
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Results and discussion Products of HPAM biodegradation Total gas production in both C-HAPM and N-HPAM enrichment cultures after 328 days of incubation, and the CH4 and CO2 yields are shown in Fig. 1. CH4 was generated to 0.053 mmol/L in N-HPAM culture, however, no CH4 was detected in the C-HPAM culture (Fig. 1a). CO2 production was observed in both enrichment cultures, but at very different rate as 28.34 mmol/L CO2 in N-HPAM culture (Fig. 1b), and only 4.98 mmol/L CO2 in C-HPAM culture (Fig. 1c). These results implied that HPAM could be degraded either as carbon or nitrogen source by microorganisms under anaerobic conditions. Ions, such as ammonium, nitrate, sulfate, sulphite and sulfide, in the enrichment cultures were also analyzed before and after 328 days of incubation and the results are presented in Fig. 2. As shown in Fig. 2a, in N-HPAM culture, the ammonium concentration increased (p \ 0.001) to 88.69 mg/L from 11.30 mg/ L initially, the sulphite and sulfide concentration increased to 85.06 mg/L (p \ 0.001) and 45.78 mg/L (p \ 0.001), but that of sulfate decreased (p \ 0.001) to 7.19 mg/L from 274.77 mg/L, respectively. While in C-HPAM enrichment culture (Fig. 2b), the content of sulfate changed slightly during the same incubation period; the concentration of nitrate reduced (p \ 0.001) to 54.88 mg/L from 484.25 mg/L
Fig. 2 Ions change in N-HPAM (a) and C-HPAM (b) cultures
initially, and that of ammonium increased (p = 0.0156) to 23.29 mg/L from 14.15 mg/L. The notable increase in ammonium concentration in N-HPAM culture indicated that the amide bond (C– N portion) of HPAM had been hydrolyzed to release the organic form N as ammonium. Also, it was demonstrated that microbial activities and methanogenesis were stimulated after addition of PAM in anaerobic environments with nitrogen shortage (Haveroen et al. 2005). Similar results were obtained in that concentration of inorganic N was elevated in PAM-treated soils than that of the untreated ones (Kay-Shoemake et al. 1998a). These observations are
Fig. 1 Gases production in enrichment cultures amended with HPAM (filled square) compared with the control without HPAM (filled circle). Methane production in N-HPAM (a), Carbon dioxide production in N-HPAM (b) and C-HPAM (c) cultures
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due to the amidase from bacteria capable of using PAM as the sole N source. The volatile fatty acids (VFAs) in enrichment cultures were analyzed and results are summarized in Fig. 3. The profiles of VFAs in N-HPAM and C-HAM cultures were distinctively different from each other. In N-HPAM, propionic acid increased (p \ 0.001) to 24.34 mg/L from 0.97 mg/L, and formic acids and acetic acids increase a little (Fig. 3a). However, in C-HPAM culture, formic acid and acetic acid increased to 11.52 mg/L from 1.10 mg/L (p \ 0.001) and to 8.51 mg/L from 0.82 mg/L (p \ 0.001), respectively. Propionic acid and butyric acid in this culture increased slightly (Fig. 3b). Biodegradation of HPAM would produce lowermolecular-weight organics such as VFAs in enrichment cultures during incubation. In C-HPAM culture with nitrate addition, VFAs would have been produced from the cleavage of HPAM and the increase of the volatile fatty acids supported this bioprocess. In N-HPAM culture with supplementation of sucrose as a carbon source, the VFAs profile was a result of a potential co-metabolism of sucrose and HPAM. These two treatments (N-HPAM and C-HPAM) produced different profiles of VFAs suggesting potential different breakdown mechanisms mediated by different microbial communities (discussed in the following section). The incubated samples taken during incubation were subjected to HPLC analysis with acrylamide monomer as control for assessing HPAM degradation
Fig. 3 VFAs production in N-HPAM (a) and C-HPAM (b) cultures
and the result is shown in Supplementary material, Fig. S1. One peak with a retention time of 4.87 min from C-HPAM culture sample and another at 4.93 min from N-HPAM culture sample were observed which were not in agreement with the retention time of acrylamide monomer at 5.81 min. This implied that other metabolites than acrylamide monomer was produced under the enrichment culture conditions. It was also reported that PAM was degraded from a concentration of 66.7–32.9 mgL-1 by granular sludge (mostly composed by filamentous and bacilli bacteria) with main intermediate products of low molecular weight polyacrylic acid other than AMD monomer (Liu et al. 2012). Another similar research showed that polyacrylamide was degraded into lower molecular organics and the amide group was transformed into carboxyl group completely (Liu et al. 2016). Change in HPAM structure To elucidate the change in HPAM structure during anaerobic biodegradation, HPAMs before and after biodegradation were subjected to FT-IR analysis and the results are shown in Supplementary material, Fig. S2. The peaks at 3100–3500 and 1500–1700 cm-1 were the characteristic absorbing peaks of amide groups (Bao et al. 2010), and the peaks of C=O, C–N and N–H bonds appeared at 1615, 1415 and 1475 cm-1, respectively (Kas¸ go¨z et al. 2001). The typical differences in the FT-IR spectrum obtained with HPAM sample before and after biodegradation are observed mainly at peaks of 3100–3500, 2300–2400 and 1480–1700 cm-1, respectively. Comparing the profile of FT-IR spectrum of HPAM sample before biodegradation, the peaks of biodegraded HPAM became either wider (at 3100–3500 cm-1) or intensive (at 1630 cm-1) which implied the newly produced groups by biodegradation of O–H and C=O with a characteristic absorbing near these two peaks. This result demonstrates that the amide groups in HPAM molecule have been degraded to carboxyl groups, releasing N as ammonium. These results are consistent with previous reports (Liu et al. 2016; Yu et al. 2015). The molecular weight (MW) profiles of HPAM before and after biodegradation were investigated using GPC (Supplementary data, Fig. S3). Three peaks appeared in the spectra of HPAM before incubation with the retention times of 8.51, 11.98 and 12.62 min,
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Biodegradation b Fig. 4 a Phylogenetic tree of bacterial 16S rRNA gene clones
retrieved from N-HPAM and C-HPAM cultures. The distributions of OTUs and closely related sequences were from the GenBank database. The bootstrap values at the nodes of C 70% (n = 1000 replicates) were indicated. The scale bar represented 5% sequence divergence. The numbers in parentheses indicated the frequencies of appearance of identical clones of the total analyzed clones followed with the accession number in GenBank. The uncultured Methanomicrobiaceae archaeon clone A-9 was used as outgroup. HPAM was used as sole carbon source in blue and sole nitrogen source in red. b Phylogenetic tree of archaeal 16S rRNA gene clones retrieved from the biodegradation of HPAM culture. The distributions of OTUs and closely related sequences were from the GenBank database. The bootstrap values at the nodes of C 70% (n = 1000 replicates) were indicated. The scale bar represented 5% sequence divergence. The numbers in parentheses indicated the frequencies of appearance of identical clones of the total analyzed clones followed with the accession number in GenBank. The New|S001020552 Escherichia coli was used as outgroup. HPAM was used as sole carbon source in blue and sole nitrogen source in red. (Color figure online)
(Fig. S3A), respectively. The spectra of HPAM in N-HPAM culture were similar to that before biodegradation but only a slight right-shift in peaking time (Fig. S3B), and this might be caused by the change of amide groups to carboxyl groups in N-HPAM culture. However, the notable right-shift and appearance of only two peaks (at 9.28 and 13.71 min) in C-HPAM culture (Fig. S3C) demonstrated the possible production of low molecular weight HPAM, which was likely to be produced by cleavage of backbone of HPAM in C-HPAM culture. These results are in agreement with that obtained in anaerobic reactor as well as in anaerobic digestion (Dai et al. 2014; Sang et al. 2015). Microorganisms involved in HPAM degradation Bacteria Microbial community was determined by means of 16S rRNA gene clone libraries, and the bacterial phylogenetic tree was constructed using the neighbour-joining method and presented in Fig. 4a. According to the phylogenetic analysis, the relative abundance of bacterial phylum is shown in Fig. 5a. Proteobacteria was the dominant phylum (66.7%) with the groups of Thermotogae (25.9%) in the original production water sample. The most abundant bacterial phylum was shifted to Planctomycetes
(49.4%) with the groups of Proteobacteria (23.6%) and Thermotogae (14.6%) in N-HPAM culture and to Proteobacteria (89.2%, mainly Pseudomonas sp) in C-HPAM culture after 328 days of incubation, respectively. The bacterial clone library in N-HPAM culture was composed of sequences affiliated with the Planctomycetes, Proteobacteria, Thermotogae, Firmicutes, and Deferribacteres. Some members of Planctomycetes are known as ammonium-oxidizing bacteria with the ability of oxidation of NH4?–N under anaerobic conditions (Strous et al. 1999). The high occurrence of Planctomycetes in N-HPAM culture indicates that its abundance might be stimulated by the releasing of NH4? in N-HPAM culture and thus play a crucial role in down-stream biodegradation of HPAM. Deferribacteres, acetoclastic iron reducing bacteria, have been found in production water from petroleum reservoir (Pham et al. 2009; Silva et al. 2013) and methanogenic cultures (Wang et al. 2011). Sulfatereducing bacteria (SRB) are frequently encountered in oil reservoir (Mckew et al. 2013) and found to be able to degrade HPAM by using it as the only carbon source (Ma et al. 2008). Members of Desulfobulbaceae, such as Desulfurivibrio is proved to be stimulated for growth by HPAM as nitrogen source (Sewell 1987). In present culture, the decrease of sulfate concentration and increase in sulphite and sulfide concentration were observed implying that SRB were active in N-HPAM culture. However, SRB were not identified after 328 days of incubation and this might be due to their low concentration in this culture. Firmicutes were the dominant bacteria in oilfield capable of degrading high-molecular-weight n-alkanes (Cheng et al. 2013). Thermotogae were also found in oilfield with ability of using small molecular organic substrate as source of energy (Mnif et al. 2013). The bacterial clone library in C-HPAM culture was composed of sequences affiliated with the Proteobacteria, Bacteroidetes, Tenericutes and Synergistetes. Pseudomonas was mostly encountered in oilfield produced water or marine sediment (Zhao et al. 2015) and members of them were identified for its ability to utilize PAM as a N source via its production of amidase with growth (Grula et al. 1994). Pseudomonas was considered as a effective HPAM degrader using HPAM as both C and N source under aerobic conditions (Liu et al. 2016). The high occurrence of Pseudomonas in C-HPAM culture indicates
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Biodegradation Fig. 5 Changes of major bacterial phyla (a) and archaeal genus (b) in HPAM biodegradation enrichment cultures
that it might play a crucial role biodegradation of HPAM. Bacteroidetes was known as specialists for degradation of high-molecular-weight compounds and considered to play an important role in the marine carbon cycle (Bauer et al. 2006). Tenericutes and Synergistetes were also found in oilfield with ability of using small molecular organic substrate as the source of energy (Mnif et al. 2013). According to the different functions of these bacteria, Planctomycetes showed a strong competitive advantage compared with other bacteria and might play an important role in HPAM biodegradation in N-HPAM culture and Proteobacteria (mainly Pseudomonas) were considered as the main bacteria responsible for HPAM biodegradation in C-HPAM culture.
In contrast to the bacterial community, the archaeal community composition was less diverse. Methanofollis, found both in N-HPAM and C-HPAM cultures, was mostly isolated from wastewater and sludge and showed ability to convert ethanol to methane and acetate (Imachi et al. 2009). Methanoculleus was detected in N-HPAM culture which was also isolated from oilfields and showed ability to use H2/CO2, formate, 2-butanol/CO2 and cyclopentanol/ CO2 as substrates for methanogenesis (Dianou et al. 2001). Since formic acid, acetic acid, propionic acid and butyric acid were found in the HPAM biodegradation cultures, the detected Methanoculleus were responsible for CH4 production by using the VFAs produced during the upstream HPAM biodegradation process and hence could accelerate HPAM biodegradation.
Archaea The biochemical process of HPAM degradation The results of archaea analyzed are shown in Figs. 4b and 5b. Phylogenetic analysis revealed that all the 16S rRNA gene sequences were affiliated with the phylum Euryarchaeota and divided into three phylotypes in the culture of HPAM biodegradation. All clones obtained with original water sample were Methanofollis (50.0%) and Methanoculleus (50.0%). The archaeal community composition changed to Methanofollis (55.0%), Methanoculleus (33.8%), and Methanothermobacter (11.2%) in N-HPAM culture and to Methanofollis (97.5%) and Methanothermobacter (2.5%) in C-HPAM culture after 328 days of incubation.
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HPAM is believed to be strongly resistant to biodegradation due to its high molecular weight and ‘‘backbone’’ linkage structure (Chang et al. 2001; Suzuki et al. 1978). The amide groups as side chains of HPAM may be easily attacked by amidase. It has been identified that the amidase showed highly activity in numerous genera of bacteria, including Pseudomonas, Rhodococcus, Bacillus, Mycobacterium, Brevibacterium, Alcaligenes, etc. (Kay-Shoemake et al. 1998a, b). In this study, we postulated that HPAM would have been degraded via different mechanisms mediated by different microbial communities under
Biodegradation Fig. 6 The proposed process of HPAM biodegradation under anaerobic conditions
conditions of either carbon or nitrogen limited. In N-HPAM supplemented with sucrose, HPAM would undergo biodegradation via potential co-metabolism (Wen et al. 2010). At the end of this experiment, accumulation of methane, carbon dioxide, ammonium, propionic acid was observed and we also noticed an increase in sulphite and sulfide while sulfate decreased. Also the widening of peak at 3100–3500 cm-1 in FT-IR spectrum (implying O– H) was observed. These results indicate that the amide groups in HPAM molecule was degraded to carboxyl groups, releasing N as ammonium (Haveroen et al. 2005). This bioprocess would have been mediated by microbial members affiliated with Planctomycetes, Proteobacteria, Thermotogae, Firmicutes, and Deferribacteres in potential symbiotic association with Methanofollis and Methanoculleus. The decrease of sulfate concentration and increase of sulphite and sulfide concentration implied the activity of sulfatereducing microorganisms in N-HPAM culture. Similar results has been reported that PAM could be used as a nitrogen source to stimulate the growth of sulfatereducing bacteria (Grula et al. 1994) and methanogens (Haveroen et al. 2005) under anaerobic conditions. In the case of C-HPAM culture, the notable right-shift of HPAM peaks in GPC spectra (possible cleavage of backbone near the end of carbon chain) and statistically significant changes in ammonium (resulting from the attack of amide groups of HPAM by amidase from microorganisms such as Pseudomonas spp.),
formic acid and acetic acid (presumably from the degradation of the carbon chain in HPAM) were identified. The cleavage of the main carbon chain backbone of polyacrylamide caused by microbial treatment (Song et al. 2017) and the metabolism products of organic acids (Dai et al. 2015) under anaerobic conditions were also reported previously. This bioprocess was attributed to the activity of Proteobacteria, Bacteroidetes, Tenericutes, Synergistetes and Methanofollis. The high occurrence of Pseudomonas indicates that it might play a crucial role in biodegradation of HPAM under carbon-limited conditions. According to these results, the process of HPAM biodegradation under anaerobic conditions was proposed and presented in Fig. 6.
Conclusions Biodegradation of HPAM is of great concern and interest to our society. In the present study, HPAM could be degraded by anaerobic enrichment cultures established with production water from oil reservoirs. Under carbon or nitrogen source limited conditions, profile of different metabolites was observed, suggesting potential different breakdown mechanisms mediated by different microbial communities. This study is of fundamental value and practical significance, especially in bio-treatment of HPAM widely used in oilfields.
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Biodegradation Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants No. 41673084, 41530318, 41273084), NSFC/RGC Joint Research Fund (Grant No. 41161160560) and the Research Foundation of Shanghai (No. 15JC1401400).
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