Evaluation of the biomethanation potential of enriched methanogenic cultures on gelatin

Abstract

Purpose

Biomethane is an environment-friendly, economic, and alternative energy resource for a clean and green future. In the present study, we have evaluated the biomethanation potential of acetate-utilizing methanogenic culture (AUMC) and gelatin-enriched mixed culture (GEMC) with Clostridium acetobutylicum NCIM 2841 (GEMC-CA.) on gelatin as a sole carbon and nitrogen source.

Methods

We conducted experiments for examining the specific-methanogenic activity of these cultures in the metabolic assay media containing 1% gelatin. The produced methane and consumed gelatin were quantified by standard experimental methods. Exchange metabolites produced by these cultures were qualitatively analyzed by mass spectrometry.

Results

Results of our study show that the growth-associated amino acid catabolism partially or completely supported the methanogenesis of these defined cultures. AUMC and GEMC found to be suitable for enhanced methanogenic activity on gelatin but a rapid degradation of amino acid was attributed by GEMC-CA. The ammonia released from these cultures was directly proportional to gelatin degradation. Mass spectral data analysis identified some key exchange metabolites from acidogenic culture and methanogenic culture for confirming the growth-associated methanogenesis.

Conclusion

The biomethanation potential of these cultures on gelatin is coupled with the Stickland reactions-directed methanogenesis in a syntrophic manner. The present study provides the importance for the development of a starter culture for the biomethanation of protein-based industrial wastes in effective ways.

Introduction

Anaerobic digestion is a complex microbial metabolic process that can be carried out by different syntrophic organisms. It has been an economically attractive and alternative process for the treatment of different types of industrial wastes (Wu et al. 2017; Liu and Liao 2018; Perisin and Sund 2018). Lignocellulosic wastes are widely used as the raw materials for industrial production of organic solvents, organic acids, and biofuel. However, a large amount of energy consumption is needed for the pre-treatment of them in harsh conditions (Balan 2014). Lipid-based wastes are not being economical to make biofuel apart from biodiesel (Vlysidis et al. 2011). Anaerobic digestion of these wastes is a complex biogenic process and requires more retention time for biomethane production, but anaerobic bacteria can rapidly degrade the protein-based wastes.

The protein-based industry (food, meat, leather, fish, bone, etc.) typically produces wastewater containing a huge quantity of proteins and amino acids. Anaerobic digestion of these wastes has been employed successfully for industrial biomethane production. During the extraction and processing of them, a large quantity of gelatin is derived from the cleavage of collagen abundantly present in these wastes. Gelatin is noticeably a pure protein consisting of glycine (21.4%), proline (12.4%), hydroxyproline (11.9%), glutamate (10%), alanine (8.9%), arginine (7.8%), and aspartic acid (6.0%) as the major amino acids (Eastoe 1995). Tryptophan and cysteine are absent and methionine is only present at a relatively low level. Hence, gelatin is considered as a model protein substrate for the biomethanation of protein-based industrial wastes due to amino acids-coupled methanogenesis and enriched contents of glycine, alanine, and proline (Chellapandi et al. 2008, 2010; Chellapandi and Uma 2012a, b).

Acidogenic bacteria (Clostridiaceae family) are second trophic microorganisms in the anaerobic digestion process. These microorganisms degrade the gelatin into amino acids and small peptides. Furthermore, electron donor amino acids are oxidatively deaminated to volatile carboxylic acids (acetate or pyruvic acid) via the Stickland reactions (Stickland 1934; Nisman 1954). The produced acetate can be served as a carbon source for the growth and methanogenesis of the methanogenic bacteria. However, complete degradation of gelatin and efficient methanogenesis is achieved only in the presence of required species in defined methanogenic culture.

Several anaerobic mixed cultures have been developed for the biomethanation of various protein-based wastes (Ollivier et al. 1986; Vieira et al. 2001; Chellapandi et al. 2008, 2010; Chellapandi and Uma 2012a, b). A mixed culture of amino acid degraders and hydrogenotrophic methanogens has been developed for the rapid degradation of gelatin and methanogenesis (Jain and Zeikus 1989). Sasaki et al. (2011) have developed thermophilic cultures for effective degradation of casein, gelatin, and bovine serum albumin syntrophically in a methanogenic environment. Nevertheless, only a limited endeavor has been made on their metabolic stability and consistency to methane production from proteins.

In this perspective, our study was aimed to evaluate the biomethanation potential of AUMC, GEMC, and GEMC-CA growing on gelatin as a sole carbon source in batch culture. A methanogenic culture understudied would have more industrial utility as a starter culture for the biomethanation of protein-based wastewater.

Materials and methods

Growth and maintenance of C. acetobutylicum

A frozen culture of C. acetobutylicum NCIM 2841 strain was procured from the National Collection of Industrial Microorganisms, India. Clostridial spores were inoculated in 100 Ml cooked meat medium containing the composition (g/L) of beef extract, 45; dextrose, 2.0; protease peptone, 20; NaCl, 5.0; and then incubated at 37 °C in an anaerobic condition. It was maintained in the cooked meat agar medium until the next use.

Preparation of anaerobic seed culture

The anaerobically digested slurry was collected from a functional biogas plant (3 m³) at the Biogas Research Centre, Gujarat Vidyapith, Sadra, India. It was filtered through a muslin cloth and centrifuged at 5000 rpm for 10 min by a bench top centrifuge. One volume of the collected supernatant was diluted with three volumes of dilution medium (g/L) containing NaHCO₃, 5.0; NaCO₃, 10; and two or three drops of resazurin (0.001%, w/v). It was pre-activated for 24 h at 37 °C before inoculation.

Enrichment technique for methanogens

Methanogens were enriched and cultivated by the anaerobic technique described previously (Wolfe 2011). Enrichment of methanogens was carried out in a medium containing the composition (g/L): NH₄Cl, 1.0; NaCl, 0.6; NaHCO₃, 5.0; KH₂PO₄, 0.3; K₂HPO₄, 0.3; MgCl₂·6H₂O, 0.16; CaCl₂·2H₂O, 0.009; and resazurin 0.001% solution, 1.0 mL. A solution of the following vitamins (10 mg/L): p-aminobenzoic acid, nicotinic acid, calcium pantothenate, pyridoxine, riboflavin, thiamine and 5 mg each of biotin, folic acid, α-lipoic acid, and cyanocobalamine B12 were prepared and 10 mL of this solution added to the above medium. A solution of trace minerals (g/L) was prepared and 10 mL of this solution mixed with 1 L of that medium: trisodium nitrilotriacetic acid, 1.5; Fe(NH₄)₂(SO₄)₂, 0.8; NaSeO₃, 0.2; CoCl₂·6H₂O, 0.1; MnSO₄·H₂O, 0.1; Na₂MoO₄·2H₂O, 0.1; NaWO₄·2H₂O, 0.1; ZnSO₄·7H₂O, 0.1; NiCl₂·6H₂O, 0.1; H₃BO₃, 0.01; and CuSO₄·5H₂O, 0.01. The pH of the medium was adjusted to 7.4 with 0.1 M KOH. Cysteine-HCl and Na₂S·9H₂O were added separately to this medium. After that, 100 mM sodium acetate for acetate-utilizing methanogens and 1% (w/v) gelatin for gelatin-enriched cultures were added separately in the enrichment media. The pre-activated seed culture (5%, v/v) was inoculated to initiate the enrichment of methanogens and then incubated at 37 °C under N₂ headspace. Acetate was replenished everyday until the culture reached a late log phase.

Assay for specific methanogenic activity

Experiments were done in the anaerobic serum vials of 132 mL with a working volume of 50 mL phosphate-buffered basal (PBB) medium (Moench and Zeikus 1983). The PBB medium is composed (g/L) of KH₂PO₄, 1.5; K₂HPO₄·3H₂O 2.9; MgCl₂·6H₂O, 0.2; CaCl₂·2H₂O, 0.1; NaCl, 0.9; NH₄Cl, 1.0; resazurin, 0.001% solution, 1.0 ml; trace mineral solution, 10 mL; vitamin solution (µg/mL): biotin, 10; pantothenate (calcium), 25; lipoic acid, 25; folic acid, 10; thiamine, 25; riboflavin, 25; pyridoxine HCl, 50; cyanocobalamine B12, 0.05; nicotinic acid, 25 and p-amino benzoic acid, 25. Trace mineral solution has consisted of (g/L) trisodium nitrilotriacetic acid, 12.8; Na₂SeO₃, 0.02; CoCl₂·6H₂O, 0.16; Na₂MoO₄·2H₂O, 0.01; H₃BO₃, 0.1; FeSO₄, 0.1; MnCl₂, 0.1; ZnCl₂, 0.1; CuCl₂, 0.02 and NiSO₄, 2.6 mg.

After the proper enrichment, methanogenic culture (5%, v/v) was inoculated into 50 mL of the PBS medium containing 100 mM sodium acetate as an initial growth substrate. It was incubated at 37 °C until the methane production and depletion of acetate cease. After addition of 2–3 drops of titanium (III)-nitrilotriacetate reductant, the gas phase of a serum bottle was changed with N₂ by evacuation and flushing. Sterilized gelatin at 1% (w/v) was added along with the overgrown culture of C. acetobutylicum and the time course was followed by incubation at 37 °C. A serum vial containing the same assay medium without gelatin was served as a negative control. The gas phase was replaced with N₂ gas daily. Gas production was measured using an air-tight syringe. After the incubation, the entire content of the digested liquid was centrifuged at 5000 rpm for 15 min and the supernatant used for determination of pH, volatile fatty acids, and other metabolites.

Analytical techniques

Gas samples were collected from the head phase of each vial and immediately analyzed for methane content using a Gas chromatograph equipped with thermal conductivity detector (Perkin-Elmer Autosystem) as outlined previously (Chellapandi and Uma 2012a, b). One microlitre of diluted biogas sample was injected into a packed 2-m Porapak T steel column using an air-tight syringe. Nitrogen was used as a carrier gas at the flow rate of 30 mL/min. The oven, injection, and detector temperatures were adjusted to 50, 70, and 150 °C, respectively. Gelatin concentration was analyzed by using a modified Bradford dye-binding protein assay (Noble and Bailey 2009) and then calibrated with a gelatin standard. The concentration of extracellular and intracellular amino acids was calorimetrically determined with ninhydrin reagent by measuring optical density at 570 nm (Moore and Stein 1954) using glycine and alanine as standards. The liberated ammonia during gelatin degradation was estimated by spectrophotometer using sodium nitroprusside reagent at 625 nm and then calibrated with ammonium sulfate standard (Chaney and Marbach 1962).

GC–MS/MS analysis

The exchange metabolites of these cultures were detected from the digested fluid by GC–MS/MS (Perkin-Elmer Autosystem with turbo mass) with PE-5MS column (30 m; 250 micron internal diameter; 0.25 micron thickness) (Halket et al. 2005). Helium was used as a carrier gas. The oven temperature was adjusted to 80 °C for 5 min and raised to 280 °C with the rate of 10 °C/min and of 15-min hold time. Both injection and electron impact source temperatures were set at 250 °C. Split ratio was adjusted to be 1:60 and mass ranged between 20 and 650 atomic mass units. The volatile fatty acids and hydrocarbons were extracted in dichloromethane, concentrated, and then injected 1 µL sample for analysis. Metabolites present in the samples were identified from mass–ionization ratio obtained in GC–MS/MS peeks using the National Institute of Standards and Technology 11 and National Bureau of Standards Mass Spectral Library.

Results and discussion

Effect of incubation time on microbial growth

In this study, gelatin was used as a model protein substrate to find out amino acid catabolism-coupled methanogenesis of AUMC and GEMC in batch digestion (Fig. 1a). It has shown that a maximum biomass yield was attained after 11 days of incubation. GEMC-CA has shown a maximum growth compared to GEMC and AUMC. It may be resulted due to the overgrowth of C. acetobutylicum in the enriched culture and its growth might be supported by gelatin in the minimal medium (Masion et al. 1987). It indicated that gelatin was a good growth substrate for these cultures similar to a mixed defined triculture of methanogens (Ollivier et al. 1986).

Fig. 1

Effect of incubation time on the growth (left) and methanogenic activity (right) of enriched methanogenic cultures on gelatin in anaerobic batch digestion (AUMC acetate-utilizing methanogenic culture, GEMC gelatin-enriched mixed culture, GEMC-CA gelatin-enriched mixed culture with Clostridium acetobutylicum NCIM 2841)

Effect of incubation time on a methanogenic activity

A considerable methanogenic potential was achieved after 4–6 days of incubation and then gradually declined (Fig. 1b). AUMC was shown a relative methanogenic activity on gelatin as it has more potential populations of acidogenic bacteria. GEMC-CA did not favor the methanogenesis compared to GEMC and AUMC due to rapid degradation of gelatin by C. acetobutylicum. The methanogenic potential of these cultures was faintly more in the presence of acetate than gelatin used alone. Some syntrophic bacteria in the AUMC and GEMC may have an impending metabolic activity and metabolite pairing (exchange) for the bioconversion of gelatin to methane. Perhaps, it could be improved for defined cultures by enhancing the growth population of methanogens or increasing acclimatization period on gelatin (Patel and Chellapandi 2009).

Effect of incubation time on gelatin degradation

The degradation of gelatin was started after 1 day and then gradually increased by the growth period (Fig. 2a). A constant rate of gelatin degradation was observed when GEMC used as a starter culture. GEMC-CA was initially supported for the gelatin degradation and after that its consumption rate was decreased. It described the relative fraction of proteolytic acidogenic bacteria in GEMC and AUMC responsible for the efficient degradation of gelatin during the anaerobic digestion process. The rate of gelatin degradation attained by GEMC was better than AUMC owing to enriched populations of gelatin-degrading bacteria as described earlier (Orlygsson et al. 1994; Fonknechten et al. 2010).

Fig. 2

Effect of incubation time on the degradation of gelatin (left) and liberation of ammonia (right) by enriched methanogenic cultures in anaerobic batch digestion (AUMC acetate-utilizing methanogenic culture, GEMC gelatin-enriched mixed culture, GEMC-CA gelatin-enriched mixed culture with Clostridium acetobutylicum NCIM 2841)

Effect of incubation time on amino acids assimilation

Glycine and alanine from gelatin were gradually metabolized and then transformed into methane in these cultures. Alanine was excreted in the exponential phase but disappears slowly in the stationary growth (Additional file 1: Figure S1). It pointed out that extracellular glycine and alanine consumed more rapidly than intracellular glycine and alanine. The interspecies hydrogen transfer is necessary to accomplish amino acid metabolism (Nagase and Matsuo 1982; Orlygsson et al. 1994; 1995; Hartwich et al. 2012). Amino acid degradation was also enhanced by the accumulation of hydrogen gas in head phase. The intracellular catabolism of glycine and alanine in AUMC was slightly higher than GEMC. Catabolism of intracellular glycine and alanine was delayed by GEMC for oxidative deamination. It may be resulted due to rapid assimilation of such metabolites to its cellular energetic metabolism (Orlygsson et al. 1994, 1995). A significant quantity of proline is not detected in the fermentation media as a lack/low activity of proline dehydrogenase present in CAC or other acidogenic bacteria (Jain and Zeikus 1989). It pinpointed the need of another bacterial species to catabolize the proline for a complete transformation of gelatin to methane.

Effect of incubation time on ammonia liberation

Ammonia was liberated after 3 days of digestion, raised as a maximum of 5 days, and then suddenly decreased during gelatin degradation (Fig. 2b). It found a similar metabolic pattern to release ammonia from gelatin from GEMC and AUMC. Ammonia was steadily liberated at a log phase of GEMC and then slowly declined. It pointed out that increasing concentration of ammonia was directly proportional to an amino acid or gelatin degradation. This suggested that a methanogenic culture was actively maintained by accumulated ammonia (alkaline buffer), which was agreed to the earliest investigations (Masion et al. 1987; Orlygsson et al. 1994, 1995; Fonknechten et al. 2010).

Effect of incubation time on exchange metabolites

Using GC–MS/MS data, we have identified extracellular metabolites produced by these cultures (Fig. 3). Apart from methane and CO₂ formation, acetamide, ethanol, ethylamine, and acetaldehyde were found as exchange metabolites of acidogenic bacteria, when GEMC was grown on gelatin. It also reported that either acidogenic bacteria or methanogens have secreted tyrosine and methionine from their cellular metabolism. Methanogenic substrates such as formate, propionate, and acetate were identified from the digested fluid. It represented that these metabolites supported the growth-associated methanogenesis in a methanogenic environment. Butyric acid, ethanol, propanol, butanol, and acetaldehyde were end products of this digestion that may be produced by CAC. It indicated that acidogenesis and solventogenesis are active in acidogenic bacteria or CAC when gelatin is used as a growth substrate for these cultures in anaerobic condition.

Fig. 3

GC–MS/MS profiling for the identification major exchange metabolites in anaerobically digested fluid after 11 days of incubation of enriched methanogenic culture on gelatin. 1—acetamide, acetaldehyde; 2—acetic acid, formic acid; 3—succinic acid, l-tyrosine, butanedioic acid, ethylamine, l-methionine, propanedioic acid, 2-pentanoic acid, 2-propionic acid; 4—butanoic acid; 5—1-propene, 3-butene; 6—n-butanol, 1-propanol; 7—propanoic acid

The proposed pathway for Stickland reactions-coupled methanogenesis

Acidogenic bacteria can degrade the gelatin into amino acids, which are further transformed into precursors requiring for methanogenesis in two ways: (1) pairs of amino acids can be catabolized via the Stickland reactions and (2) single amino acids can be fermented in a process that requires the presence of hydrogen-utilizing methanogens (Vieira et al. 2001; Stickland 1934; Nisman 1954; Ollivier et al. 1986). Glycine/alanine or ornithine/proline or ornithine/lysine was used as an amino acid pair in the Stickland reactions of many Clostridia (Nagase and Matsuo 1982; Orlygsson et al. 1995; Fonknechten et al. 2010; Hartwich et al. 2012). However, uncoupled degradation of amino acids (10% of total amino acids) only occurs in mixed culture if there was a shortage of amino acids as electron acceptors (Orlygsson et al. 1994). Based on the growth of kinetic and GC–MS/MS data, we proposed the amino acid-catabolism-directed methanogenesis in a methanogenic culture (Fig. 4). In the proposed system, the presence of glycine reductase has reduced glycine alone to acetate with the release of ammonia via intermediate acetyl phosphate. Amino acid dehydrogenase and glycine reductase could be active in these methanogenic cultures during anaerobic digestion of gelatin. Clostridiale family has developed the specific pathways to degrade amino acids by fermentation processes (Nagase and Matsuo 1982; Fonknechten et al. 2010; Sangavai and Chellapandi 2017). Acetate probably arises from the oxidation of amino acids such as alanine or the reduction of glycine similar to the earlier work (Tang et al. 2005). Methanogenesis of these cultures depends on the growth environment where acetate is not necessarily a preferred substrate for all methanogenic bacteria. After depleting the acetate, acetoclastic or hydrogenotrophic methanogens in these cultures consume other methanogenic substrates such as propionic acid, pyruvic acid, hydrogen, and carbon dioxide produced during anaerobic fermentation of gelatin (Ramasamy and Pullammanmappallil 2001). AUMC and GEMC consisted of a considerable population of acidogenic bacteria but have no balanced tropical anaerobic bacteria.

Fig. 4

The proposed pathway for the Stickland reactions-coupled methanogenesis in enriched methanogenic culture. ACE acetate, APHO acetyl phosphate, ACOA acetyl-CoA, MCP a[methyl-Co(III) corrinoid Fe–S protein, MTHS 5-methyl-tetrahydrosarcinapterin, MCOM methyl-CoM, CFP a[Co(I) corrinoid Fe–S protein], OFD oxidized ferredoxin, RFD reduced ferredoxin, COA coenzyme A, THM tetrahydromethanopterin, COM coenzyme M, THS tetrahydrosarcinapterin, COB coenzyme B, COMB coenzyme MB, CO₂ carbon dioxide, CH₄ methane

Conclusions

Studying the growth of physiology and metabolites sharing is essential for evaluation of the biomethanation potential of defined methanogenic cultures on gelatin in batch digestion. Biosynthesis of methane can be directed partially or completely by growth-associated catabolism of amino acids in methanogenic cultures during anaerobic digestion of gelatin. The methanogenic activity of these cultures is enhanced with suitable amino acid pairs despite single amino acids. The biomethanation of these cultures on gelatin depends on the metabolic rate of the Stickland reactions-coupled methanogenesis and availability of methanogenic precursors. Conceivably, the present methanogenic cultures are suitable candidates for the bioconversion of protein-based wastes to methane in anaerobic batch digesters. The metabolic and growth stability of these enriched cultures should be further evaluated by biochemical and metabolic kinetic experiments. Such data would provide the importance to understand the metabolic behaviors of syntrophic parterres in the future.