Relationship Between Rumen Microbial Composition and Fibrolytic Isozyme Activity During the Biodegradation of Rice Straw Powder Using Rumen Fluid.

Rumen fibrolytic microorganisms have been used to increase the rate of lignocellulosic biomass biodegradation; however, the microbial and isozymatic characteristics of biodegradation remain unclear. Therefore, the present study investigated the relationship between rumen microorganisms and fibrolytic isozymes associated with lignocellulosic biomass hydrolysis. Rice straw, a widely available agricultural byproduct, was ground and used as a substrate. The biodegradation of rice straw powder was performed anaerobically in rumen fluid for 48 h. The results obtained revealed that 31.6 and 23.3% of cellulose and hemicellulose, respectively, were degraded. The total concentration of volatile fatty acids showed a 1.8-fold increase (from 85.4 to 151.6| |mM) in 48 h, and 1,230.1| |mL L-1 of CO2 and 523.5| |mL L-1 of CH4 were produced. The major isozymes identified by zymograms during the first 12| |h were 51- and 140-kDa carboxymethyl cellulases (CMCases) and 23- and 57-kDa xylanases. The band densities of 37-, 53-, and 58-kDa CMCases and 38-, 44-, and 130-kDa xylanases increased from 24 to 36 h. A microbial ana-lysis indicated that the relative abundances of Prevotella, Fibrobacter, and Bacteroidales RF16 bacteria, Neocallimastix and Cyllamyces fungi, and Dasytricha and Polyplastron protozoa were related to fibrolytic isozyme activity. The present results provide novel insights into the relationships between fibrolytic isozymes and rumen microorganisms during lignocellulose biodegradation.

Bacterial and fungal gut microbiota of supralittoral talitrid amphipods feeding on brown macroalgae and paper.

Some macroalgae drift on the ocean and are stranded on coasts, and these stranded brown macroalgae are regarded to be degraded by organisms. Alginate is a major component of brown macroalgae. An uncovering of how carbon is cycled through brown macroalgae is needed to deeply understand coastal ecosystems. In this study, to gain insights into metabolism of brown macroalgae and alginate in the organisms, we initially confirmed that supralittoral talitrid amphipods (beach fleas or sandhoppers collected on the Shibagaki coast in Ishikawa Prefecture, Japan) fed on the brown macroalgae. We then isolated bacteria such as Vibrio sp. with alginate-assimilating capability from the gut of the amphipods. Metagenomic analysis of the gut of amphipods housed in several conditions (e.g. macroalgae or paper as feed, non-sterilized or sterilized environment) showed no condition-dependent compositions of bacteria and fungi, but Vibrio sp. were detected at high frequency, in good agreement with the isolation of Vibrio sp. An intervention study using antibiotics showed that amphipods fed on algae or paper at about the same rate in the presence or absence of antibiotics, and that the antibiotics had no effects on the life span. Moreover, intervention with antibiotics completely killed Vibrio sp. and some other bacteria, and had significant effects on the composition of the flora in the gut, with elimination of the variations observed in the guts of amphipods housed without antibiotics. These data suggest that microbes that were killed by antibiotics, including Vibrio sp., in the gut of talitrid amphipods are not essential for assimilation of brown macroalgae.

Administration of Aspergillus oryzae suppresses DSS-induced colitis.

Aspergillus oryzae, a filamentous fungus, has long been used for the production of traditional Japanese foods. Here, we analyzed how A. oryzae administration affects the intestinal environment in mice. The results of 16S rRNA gene sequencing of the gut microbiota indicated that after the administration of heat-killed A. oryzae spores, the relative abundance of an anti-inflammatory Bifidobacterium pseudolongum strain became 2.0-fold greater than that of the control. Next, we examined the effect of A. oryzae spore administration on the development of colitis induced by dextran sodium sulfate in mice; we found that colitis was alleviated by not only heat-killed A. oryzae spores, but also the cell wall extracted from the spores. Our findings suggest that A. oryzae holds considerable potential for commercial application in the production of both traditional Japanese fermented foods and new foods with prebiotic functions.

De novo transcriptome assembly of the midgut glands of herbivorous land crabs, Chiromantes haematocheir, and identification of laccase genes involved in lignin degradation.

Herbivorous land crabs such as Chiromantes haematocheir and C. dehaani show biomass-degrading activities. In this study, we performed RNA-seq analysis to detect biomass-degrading enzymes. A de novo transcriptome assembly in the midgut glands of molting and non-molting C. haematocheir crabs was constructed using RNA sequencing. Illumina sequencing generated 44,937,002 and 44,394,310 reads from the two midgut glands. In total, 178,710 contigs with an average length of 750 bp and an N50 value of 1,235 bp were assembled, of which 37,890 contigs were annotated using BLASTx search against the NCBI database. We identified 22 contigs (11 genes) belonging to the laccase family and 44 contigs (22 genes) belonging to the peroxidase family. Sixteen contigs (three genes) belonging to the GH9 cellulase family were also detected. We selected the gene accounting for the majority of expressed laccase and analyzed its properties. The 24131-laccase transcript (2465 bp) had one complete open reading frame, nt 149-1987, encoding a protein of 613 amino acids with a deduced molecular mass of 67.708 kDa. The enzyme was shown to belong to the multicopper oxidase family. The 24131-laccase protein was confirmed to have oxidation activity against 2,6-dimethoxyphenol by ectopic expression in Escherichia coli. Laccase activity was significantly enhanced by feeding land crabs with plant diets. These data suggest that the enzyme plays an important role in the digestion of lignin in the guts of land crabs.

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid.

Treatment with rumen fluid improves methane production from non-degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community's structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium, Diploplastron, and Eudiplodinium) and fungal (e.g., Neocallimastix, Orpinomyces, and Olpidium) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.

Characteristics of various fibrolytic isozyme activities in the rumen microbial communities of Japanese Black and Holstein Friesian cattle under different conditions.

Rumen microorganisms produce various fibrolytic enzymes and degrade lignocellulosic materials into nutrient sources for ruminants; therefore, the characterization of fibrolytic enzymes contributing to the polysaccharide degradation in the rumen microbiota is important for efficient animal production. This study characterized the fibrolytic isozyme activities of a rumen microbiota from four groups of housed cattle (1, breeding Japanese Black; 2, feedlot Japanese Black; 3, lactating Holstein Friesian; 4, dry Holstein Friesian). Rumen fluids in all cattle groups showed similar concentrations of total volatile fatty acids and reducing sugars, whereas acetic acid contents and pH were different among them. Predominant genera were commonly detected in all cattle, although the bacterial compositions were different among cattle groups. Zymograms of whole proteins in rumen fluids showed endoglucanase activities at 55 and 57 kDa and xylanase activity at 44 kDa in all cattle. Meanwhile, several fibrolytic isozyme activities differed among cattle groups and individuals. Treponema, Succinivibrio, Anaeroplasma, Succiniclasticum, Ruminococcus, and Butyrivibrio showed positive correlations with fibrolytic isozyme activities. Further, endoglucanase activity at 68 kDa was positively correlated with pH. This study suggests the characteristics of fibrolytic isozyme activities and their correlations with the rumen microbiota.

Identification of bacteria involved in the decomposition of lignocellulosic biomass treated with cow rumen fluid by metagenomic analysis.

We had developed a new pretreatment system using cow rumen fluid to improve the methane production from lignocellulosic substrates. However, the pretreatment conditions differ from the in-situ rumen environment, therefore different microbes may be involved in plant cell wall decomposition. In the current study, shotgun metagenomic analysis using MiSeq platform was performed to elucidate the bacteria which produce cellulase and hemicellulase in this pretreatment system. The rumen fluid which contained waste paper pieces (0.1% w/v) were incubated at 37°C during 120 h. The fluid samples were collected from the reactor at each time-point and analyzed for chemical properties. Rumen microbial DNA was extracted from 0-h and 60-h samples and subjected to shotgun-metagenomic analysis. After pretreatment, approximately half of cellulose and hemicellulose contents of the waste paper were decomposed and some volatile fatty acids were accumulated. Clostridia (e.g., Ruminococcus and Clostridium) were the predominant bacteria before and after 60-h pretreatment, and their relative abundance was increased during pretreatment. However, Prevotella and Fibrobacter, one of the most dominant bacteria in-situ rumen fluid, were observed less than 3% before incubation and they were decreased after pretreatment. Genes encoding cellulase and hemicellulase were mainly found in Ruminococcus, Clostridium, and Caldicellulosiruptor. Calicellulosiruptor, which had not been previously identified as the predominant genus in lignocellulose decomposition in in-situ rumen conditions, might be considered as the main fibrolytic bacterium in this system. Thus, this study demonstrated that the composition of fibrolytic bacteria in this system was greatly different from those in the in-situ rumen.

Administration of Enterococcus faecium HS-08 increases intestinal acetate and induces immunoglobulin A secretion in mice.

A probiotic is considered a live microbial feed supplement that has beneficial effects on the host. In this study, the probiotic property by which Enterococcus faecium HS-08 strengthens the immune system was investigated. Using a murine model, we evaluated the abilities of this strain to increase intestinal short-chain fatty acid contents and to induce the production of mucosal immunoglobulin A (IgA), which are crucial for mucosal immune systems. Various amounts (0%, 0.0038%, 0.038%, or 0.38%) of strain HS-08 cells were administered to BALB/cAJcl mice, which resulted in a dose-dependent increase of fecal IgA levels. A qRT-PCR analysis of Peyer's patch cells revealed that the gene expression of retinal-dehydrogenase, interleukin 6, B-cell-activating factor, and a proliferation-inducing ligand were increased, which leads to IgA secretion via a T-cell-independent mechanism. The administration of 0.038% and 0.38% of strain HS-08 cells also increased fecal acetate levels, which plays an important role for maintaining immune functions. This cecal floral analysis and the stability of strain HS-08 against gastrointestinal digestion suggest that this strain can inhabit the host intestine. In conclusion, the administration of E. faecium HS-08 increased intestinal acetate levels and enhanced IgA secretion, which may result in strengthening of the mucosal immune system.

Change of Endoglucanase Activity and Rumen Microbial Community During Biodegradation of Cellulose Using Rumen Microbiota.

Treatment with rumen microorganisms improves the methane fermentation of undegradable lignocellulosic biomass; however, the role of endoglucanase in lignocellulose digestion remains unclear. This study was conducted to investigate endoglucanases contributing to cellulose degradation during treatment with rumen microorganisms, using carboxymethyl cellulose (CMC) as a substrate. The rate of CMC degradation increased for the first 24 h of treatment. Zymogram analysis revealed that endoglucanases of 52 and 53 kDa exhibited high enzyme activity for the first 12 h, whereas endoglucanases of 42, 50, and 101 kDa exhibited high enzyme activities from 12 to 24 h. This indicates that the activities of these five endoglucanases shifted and contributed to efficient CMC degradation. Metagenomic analysis revealed that the relative abundances of Selenomonas, Eudiplodinium, and Metadinium decreased after 12 h, which was positively correlated with the 52- and 53-kDa endoglucanases. Additionally, the relative abundances of Porphyromonas, Didinium, unclassified Bacteroidetes, Clostridiales family XI, Lachnospiraceae and Sphingobacteriaceae increased for the first 24 h, which was positively correlated with endoglucanases of 42, 50, and 101 kDa. This study suggests that uncharacterized and non-dominant microorganisms produce and/or contribute to activity of 40, 50, 52, 53, and 101 kDa endoglucanases, enhancing CMC degradation during treatment with rumen microorganisms.

Pretreatment of Lignocellulosic Biomass with Cattle Rumen Fluid for Methane Production: Fate of Added Rumen Microbes and Indigenous Microbes of Methane Seed Sludge.

The pretreatment of lignocellulosic substrates with cattle rumen fluid was successfully developed to increase methane production. In the present study, a 16S rRNA gene-targeted amplicon sequencing approach using the MiSeq platform was applied to elucidate the effects of the rumen fluid treatment on the microbial community structure in laboratory-scale batch methane fermenters. Methane production in fermenters fed rumen fluid-treated rapeseed (2,077.3 mL CH4 reactor-1 for a 6-h treatment) was markedly higher than that in fermenters fed untreated rapeseed (1,325.8 mL CH4 reactor-1). Microbial community profiling showed that the relative abundance of known lignocellulose-degrading bacteria corresponded to lignocellulose-degrading enzymatic activities. Some dominant indigenous cellulolytic and hemicellulolytic bacteria in seed sludge (e.g., Cellulosilyticum lentocellum and Ruminococcus flavefaciens) and rumen fluid (e.g., Butyrivibrio fibrisolvens and Prevotella ruminicola) became undetectable or markedly decreased in abundance in the fermenters fed rumen fluid-treated rapeseed, whereas some bacteria derived from seed sludge (e.g., Ruminofilibacter xylanolyticum) and rumen fluid (e.g., R. albus) remained detectable until the completion of methane production. Thus, several lignocellulose-degrading bacteria associated with rumen fluid proliferated in the fermenters, and may play an important role in the degradation of lignocellulosic compounds in the fermenter.

Bicarbonate and unsaturated fatty acids enhance capsular polysaccharide synthesis gene expression in oral streptococci, Streptococcus anginosus.

We recently reported on the capsular polysaccharide (CP) synthesis (cps) genes of the oral streptococci, Streptococcus anginosus. In this study, we investigate the effects of carbon dioxide (CO2), bicarbonate (HCO3-) and unsaturated fatty acids (UFAs) on CP synthesis of S. anginosus. We found that CP production increased when bacteria were exposed to high concentrations of CO2. This increase was similarly observed in the presence of sodium bicarbonate (NaHCO3) under atmospheric condition. Since ectopic expression of carbonic anhydrase, which converts CO2 to HCO3-, eliminated the requirement for CO2 in CP production and growth of S. anginosus lacking this enzyme, it seemed that HCO3- is an essential factor for CP production. Furthermore, UFAs also stimulated the CP production. Promoter-reporter assay and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis confirmed that stimulation of CP production occurs at the transcription level. The results of the promoter assays suggest that the expression and stimulation of cps genes by HCO3- or UFAs require the cpsA gene, which is located in the first position of the cps operon. With respect to the relationship between HCO3-and UFAs, HCO3- may stimulate UFA synthesis pathway at transcription level. Therefore, it is possible that UFAs are definitive signals for the CP production in S. anginosus.

Preservation of rumen fluid for the pretreatment of waste paper to improve methane production.

It is necessary to preserve rumen fluid for transport from slaughterhouses to the pretreatment facilities for use in treating lignocellulosic biomass. In this study, we investigated how the preservation of rumen fluid at various temperatures affects its use in hydrolysis of waste paper. Rumen fluid was preserved anaerobically at 4, 20, and 35 °C for 7 days. The number of protozoa and fibrolytic enzyme activity after preservation at 4 °C were significantly higher than that after preservation at either 20 or 35 °C. Waste paper was subsequently treated with preserved rumen fluid at 37 °C for 48 h. Preservation at 20 °C remarkedly decreased the hydrolysis of waste paper. Xylanase activity in rumen fluid preserved at 35 °C increased during the treatment, which enhanced the solubilization of waste paper as comparable to the control and preservation at 4 °C. Pretreatment of waste paper with rumen fluid preserved at 4 °C showed that the fluid retained high fibrolytic activity, and reduced the loss of organic carbon as substrate for methanogens. Our results suggest that preservation of rumen fluid at 4 °C is most suitable for efficient pretreatment and methane fermentation of waste paper.

Guaiacol oxidation activity of herbivorous land crabs, Chiromantes haematocheir and Chiromantes dehaani.

The land crabs, Chiromantes haematocheir (Akate-gani) and Chiromantes dehaani (Kurobenkei-gani) inhabit seaside forests in Japan. The crabs mainly consume plant material and its detritus. Therefore, they are expected to possess the ability to degrade the major components of biomass, cellulose and lignin in order to digest plant materials. In this study, we analyzed biomass-degrading activities of the land crabs, especially guaiacol oxidation activity, which seems to be related to lignin degradation. Cellulase activity was detected from almost all gut samples including the stomach, midgut gland and intestine of all dissected crabs. Conversely, high guaiacol oxidation activity was detected in the midgut gland of all C. dehaani and several female C. haematocheir crabs. This is consistent with a previous study showing that female crabs were more herbivorous than male crabs were and observation that C. dehaani crabs are more herbivorous than C. haematocheir. Guaiacol oxidation activity might play an important role in the herbivorous behavior of land crabs.

Effects of molecular hydrogen-dissolved alkaline electrolyzed water on intestinal environment in mice.

Increasing evidence indicates that molecular hydrogen-dissolved alkaline electrolyzed water (AEW) has various physiological activities such as antioxidative activity. Gut microbiota are deeply associated with our health through a symbiotic relationship. Recent reports have described that most gastrointestinal microbial species encode the genetic capacity to metabolize molecular hydrogen, meaning that molecular hydrogen might affect the gut microbial composition. Nevertheless, AEW effects on gut microbiota remain unknown. This study investigated AEW effects on the intestinal environment in mice, including microbial composition and short-chain fatty acid contents. After mice were administered AEW for 4 weeks, 16S rRNA gene sequencing analyses revealed their fecal microbiota profiles. Organic acid concentrations in cecal contents were measured using an HPLC system. Compared to the control group, AEW administration mice had significantly lower serum low-density lipoprotein cholesterol level and alanine aminotransferase activity. Organic acid concentrations of propionic, isobutyric, and isovaleric acids were higher in AEW-administered mice. Results of 16S rRNA gene sequencing analyses showed that the relative abundances of 20 taxa differed significantly in AEW-administered mice. Although the definitive role of gut microbes of AEW-administered mice remains unknown, our data demonstrate the possibility that AEW administration affects the gut microbial composition and that it has beneficial health effects in terms of cholesterol metabolism and liver protection.

Pretreatment with rumen fluid improves methane production in the anaerobic digestion of paper sludge.

Because paper sludge discharged from the waste paper recycling process contains high levels of lignin and ash, it is not hydrolyzed effectively during anaerobic digestion. In this study, we investigated the effects of pretreatment with rumen fluid on paper sludge and on the methane fermentation process. Paper sludge was pretreated with rumen fluid at 37 °C for 6 h. Following pretreatment, 4.5% of the total solids in paper sludge were degraded and converted, and the dissolved chemical oxygen demand and volatile fatty acid concentration increased. Batch methane fermentation was conducted at 37 °C for 20 days. During methane fermentation, the degradation and hydrolysis of paper sludge were enhanced by pretreatment with rumen fluid. The amounts of total methane production from pretreated paper sludge (excluding methane generated from rumen fluid), rumen fluid and untreated paper sludge were 650.4, 819.9 and 190.8 ml, respectively. The volume of methane gas produced from pretreated paper sludge was 3.4 times larger than that from untreated paper sludge. These results indicate that pretreatment with rumen fluid enhances methane production from paper sludge.

Agaro-Oligosaccharides Regulate Gut Microbiota and Adipose Tissue Accumulation in Mice.

Gut microbiota are deeply associated with the prevalence of obesity. Agarose is hydrolyzed easily to yield oligosaccharides, designated as agaro-oligosaccharides (AGO). This study evaluated the effects of AGO on obese phenotype and gut microbial composition in mice. Mice were administered AGO in drinking water (AGO-receiving mice). 16S rRNA gene sequencing analyses revealed their fecal microbiota profiles. Serum bile acids were ascertained using a LC-MS/MS system. Compared to the control group, AGO administration significantly reduced epididymal adipose tissue weights and serum non-esterified fatty acid concentrations, but the cecal content weights were increased. Data from the serum bile acid profile show that concentrations of primary bile acids (cholic acid and chenodeoxycholic acid), but not those of secondary bile acids (deoxycholic acid, lithocholic acid, and ursodeoxycholic acid), tended to increase in AGO-receiving mice. 16S rRNA gene sequencing analyses showed that the relative abundances of 15 taxa differed significantly in AGO-receiving mice. Of these, the relative abundances of Rikenellaceae and Lachnospiraceae were found to be positively correlated with epididymal adipose tissue weight. The relative abundances of Bacteroides and Ruminococcus were correlated negatively with epididymal adipose tissue weight. Although the definitive role of gut microbes of AGO-received mice is still unknown, our data demonstrate the possibility that AGO administration affects the gut microbial composition and inhibits obesity in mice.

Pretreatment of lignocellulosic biomass by cattle rumen fluid for methane production: Bacterial flora and enzyme activity analysis.

We attempted to develop a pretreatment method for methane fermentation of lignocellulosic biomass using cattle rumen fluid, treated as slaughterhouse waste. When rapeseed (Brassica napus L.) was added to the methane fermentation after being solubilized with rumen fluid, 1.5 times more methane was produced compared with untreated rapeseed. Analysis of the bacterial flora during rumen fluid treatment using the MiSeq next-generation sequencer showed that the predominant phylum shifted from Bacteroidetes, composed of amylolytic Prevotella spp., to Firmicutes, composed of cellulolytic and xylanolytic Ruminococcus spp., in only 6 h. In total, 7 cellulolytic, 25 cello-oligosaccharolytic, and 11 xylanolytic bacteria were detected after investigating the most abundant sequences of detected taxa. The relative abundance of two Ruminococcus species (Ruminococcus albus and R. flavefaciens), known as cellulolytic, cello-oligosaccharolytic, and xylanolytic bacteria, increased with increasing cellulose and hemicellulose degradation rates, and, finally, comprised 48% of all operational taxonomic units. The chronological observation of enzyme activities showed that cellulolytic and xylanolytic activities increased 6 h later, and that oligosaccharolytic activity increased 24 h later. This study detected six bacteria that participate in the degradation of aromatics derived from lignin, which have rarely been reported in rumen fluid. The constitution of the detected bacteria suggests that the aromatics were converted into acetate via benzoate. The list of microbes that cover all lignocellulose-degrading candidates will provide fundamental knowledge for future studies focusing on rumen microbes.

Enhancing methane production during the anaerobic digestion of crude glycerol using Japanese cedar charcoal.

The use of Japanese cedar charcoal as a support material for microbial attachment could enhance methane production during anaerobic digestion of crude glycerol and wastewater sludge. Methane yield from a charcoal-containing reactor was approximately 1.6 times higher than that from a reactor without charcoal, and methane production was stable over 50 days when the loading rate was 2.17 g chemical oxygen demand (COD) L(-1) d(-1). Examination of microbial communities on the charcoal revealed the presence of Uncultured Desulfovibrio sp. clone V29 and Pelobacter seleniigenes, known as 1,3-propandiol degraders. Hydrogenotrophic methanogens were also detected in the archaeal community on the charcoal. Methanosaeta, Methanoregula, and Methanocellus were present in the charcoal-containing reactor. The concentration of propionate in the charcoal-containing reactor was also lower than that in the control reactor. These results suggest that propionate degradation was enhanced by the consumption of hydrogen by hydrogenotrophic methanogens on the charcoal.

Anaerobic digestion of crude glycerol from biodiesel manufacturing using a large-scale pilot plant: methane production and application of digested sludge as fertilizer.

This report is the first to consider methane production energy balance from crude glycerol at a practical rather than a laboratory scale. Crude glycerol was added to the plant progressively at between 5 and 75 L glycerol/30 m(3)-day for 1.5 years, and the energy balance was positive at a loading rate of 30 L glycerol/30 m(3)-day (1 ml/L-day). At this loading rate over one year, an energy output equivalent to 106% of the energy input was achieved. The surplus energy was equivalent to transport for 1200 km, so the proper feedstock-transportation distance was within a 12.5-km radius of the biogas plant. In addition, the digested sludge contained fertilizer components (T-N: 0.11%, P2O5: 0.036%, K2O: 0.19%) that increased grass yield by 1.2 times when applied to grass fields. Thus, crude glycerol is an attractive bioresource that can be used as both a feedstock for methane production and a liquid fertilizer.