Isolation and phylogenetic analysis
The results of the present study showed the existence of a multiple bacterial community in the studied sample. A total of 30 bacteria were isolated from all culture media used (Table 1) except for the guaiacol-containing (which did not show bacterial growth). Among the culture media to isolate hydrolytic bacteria, the NA medium had the highest number of bacterial colonies (n = 10), followed by the LE medium (n = 5). Regarding the culture medium for isolation of anaerobic bacteria, the acidogenic medium (ACD) did not recover any bacteria, while the acetogenic (ACT) and methanogenic (MET) recovered 2 and 3 isolates, respectively. Biochemical analyzes in CLED culture medium and the use of the 4 distinct groups of culture media, simulating the 4 phases of anaerobic digestion (hydrolytic, acidogenic, acetogenic and methanogenic), suggested the presence of 16 distinct ribotypes from the 30 isolates recovered from starter, with the vast majority being Gram positive bacteria (Table 1).
Table 1
Microbial groups recovered from the inoculum (starter) derived from the anaerobic digestion process.
Bacteria recovered from hydrolytic medium
|
Code
|
Substrate
|
Nº colonies
|
Enzyme
|
Lactose Ferm.
|
Morphotype (n= nº isolates)
|
Morfology
|
Gram
|
16S rDNA ID
|
Culture Medium
|
CLED
|
LE3
|
Milk
|
5
|
protease
|
-
|
1 (n=5)
|
Rod
|
+
|
Lisinibacillus sp.
|
Bright and transparent colony /blue medium
|
NA1
|
NA
|
10
|
-----
|
2 (n=4)
|
-
|
Luteimonas sp.
|
Bright yellow colony / blue medium
|
NA2
|
3 (n=3)
|
+
|
N.A.
|
Opaque White colony / blue medium
|
NA3
|
4 (n=3)
|
-
|
N.A.
|
Bright greenish colony / blue medium
|
RBBR1
|
RBBR
|
1
|
ligninase
|
5 (n=1)
|
Priestia megaterium
|
Opaque yellow colony / blue medium
|
AM1
|
Starch
|
4
|
amylase
|
+
|
6 (n=2)
|
+
|
Bacillus sp.
|
Dark cream colony with exsudate / yellow medium
|
AM4
|
7 (n=2)
|
N.A.
|
Opaque yellow colony / yellow medium
|
CMC1
|
CMC
|
3
|
cellulase
|
-
|
8 (n=1)
|
Cocos
|
+
|
Rhodococcus sp.
|
Opaque light brown colony / blue medium
|
CM2
|
9 (n=2)
|
N.A.
|
Bright white colony / blue medium
|
OL1
|
Olive oil
|
2
|
lipase
|
+
|
10 (n=1)
|
Rod
|
N.A.
|
Opaque yellow colony / yellow medium
|
OL2
|
-
|
11 (n=1)
|
Niallia circulans
|
Greenish colony / blue colony
|
Bacteria recovered from acetogenic medium
|
ACT
|
ACT2
|
ACT
|
2
|
N.A.
|
N.A.
|
12 (n=1)
|
Cocos
|
+
|
N.A.
|
Cream colony
|
ACT 3
|
13 (n=1)
|
Rod
|
Robertmurraya siralis
|
Cream colony
|
Bacteria recovered from methanogenic medium
|
MET
|
MET1
|
MET
|
3
|
N.A.
|
N.A.
|
14 (n=1)
|
Rod
|
+
|
Vagococcus acidifermentans
|
Cream colony
|
MET2
|
15 (n=1)
|
Bacillus sp.
|
Yellowish colony
|
MET3
|
16 (n=1)
|
Proteiniclasticum sp
|
Cottony colony
|
N.A. Not analyzed. |
Ten of the 10 isolates recovered were sequenced. From the hydrolytic culture media, the following genera and/or species were identified: Lisinibacillus capsici., Luteimonas sp., Priestia sp., Bacillus sp., Rhodococcus sp., Niallia circulans, for the mediums LE, NA, RBBR, AM, CMC and OL, respectively. Regarding the culture medium presenting acetogenic conditions (ACT), a Robertmurraya siralis strain was identified, while the culture medium showing methanogenic conditions (MET) recovered the strains Vagococcus acidifermentans, Bacillus sp., and Proteinclasticum sp. (Table 1, Figure 1).
The taxonomic groups identified in this study have already been reported in the literature, with strains involved in hydrolysis processes of compounds present in the metabolism and production of biogas. The genus Lysinibacillus have already been identified in a study of the characterization of the methanogenic microbial community in brewery wastewater samples (Murunga et al. 2016) as well as in samples of digestate associated with digestion processes using animal manure and food waste (Sun et al. 2020). The specie Lysinibacillus sphaericus has been reported as a strain capable of breaking down the complex structure of lignin (Persinoti et al. 2018; Rashid et al. 2017). Luteimonas species have been reported with activity of Esterase (C4), β-Galactosidase, α and β-Glucosidase as well as strains were present in samples of biogas waste and organic manure (Pu et al. 2018; Roh et al. 2008).
Several Bacillus species including B. megaterium (currently known as Priestia megaterium), B. licheniformis, B. pumilus, B. brovis and B. alvei, have already been recovered from samples obtained of anaerobic digestion processes for biogas production (Biedendieck et al. 2021; Rabah et al. 2010). Bacillus genus is represented by mandatory or facultative aerobic species, and the species B. halodurans has been reported as a carbohydrate fermenter in high temperature environments in an anaerobic biodigestion process in acidic phases (Shah et al. 2014).
Yoon (2021), suggested a new potential species of the genus Bacillus (or proposed new genus Niallia), with the new species Niallia circulans, and no reports were found of the association of this new species with processes of anaerobic digestion and biogas production. Likewise, according to Gupta et al. (2020), representatives of the new genera Niallia gen. nov., Priestia gen. nov., Robertmurraya gen. nov, were reclassified from several Bacillus species after strong phylogenetic and molecular evidence using multiple phylogenetic trees on a genomic scale. Proteiniclasticum sp. and Clostridium sp. were observed in a study involving the use of peat soil, digested sludge, and ruminal fluid for simultaneous consumption of carbon dioxide and production of acetic acid in a biogas production process (Chaikitkaew et al. 2021).
The species Rhodococcus opacus PD630 has catabolic pathways and tolerance mechanisms for aromatic compounds present in ligninocellulosic material, including hexoses and pentoses, and can be considered a good candidate for hydrolysis of the material found in the starter (Anthony et al. 2019). Representatives of the genus Vagococcus were identified in a study addressing genomic analysis of 16S rRNA in anaerobic digestion processes and were correlated with ammonia inhibition (Poirier et al. 2020). In addition, the first-time reported species Vagococcus acidifermentans was isolated from an acidogenic fermentation bioreactor in Naju province, South Korea, with the ability to ferment different sugars (Wang et al. 2011).
Metabarcoding analyzes
The initial metabarcoding analyzes of the sample resulted in a total of 20.652 reads and, after quality processing (filtering, denoising, reads merging, and chimera removal), 16.377 final sequences were obtained. The rarefaction curve of the observed ASVs richness showed that it reached saturation, indicating that the sampling was efficient and capable of revealing almost all the prokaryote microbial species of the samples (Figure 2). Shannon and Simpson’s index values 97 and 0.96, respectively, indicated great diversity in the samples.
Metabarcoding results revealed great prokaryotic diversity and showed the presence of representatives of 16 different phyla, 14 from Bacteria and 2 from Archaea domain (Figure 3). The most abundant phylum was Firmicutes (42.60%), followed by Bacteroidota (32.41%), Chloroflexi (11.38%) and Synergistota (4.07%). The archaea domain was represented by Halobacterota (77.71%) and Euryarchaeota (22.29%). The low archaeal diversity observed may be a result of the inability of prokaryotic primers to amplify archaea, as they are specific for the bacterial rRNA genes.
The main phyla found in our work have already been reported in other studies that evaluated the diversity in anaerobic digestion processes. In the work carried out by Nordgård et al. (2017), the authors observed that the Firmicutes phylum was more abundant in swine manure samples. Brandt et al. (2020), observed a greater abundance of representatives of the phyla Bacteroidota and Firmicutes in complex microbial communities associated with anaerobic digestion processes in different biogas and wastewater treatment plants. Representatives of the phylum Chloroflexi were found in large-scale anaerobic digesters with excess sludge capture from wastewater treatment plants (Petriglieri et al. 2018), while in processes used to understand the regulatory role of H2 in methane production in anaerobic digestion processes the presence of members of the phylum Synergistota was described (Kakuk et al. 2021). Representatives of methanogenic archaea are well known in biogas production processes. The archaeal phyla found in our study, Halobacterota and Euryarchaeota, have also been reported in other works involving anaerobic digestion and biogas production (Heitkamp et al. 2021; Zhang et al. 2020).
Firmicutes phylum have been well described in the literature as an important taxonomic group associated with anaerobic digestion processes as its representatives can express metabolic pathways involved in this process, such as at acetogenic phase, in the degradation of cellulosic compounds, with formation of volatile acetic acid, CO2 and hydrogen (Mukhuba et al. 2020; Nordgård et al. 2017; Zhou et al. 2017). The most abundant genera belonging to the Firmicutes phylum observed in the sample were Enterococcus (17.27%), HN-HF0106 (15.32%), Clostridium sensu stricto 1 (15.28%), Syntrophomonas (4.4%) and a large abundance of unaffiliated bacteria NA (23.67%) (Figure 4).
Watcharasukarn et al. (2009), performed a study where the ability to reduce pathogens from biogas plants was evaluated. In this study Enterococcus species were used as biological indicators in treatments where the temperature exceeds 55°C. The authors concluded that Enterococcus spp. can be resistant to different types of waste treatments, serving as biological indicators in biogas plants. Regarding the genus HN-HF0106, members of this group have been associated with cellulolytic activity, being able to use cellulose as a substrate with production of H2 and acetate (Xie et al. 2021). In a work by Hahnke et al. (2014), carried out from a biogas production reactor fed with corn silage and wheat straw, found a new anaerobic hydrogen-producing mesophilic bacterium affiliated to the genus Clostridium sensu stricto (cluster I of the clostridia). This strain, cultivated in the presence of glucose, was able to produce H2, CO2, formate, lactate, and propionate, which are intermediate compounds to produce methane. In another work, the structures of microbial community in biogas digesters with different types of waste, including cow, pig, sheep manure and human feces, were evaluated. Clostridium sensu stricto 1 represented the highest abundance in the digester with mixed raw materials including dairy cattle manure, sheep manure, and human feces (Han et al. 2021). Wongfaed et al. (2020) evaluated the effect of the presence of oil and its derivatives (long chain fatty acids) in palm oil factory effluent destined for methane production, as well as the structure of the microbial community. The authors observed a cooperation between fatty acid degrading bacteria including Syntrophomonas sp. (strain capable of using long-chain fatty acids with more than 12 carbon atoms) and Acinetobacter sp., with H2- consuming methanogenic bacteria, including Methanococcus sp. and Methanogenium sp. The authors point out that the occurrence of this association in the normal AD process plays an important role in the degradation of oil and derivatives present in palm oil mill effluent.
The most abundant genus belonging to the phylum Bacteroidota was Ruminofilibacter (20.87%), while the main Chloroflexi genus was Longilinea (54%) and, for the phylum Synergistota, the most abundant group was Acetomicrobium (100%). There are no reports in the literature on the association between these genera in the production of biogas. In a work developed by Dong et al. (2019), genes from representatives of the genus Ruminofilibacter (related to cellulose degradation) were found in large quantities in the digestate after anaerobic digestion of cattle manure for biogas production. Yıldırım et al. (2017) evaluated the effects of bioaugmentation using anaerobic ruminal fungi on biogas production in anaerobic digesters fed with animal manure. In the study, the genera Clostridium and Longilinea were some of the most abundant observed in digesters, and the genus Clostridium has been reported to be important in the production of butanol, butyric acid, acetone and iso-propanol, intermediate compounds in this bioprocess. The authors also reported that these two genera were the ones with the greatest capacity to degrade animal waste, which provided higher methane yields. Zhao et al. (2013) evaluated the dynamics of the microbial community in composting systems using biogas slurry compost and cow manure compost for biogas production. The authors adopted th denaturing gradient gel electrophoresis (DGGE) and gene clone library approaches, finding sequences associated with the Acetomicrobium genus after sequencing the clones. Representatives of the Acetomicrobium genus were reported as dominant in a dark fermentation process of fats and protein, using proteins as substrate (Litti et al. 2020). However, it is important to highlight that a large quantity of bacteria was not affiliated to any taxonomic group (NA = 42.16%), showing that a lot of information remains unknown and reinforcing the need for further studies to characterize the taxonomic groups associated with the starter studied here.
Regarding the archaeal sequences, representatives were found for the genera Methanosaeta and Methanobacterium respectively fof the phyla of the Halobacterota and Euryarchaeota phyla in the starter, which have already been related to other processes of anaerobic digestion and biogas production. Representatives of the Methanosaeta genus maintained their dominance over other methanogenic groups in a study where acetoclastic methanogen groups able to act at low pH were acclimated to replace the use of NaOH to regulate buffer pH, a procedure that can inhibit methanogenic microorganisms (Ali et al. 2019). The acetoclastic methanogenic genus Methanosaeta has also been observed in other studies to improve biogas production (Zamorano et al. 2020; Chen et al. 2017). Concerning the genus Methanobacterium, representatives of this group were reported in a study that evaluated the production of biogas containing hydrogen and methane using Microbial Electrolysis Cell (He et al. 2021). In this work, the authors observed that through hydrogenotrophic methanogenesis, the group could synthesize CH4 using H2 and CO2.
The diversity of the microbial community found in anaerobic digestion processes is very diverse, and a large group of bacteria can be found in the organic substrates used in the system. From the beginning of the process, with the anaerobic degradation of organic substances, to the formation of biogas, there is the participation of a diverse microbial consortium, which includes fermentative bacteria, hydrogen-producing acetogenic bacteria, hydrogen-consuming acetogenic bacteria, carbon dioxide-reducing methanogens and acetoclastic methanogenic archaea (Lohani and Havukainen 2018).
The hydrolytic metabolism performed by enzymes such as amylases, lipases, ligninases, cellulases and proteases breaks down organic matter into simpler compounds, including sugars, amino acids, fatty acids, and peptides. This hydrolysis is generally carried out by the metabolic activity of anaerobic bacteria associated to the genera Streptococcus and enterobacteria (Kunz et al. 2019; Shah et al. 2014), and these groups were found in our work, Enterococcus representing the most abundant enterobacteria, and Streptococcus in lesser abundance (0.42% of Firmicutes).
Metabolites formed by enzymatic hydrolysis are converted to other compounds in the acidogenic step. Glucose can be converted into lactic acid by Lactobacillus, and fatty acids can be degraded by Acetobacter species via β-oxidation, forming acetate. Likewise, amino acids are degraded by Clostridium species to form acetate, ammonia, carbon dioxide and hydrogen sulfide (Kunz et al. 2019). In our study, we found Clostridium, but it was not possible to observe Lactobacillus and Acetobacter. However, a relative abundance of Acetomicrobium was found, which can ferment glucose to acetate, CO2 and H2 (Hania et al. 2016), as well as the genus gene HN-HF0106 (Xie et al. 2021).
During the acidogenic step, further short-chain organic acids can be formed including formic, acetic, propionic, butyric and pentanoic acids, as well as alcohols (methanol, ethanol), aldehydes, carbon dioxide and hydrogen (Shah et al. 2014). In our work, it was possible to isolate 3 distinct lactose fermenting morphotypes, two isolates recovered from the culture medium enriched with starch (01 Bacillus sp.) and one isolated from the culture medium enriched with olive oil, which proves that they are bacteria capable of fermenting simpler sugars and lipids via enzymatic hydrolysis. According to Westerholm and Schnürer (2019), the degradation of proteins and amino acids in anaerobic digesters has been shown to be carried out by several genera within the Firmicutes phylum (predominant in our work), which include Gram-positive bacilli.
In the methanogenesis stage (strictly anaerobic), the carbon contained in the biomass is converted into carbon dioxide and methane by methanogenic archaea. Acetoclastic methanogenic archaea, such as the genus Methanosarcina, convert acetate to methane, and the hydrogenotrophic methanogenic archaea, such as the genus Methanobacterium and Methanospirillum, convert hydrogen and carbon dioxide to methane (Kunz et al. 2019). Our findings corroborate those reported by Kunz et al. (2019) in view of the methanogenic representatives, including Methanobacterium in the inoculum sample studied in the present work.
The analysis of parameters found for volatile solids, volatile organic acids (FOS) and total inorganic carbon (TAC) show the rich nutritional composition of the evaluated substrate (carbon sources) for the development of the microbial community studied (Cerqueira et al. 2011). The concentrations of volatile solids, FOS, and TAC, found in the inoculum were 659.10 g kg-1, 717.70 g kg-1, 70005.0 g kg-1, respectively, which correspond to a large amount of material, including volatile organic acids (acetic, propionic, and butyric acids) and inorganic carbon (Cerqueira et al. 2011). pH can influence microbial growth inside the biodigester. On the day of inoculum collection, the pH was 7.6, which may favor the growth of methanogenic archaea, whose optimal pH for development is 6.7 to 7.5. However, fermentative bacteria can adapt to pH variations between 4.0 and 8.5 (Shah et al. 2014).
Thus, we can say that the methodology adopted in this study was able to recover hydrolytic bacteria, such as proteolytic, ligninolytic, amylolytic and cellulolytic bacteria, capable of hydrolyzing protein, lignin, starch, and cellulose that may be present in the inoculum composition, as well as bacteria of the acetogenic phase. However, it was not possible to isolate methanogenic archaea using the media defined for this purpose. This limitation was overcome by using the combination of culture-dependent (enrichment and isolation) and culture-independent (metabarcoding) methods, which allowed access to a greater amount of information about the microbial diversity associated with the anaerobic digestion process (starter). The methods were complementary, as with culture-dependent methods it was possible to isolate representative strains of AD, which were not observed in the culture-independent method and vice versa. Thus, we can conclude that the adoption of both approaches to characterize the microbial community in samples of AD processes is integrative and provides information of great relevance for understanding the microbial function and dynamics in the different stages of biogas production.