Cultured and Uncultured Microbial Community Associated With Biogas Production

Júlia Ronzella Ottoni Universidade Federal da Integracao Latino-Americana Suzan Prado Fernandes Bernal Universidade Federal da Integracao Latino-Americana Tiago Joelzer Marteres CIBiogas Franciele Natividade Luiz CIBiogas Viviane Piccin dos Santos UNICAMP: Universidade Estadual de Campinas Ângelo Gabriel Mari CIBiogas Juliana Gaio Somer CIBiogas Valéria Maia de Oliveira UNICAMP: Universidade Estadual de Campinas Michel Rodrigo Zambrano Passarini (  michel.passarini@unila.edu.br ) Universidade Federal da Integracao Latino-Americana https://orcid.org/0000-0002-8614-1896


Introduction
Population growth has increased the demand for limited supplies of food and fuel by nations around the world. However, petroleumderived fuels, which, in addition to being composed of highly polluting substances to the environmental, are originated from nite sources of energy, factors that indicate the need to replace these fuels in the medium term with those produced from renewable sources. (Gielen et al. 2019). Innovative technologies are being developed to e ciently convert biomass into valuable products such as biogas (Vale et al. 2019). Biogas derived from animal manure and plant residues (lignocellulosic biomass) has been considered an alternative and renewable biofuel with ample energy capacity and can be a sustainable option over the use of fossil fuels (Anthony et al. 2019; Gulhane et al. 2017). One of the major limitations that biogas production still has is the lack of a higher yield in the anaerobic digestion (AD) process, so that the technology can be transferred to large-scale biodigesters. This de ciency can be overcome with greater knowledge of the microbial communities involved in anaerobic digestion (Muturi et al., 2021). Biogas production from renewable and sustainable resources is becoming a prominent alternative in most developed and developing countries (Murunga et al. 2016), which has increased its use, a more viable option for modern society.
AD is a process derived from microbial metabolism that produces biogas (methane) from the conversion of organic matter (Orhorhoro et al. 2017). This process, which occurs in the absence of oxygen, involves different microbial groups, each one being responsible for the degradation of a category of organic compound present in the system. In AD, methane (CH 4 ) production occurs over 4 stages, which are hydrolysis, acidogenesis, acetogenesis and methanogenesis (Vrieze and Verstraete 2016). In hydrolysis, carbohydrates, proteins and fats from animal manure and food residues are broken down into soluble compounds, such as monosaccharides, amino acids, and fatty acids, by the action of enzymes produced by hydrolytic bacteria of genera such as Bacillus, Bacteroides and Eubacterium (Soares et al. 2017). The products of hydrolysis are transformed into volatile fatty acids and alcohols by acidogenic bacterial genera (acidogenesis), including Clostridium and Bacteroides (Valijanian et  Therefore, the implementation of AD processes in biodigesters improves the production of biogas, since in these systems the conditions are controlled, and the processes can be standardized according to the market needs for this biofuel. In addition, the characterization of microbial communities in environmental samples using culture-dependent and culture-independent methods are widely used technologies (Arguita-Maeso et al. 2020; Wei et al. 2021), and essential for the optimization of the process. Given the importance of the composition of the microbiota associated with the inoculum (starter), this work aimed to analyze an inoculum used by the company CIBiogás -International Center for Renewable Energies, to optimize the anaerobic digestion tests developed by the company. Culture-dependent and independent methods were used for taxonomic characterization of the communities present in the inoculum, and the main groups involved in AD processes were identi ed. Such information will be used to optimize and develop strategies for a better understanding of biomethane production.

Sampling and isolation
An inoculum sample (starter) produced in a biodigester was provided by the company CIBiogás located in the city of Foz do Iguaçu and was used in the present work to assess the functional and taxonomic diversity of associated microorganisms. Sampling was carried out on 1/20/2020 in the laboratory of the company CIBiogás. Forty mL of the contents of the biodigester were collected, properly placed in 50 mL asks under sterile conditions. The sample was homogenized in an automatic shaker and serial dilution (10 −1 , 10 −2 and 10 −3 ) was performed. Aliquots of 50 µL of each dilution were inoculated to the respective culture media, described as follows: Hydrolytic culture media (HM), composed by the inorganic salts (SI) KH 2 PO 4 10 g.L −1 ; MgCl 2 .6H 2 O 6.6 g.L −1 ; NaCl 8 g.L −1 ; Na 2 SO 4 0.28 g.L −1 ; NH 4 Cl 8 g.L −1 and CaCl 2 . skimmed milk 10%; vi) for isolation of distinct bacteria (NA-nutrient agar): meat extract 3 g.L −1 ; peptone 5 g.L −1 , pH 6.8. The different culture media containing the sample inoculum were incubated at 37 ºC for 5 to 7 days. Morphologically distinct colonies were puri ed and preserved at -80 ºC in 20% glycerol.

Physicochemical analyzes
The physicochemical analyzes were performed by evaluating the following parameters: total solids, xed solids, volatile solids, volatile organic acids (VOA), total inorganic carbon (TIC), temperature and pH. All these parameters were determined in the CIBiogás laboratory.

DNA extraction
The DNA extraction from 10 distinct isolates was performed according to Aamir et al. (2015). Cells were extracted with 900 µL of phenol in a tube containing a small amount of glass beads followed by incubation at 65°C for 20 minutes. Samples were centrifuged at 16,000xg for 10 minutes at 4°C. The supernatants were added with 800 µL of phenol, brie y homogenized and centrifuged at 16,000xg for 5 minutes at 4°C. Phenol, in a 1:1 ratio, was added to the supernatant, followed by brief homogenization and centrifugation at 16,000xg for 5 minutes at 4°C. A volume of 600 µL of isopropanol was added to the supernatants, followed by homogenization and incubation at -20°C for 20 minutes. Samples were centrifuged at 16,000xg for 10 minutes at 4°C and supernatants were discarded. A volume of 100 µL of 70% ethanol was added to the pellets and, after 1 minute, the ethanol was discarded. Pellets were dried at room temperature and then suspended in 50 µL of sterile MilliQ water. The extracted DNA was quanti ed in 0.8% agarose gel and visualized in a photodocumenter.

PCR and puri cation
The DNAs of the isolates were subjected to PCR for ampli cation of the 16S rRNA gene. Reactions were performed with Buffer

Sequencing and Phylogenetic analysis
Ampli ed products puri ed were sequenced using Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems™) for ABI 3500 Genetic Analyzer (Applied Biosystems™), according to the manufacturer's guideline. Partial gene sequences obtained from isolates were assembled in a consensus sequence using the BioEdit program and further compared to sequences obtained from reference and type strains in the public database GenBank (www.ncbi.nlm.nih.gov). Sequence alignment was performed using the BioEdit program and analyzed with MEGA X software by using the Kimura Evolutionary distances substitution model (Kimura 1980).
Phylogenetic reconstruction was performed using the neighbor-joining (NJ) algorithm (Saitou and Nei 1987) with bootstrap values calculated from 1000 replicate runs.

Sequence accession numbers of the 16S rDNA strains
The sequences obtained from the isolates sequencing were deposited in GenBank under the following accession codes: D2

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).  Figure 1). The most abundant genus belonging to the phylum Bacteroidota was Rumino libacter (20.87%), while the main Chloro exi 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 Rumino libacter (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, nding 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 a liated 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.

The taxonomic groups identi ed in this study have already been reported in
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 CH 4 using H 2 and CO 2 .
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 ) 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 ndings 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 in uence 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 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 de ned 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 culturedependent 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. Figure 1 Phylogenetic analysis based on partial bacterial 16S rRNA sequences of isolates from starter sample. Bootstrap values (1000 replicate runs, shown as %) greater than 70% are listed. GenBank accession numbers are listed after species names.

Figure 2
Rarefaction curve of the prokaryote 16S rRNA gene sequences obtained from inoculum (starter) from CIBiogás anerobic processes using the metabarcoding method. ASVs calculated at 99% identity.

Figure 3
Number of bacterial and archaeal phyla obtained through metabarcoding method from inoculum (starter) from CIBiogás anerobic processes.