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Ordaz-Díaz, L. A., and Bailón-Salas, A. M. (2020). "Molecular identification of microbial communities in the methane production from vinasse: A review," BioRes. 15(2), 4528-4552.


Sugars, starches, and cellulose materials are used for ethanol production. When producing a liter of alcohol, 10 to 15 liters of liquid waste are generated. This waste is called vinasse, and it generates negative impacts on the environment. The process of storing and disposing vinasse in soils generates emissions to the atmosphere, mainly methane. Anaerobic treatment allows for the capture and generation of more biogas, therefore allowing mitigation of the environmental impacts. The microbial diversity present in the anaerobic digestion (AD) of vinasse is strongly related to the efficiency and quality of methane production. The gene 16s rDNA-based molecular techniques have been the most commonly used techniques for monitoring microbial communities present in the digesters. However, the identification is not enough. Rather, it is necessary to know the metagenomic functionality in this type of habitat. This review provides a comprehensive overview of methods to identify the microorganisms in the anaerobic digestion of vinasse. In addition, microbial community identification in vinasse reactors and their relationship with methane production are reviewed.

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Molecular Identification of Microbial Communities in the Methane Production from Vinasse: A Review

Luis A. Ordaz-Díaz and Ana M. Bailón-Salas *

Sugars, starches, and cellulose materials are used for ethanol production. When producing a liter of alcohol, 10 to 15 liters of liquid waste are generated. This waste is called vinasse, and it generates negative impacts on the environment. The process of storing and disposing vinasse in soils generates emissions to the atmosphere, mainly methane. Anaerobic treatment allows for the capture and generation of more biogas, therefore allowing mitigation of the environmental impacts. The microbial diversity present in the anaerobic digestion (AD) of vinasse is strongly related to the efficiency and quality of methane production. The gene 16s rDNA-based molecular techniques have been the most commonly used techniques for monitoring microbial communities present in the digesters. However, the identification is not enough. Rather, it is necessary to know the metagenomic functionality in this type of habitat. This review provides a comprehensive overview of methods to identify the microorganisms in the anaerobic digestion of vinasse. In addition, microbial community identification in vinasse reactors and their relationship with methane production are reviewed.

Keywords: Biogas; Vinasse; Anaerobic biological treatment; Wastewater; Digester

Contact information: Ingeniería en Tecnología Ambiental, Universidad Politécnica de Durango, Carr. Dgo-Mex Km 9.5, Col. Dolores Hidalgo, 34300, Durango, Dgo., México;

* Corresponding author:


There are three main types of raw materials used in the manufacture of ethanol via fermentation: sugars, starches, and cellulose materials (Lin and Tanaka 2006). The distillation stage generates up to 10 to 15 L liquid waste per liter of ethanol (García et al. 1997; Moraes et al. 2015). This acidic waste liquid is very aggressive to the environment due to its high content of toxic and recalcitrant organic matter (Robles-González et al. 2012). Vinasse has the following characteristics: a pH between 3.9 to 5.1, a chemical demand of oxygen (COD) in the range of 50,000 to 95,000 mgL-1, a high concentration of total solids (TS) (79,000 to 37,500 mg L-1), and a high level of volatile solids (VS) (79,000 to 82,222 mgL-1) (Moran-Salazar et al. 2016).

Vinasses are effluents that are difficult to treat; soil fertilization has been a common technique for final disposal (Moran-Salazar et al. 2016). During such disposal, the waste is not actually treated. It has been reported that this disposition, storage, and fertilization generates CO2 and methane emissions (do Carmo et al. 2012; de Oliveira et al. 2013; Moraes et al. 2017), as well as negative environmental impacts (Cruz et al. 1991). In addition, methane is the second most important greenhouse gas induced by man after carbon dioxide (Saunois et al. 2016).

The anaerobic digestion (AD) allows for the capture and generation of biogas. The anaerobic treatment of the vinasse also generates a low production of sludge and can be used to reduce the contamination while producing biogas, which can be used as a source of renewable energy (Belhadj et al. 2013; Fuess et al. 2018; Volpini et al. 2018). In the anaerobic treatment of vinasse, the production of biogas is between 400 and 600 L per kg of COD, which is eliminated with a methane content of 60 to 70% (Moletta 2005). The advantage of methane is that it is odorless, colorless, and non-poisonous. Furthermore, it is easily separated from the liquid phase, which can contribute to the reduction of the process costs (Marty et al. 2001; Lu et al. 2009). Due to the presence of mezcal, vinasses have similar physiochemical characteristics with tequila, sugarcane, and beet vinasse (Moran-Salazar et al. 2016). These could also be used for the generation of renewable energy and reduce the polluting potential (Leme and Seabra 2017).

The efficiency and quality of the process depends on the composition and activity of the microbial community that is present (Christy et al. 2014; Bailón-Salas et al. 2017a), i.e., temperature and pH (Basu 2010). So, the lack of knowledge of the microbial communities present in AD of vinasse limits the capacity to maximize the methane production. In AD of vinasse, changes in the structure of microbial communities have been rarely studied (Jiménez et al. 2018). In the next sections, molecular techniques for the microorganism identification in diverse vinasses are summarized. The National Center for Biotechnology Information (NCBI) database is an important resource relevant to biotechnology and has been used in this review. Besides, a search about microorganisms identified in several studies was made. The methane yield also depends on using varied inoculum sources in varied vinasse types.

Vinasse Types of and Methane Yield

The vinasses composition varies depending on the biomass used for the ethanol production (España-Gamboa et al. 2011). Many feedstocks have been used for alcohol production, and these confer unique characteristics to each vinasse generated. The feedstocks include sugar crops (sugarcane, sugar beet, molasses, and sweet sorghum), starch crops (corn, wheat, rice, cassava, and barley), cellulosic material (harvesting crop residues, bagasse, and wood), fruit sources and Agavaceae family plant. Tables 1 to 4 detail physicochemical properties and composition of vinasses.

The distillation of sugar crops for the production of alcohol generates an effluent with a high organic matter (COD 109,700 to 57,600 mg L-1) (Table 1).

Sugarcane vinasse is a liquid produced in the unit of rectification and distillation in bioethanol production (Parsaee et al. 2019). Some of the main parameters of sugarcane vinasse characterization are given in Table 1. Low pH (3.34), COD greater than 58,000 mg L-1, and a BOD of 23,182 mg L-1 were reported.

The waste from solid-state fermentation of sorghum, corn and/or wheat is called solid vinasse (Wang et al. 2010; Ao et al. 2019). The shown value of pH (4.36) (Ao et al. 2019) was higher compared to sugarcane vinasse.

The most important source of starch for bioethanol is cassava. This is due to its abundance and low cost (Zhang et al. 2016). The cassava vinasses characterization is shown in the Table 2. The pH near 4, COD, BOD, suspended solids, total nitrogen, and total phosphorus of up to 70,000, 35,000, 45000, 900 and 400 mg L-1, respectively, was reported (Luo et al. 2009). Rice wine vinasse also have low pH (3.8) (El-Zaiat et al. 2019) and lower concentrations of organic material (Table 2).

Table 1. Characteristics of Vinasse from Sugar Crops

All values, except pH are expressed in mg L−1.

Many cellulosic materials have been used in ethanol production (Lu-Chau et al. 2019). These cellulosic materials include sugarcane bagasse (Liu et al. 2015; Joppert et al. 2017), agave bagasse (Aguilar et al. 2018), newspaper (Wu et al. 2014), and coffee husks (Gouvea et al. 2009), etc. However there are few studies on physicochemical characteristics of the cellulosic vinasses. A study about the production of 2G ethanol from sugarcane bagasse reported COD values of 38,800 mg L-1 in the vinasse (Tian et al. 2013). Chemical characterization of cotton vinasse gave the following results: pH 4.7, nitrate 350 mg L-1, and ammonium 90 mg L-1 (Diaz et al. 2003). Wheat straw processing for ethanol resulted in pH 3.6, COD 150,000 mg L-1, ammonium 160 mg L-1, and phenols 61 mg L-1 (Kaparaju et al. 2010).

Table 2. Vinasse Characterization from Some Starch Crops

All values, except pH are expressed in mg L−1.

In the fruit wine production, large amounts of water are used in the cleaning and distillation stages (Pap et al. 2004). The vinasses are complex effluents with variable physicochemical properties (Sousa et al. 2019). Table 3 shows high levels of organic compounds, principally polyphenols, as well as other parameters. It has been reported that the phenolic compounds are toxic and can inhibit the bacterial activity (Borja et al. 1993).

Table 3. Physicochemical Properties and Composition of Fruit Vinasses

All values, except pH are expressed in mg L−1.

Some Agave species are used for liquor production (Ramírez-Malagón et al. 2008). Sotol is obtained from the genus Dasylirion, whereas tequila is produced exclusively from Agave tequilana and mezcal from several species of Agave (Gentry 1982; Pardo-Rueda et al. 2015; CRM 2018). The physicochemical characteristics of tequila and mezcal vinasses are shown in Table 4. Based on this review, mezcal vinasse have more sulfate content than sugarcane vinasse.

Table 4. Tequila and Mezcal Vinasse Characterization

All values, except pH are expressed in mg L−1.

All types of biomass can be used as substrates for biogas production (Braun 2007). However the anaerobic digestion of wood is not suitable due to the slow decomposition (Weiland 2010).

The pH values of all vinasses are very low (Tables 1 to 4). So the pH must be adjusted before starting anaerobic digestion. Weiland (2010) recommended an initial pH in the digestion systems in the range 7.0 to 8.0.

To avoid process failure by ammonia accumulation, the C/N ratio should be between 15 and 30 (Zubr 1986; Weiland 2010), and the macronutrients phosphorus and sulfur are necessary in a ratio of 15:5:1 (Weiland 2010).

Moreover, the inoculum selection as well is used to increase the methane production from vinasse (Ordaz-Díaz and Bailón-Salas 2019). Table 5 shows the methane yield using varied inoculum sources and vinasse types. In methane production from vinasses, different types of inoculum have been used, such as brewery sludge, sludge from a wastewater plant, rumen waste, sludge from poultry slaughterhouse reactor, pulp and paper wastewater, swine wastewater, sludge from distillery waste, and sludge from anaerobic reactor. Based on Table 5, the maximum methane yield was obtained using brewery sludge as the inoculum.

Furthermore regarding the inoculum selection, a mesophilic and constant process is recommended. Fluctuations have been found to affect the biogas production negatively (Levén et al. 2007).

Table 5. Methane Yield Using Varied Inoculum Source

aL kg-1 TS, bm3 m−3 day−1.


Anaerobic digestion is the fermentation of organic waste in the absence of oxygen (Abbasi et al. 2012). In the anaerobic wastewater treatment, microorganisms carry out the degradation of the organic matter to produce methane, carbon dioxide, and nutrient-rich sludge (Tabatabaei et al. 2010).

Stages and Microorganisms Involved in Methane Production

The stages of AD are hydrolysis, acidogenesis, acetogenesis, and methanogenesis, where the archaea and bacteria kingdoms participate in the process (Dugba and Zhang 1999).

Fig. 1. Flowchart of AD process for the methane production

In the hydrolysis step (Fig. 1), the organic matter (large chains of organic polymers) is hydrolyzed to simpler compounds or monomers by the action of extracellular enzymes produced by hydrolytic bacteria. The saccharolytic and proteolytic microorganisms decompose the sugars and proteins, respectively (Demirel and Scherer 2008). The monomers can be used as a carbon source by other bacteria and by the same hydrolytic bacteria. At this stage, obligate or facultative anaerobic bacteria participate (Vavilin et al. 1996).

Acidogenesis (fermentation) is the quickest reaction, where the hydrolyzed products are further transformed into simpler organic compounds. The sugars, long-chain fatty acids, and amino acids from hydrolysis are used by fermentative microorganisms that produce organic acids (Kalyuzhnyi et al. 2000; Demirel and Scherer 2008). This stage is of great importance, because mainly acetic and butyric acids are precursors for the formation of methane (Hwang et al. 2001). The biotransformation of organic matter to organic acids causes a decrease in pH in the system. This environment favors acidogenic and acetogenic bacteria (Demirel and Yenigün 2002).

In the acetogenesis, strict anaerobic bacteria participate, and these microorganisms grow slowly (Xing et al. 1997). Acetogenic bacteria produce intermediate products such as ethanol, propionate, and others. The intermediate products are converted to simpler organic acids such as CO2 and H2. Microorganisms that produce and consume hydrogen are possible under this condition. The monitoring and reduction of acetogenic microorganisms in addition to the constant elimination of hydrogen are essential to ensure that acetate production is not interrupted or drastically reduced (Demirel and Scherer 2008; Schuchmann and Müller 2016). At this stage, the methane production could be improved by injecting CO2, which produces more acetic acid yield in this stage. This is due to the fact that acetic acid is the direct substrate for methanogenic microorganisms (Li et al. 2019).

Subsequently, methanogen microorganisms consume organic acids and generate biogas. The CH4 is produced by two major pathways: the acetoclastic pathway where approximately two-thirds of the methane is produced, and by CO2 reduction where CO2 reducing methanogens produce the remaining amount of methane. The sulfate content in the vinasse can inhibit methanogenic archaea richness, since the sulfate-reducing bacteria are competing for the carbon sources (Moestedt et al. 2013; Buitrón et al. 2019). Acetotrophic methanogens convert acetate into biomethane and CO2, where 70% of methane is formed from acetate (Demirbas et al. 2006; Demirel and Scherer 2008). At the end of the process, the biogas produced contains 60% methane, 40% carbon dioxide, water vapor, and minimum amounts of hydrogen sulfide (Christy et al. 2014).

As can be seen in Fig. 1, the microorganisms that participate in each stage of methane production are classified at the class level. Microbial consortia composition has been studied for the production of methane from sugarcane vinasse (dos Reis et al. 2015; Dias et al. 2016; de Barros et al. 2017; Iltchenco et al. 2019), brewery vinasse (Enitan et al. 2014), and synthetic vinasse (Rodríguez et al. 2012). These studies just focused on microbial composition identification at initial and/or final times. There has been a lack of available information about other stages that are crucial for methane production. Li et al. (2019) mentioned that the improvement of methane production requires the improvement of each step of anaerobic digestion. Moreover, this cannot be improved if the changes in the microbiota that are responsible for performing a specific function are not known.

Molecular Techniques for the Identification and Monitoring of Microorganisms in the Anaerobic Digestion of Vinasse

Metagenomics allows the study of microbial communities without the necessity of obtaining pure cultures (Ghosh et al. 2019). Instead, nucleic acids are isolated directly from the sample (Haynes 2008). The basic stages in the study of microbial communities using molecular techniques involves the metagenomic DNA extraction, amplification and sequencing. Other molecular tools are fingerprint methods, such as denaturing gradient gel electrophoresis (DGGE).

The molecular identification of microbial communities is mainly based on the sequence of 16s ribosomal DNA (rDNA) amplified by the polymerase chain reaction (PCR) (Takami 2019). The V3 and V4 region of the 16s gene has been studied to compare the structures of microbial communities due to the precision in taxonomic assignments (Liu et al. 2007). However, universal single-copy “marker” genes are also ideal candidates for taxonomic analysis of environmental samples (Segata et al. 2012). For example, the rpoB gene can be used to calculate relative abundances and provide better bootstrap support for phylogenetic reconstruction (Walsh et al. 2004; Adékambi et al. 2009).

DNA extraction

Traditional techniques for DNA extraction are based on the use of hazardous chemicals including phenol and chloroform (Griffiths et al. 2000; Nwokeoji et al. 2016) and on the guanidine thiocyanate method (Godon et al. 1997). However, for complex samples of wastewater it has been recommended to use the QIAamp DNA Mini Kit and MO BIO Power Soil DNA Isolation Kit due to the high integrity in terms of diversity (Martínez et al. 2014; Dias et al. 2016; Walden et al. 2017).

Nucleic acid amplification methods

In studies based on specific genes, amplification is necessary. The PCR allows generating multiple copies of a specific fragment of DNA or RNA (Hoy 2013). The advantage of PCR-based methods is that they are fast and accurate (Tong 2014). However, there are modified methods such as Real-Time PCR (RT-qPCR) or alternative methods based on isothermal amplification.


In Real-Time PCR DNA, amplification is detected when the reaction is progressing through a fluorescent reporter, where the intensity of the signal is proportional to the number of amplified DNA molecules (Jia 2012). In the microbial community studies in the AD of tequila vinasse, the primers W49F / W104R and W274R / W275F were used to amplify the V3 region of the 16S rRNA gene (Jáuregui-Jáuregui et al. 2014; Toledo-Cervantes et al. 2018).

Isothermal amplification of specific sequences

In isothermal amplification, specialized equipment is not required, such as a thermocycler. Various proteins help DNA polymerase to replicate the DNA (Gill and Ghaemi 2008). There are several types of isothermal amplification methods such as loop-mediated isothermal amplification (LAMP). This amplification method allows for the amplification of six different regions. It is suitable for Sanger and pyrosequencing sequencing (Nagamine et al. 2002; Gill and Ghaemi 2008; Fakruddin and Chowdhury 2012). Other technologies such as strand displacement amplification (SDA), cross priming amplification (CPA), Nicking Enzyme Amplification Reaction (NEAR), and Nicking enzyme-mediated amplification (NEMA) require an additional enzyme, such as a restriction endonuclease or a nicking enzyme. A disadvantage of isothermal amplification of specific sequences is that the components of the reaction mixture and the primer design are more complicated compared to conventional PCR (Tong 2014).

Sequencing and Analysis

The microbial communities can be identified by high-throughput sequencing, which allows sequencing of the amplicon library for rDNA (Haynes 2008). Other technology includes the single molecule real time (SMRT) that allows for the generation of a full sequence data of 16s rRNA genes. The objective of SMRT is to identify bacterial diversity and community structure at the species level (Yang et al. 2018).

Once the sequence is obtained, they are submitted to a database such as BLAST (Altschul et al. 1990), HBLAST (O’Driscoll et al. 2015), or to the Metabolic and Physiological Potential Evaluator (MAPLE) system using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Takami 2019). DNA extraction, 16s rDNA amplification, sequencing, and later analysis of the sequences by means of software are required. For example, the Quantitative Insights into Microbial Ecology (QIIME) package allows the analysis of the microbial community based on data from DNA sequences (Navas-Molina et al. 2013).

DNA Fingerprinting Techniques

Molecular fingerprinting techniques based on the amplification of the 16s rDNA are powerful tools for the study of microbial communities in environmental samples (Kuhn et al. 2017). The PCR-DGGE allows the separation of DNA fragments (same size) previously amplified and the analysis of resulting banding patterns. The double-stranded fragments are separated in polyacrylamide gel with urea-formamide depending on their nucleotide sequence (Myers et al. 1987; Muyzer et al. 1993). In PCR-DGGE, P338f-GC and P518r (Jiménez et al. 2018), 1055F / 1392R-GC (Dias et al. 2016), and 968FGC-1401R (Rodríguez et al. 2012; dos Reis et al. 2015) primers have been used for the study of bacterial communities in the AD of vinasse. For the Domain Archaea, Parch519fGC / Arch915r and A109 (T) -F / 515-GC-R primers have been used (Rodríguez et al. 2012; dos Reis et al. 2015). Also, denaturing gradients ranging from 45% to 60% (Jiménez et al. 2018) and 30% to 70% (Dias et al. 2016) have been used. Other studies have used a gradient of 42% to 67% and 30% to 60% for bacterial and archaeal communities. The DGGE profiles have been analyzed with the Bionumerics software 5.1 (Applied Maths, Kortrijk, Belgium) (Jiménez et al. 2018), BioNumerics 7.1 (Dias et al. 2016) and BioNumerics 2.5 (dos Reis et al. 2015). The bands obtained are excised, crushed, eluted in milliQ water, re-amplified, purified, and sequenced (Bailón-Salas et al. 2017b). In some studies, bands are not sequenced (Jiménez et al. 2018).


The objective of metagenomic analysis is to know the function that microbial communities perform in different environments (Takami 2019). However, all the reports of microbial communities related to the production of methane from vinasse and other environments are based on the 16s rDNA gene.

In AD, there has been a limited understanding of the relationship between microbial community structure and function (Venkiteshwaran et al. 2015). To evaluate the metagenomic functionality of microbial communities, Takami et al. (2012, 2014) developed a method. Subsequently Takami et al. (2016) and Arai et al. (2018), respectively, developed and improved a system to evaluate metagenome functionality. The system was called MAPLE, which allows a homology to search much faster than the Basic Local Alignment Search Tool (BLAST) (Takami 2019). In the KEGG module the methane metabolism is available (Takami 2019).


Molecular techniques for the microbial identification in reactors for the production of methane from vinasse have been studied in samples of sugarcane vinasse (Martínez et al. 2014; dos Reis et al. 2015; Dias et al. 2016; de Barros et al. 2017; Jiménez et al. 2018), synthetic vinasse (Rodríguez et al. 2012), and tequila vinasse (Jáuregui-Jáuregui et al. 2014; Toledo-Cervantes et al. 2018).

Table 5. Microorganisms Identified in the Production of Methane from Vinasse

Few studies have been conducted with a metagenomic analysis in methane production from vinasses (Rodríguez et al. 2012; Enitan et al. 2014; dos Reis et al. 2015; Dias et al. 2016; de Barros et al. 2017; Iltchenco et al. 2019). In general such approaches are not able to identify microorganisms at the species level, so the specific function in the process is uncertain. Besides, present attempts have not been made to understand the microbial community structure in AD of mezcal vinasses.

The microorganisms identified in the methane production from vinasse are shown in Table 6. Some references are available in the public database of the NCBI and others was made based in reports of journals.

Sporomusa sp. is strictly an anaerobic bacterium (Möller et al. 1984), isolated from the alcohol distillation industry (Ollivier et al. 1985), and synthetic vinasse (Rodríguez et al. 2012). In the acidogenesis, sugars and fatty acids are converted to organic acids such as acetic, propionic, and butyric acids. In the AD process, butyrate and propionate are important intermediate compounds (Schink and Stams 2006). It has been reported that some species of the genus Anaerostipes are butyrate producing bacteria (Eeckhaut et al. 2010). Clostridium sp. is a solvent producing bacterium that has the capacity to convert a range of carbohydrates to end products such acetone, butanol, and ethanol. Specifically, Clostridium beijerinckii produces butyric acid and acetic acid (Mitchell 1997; Nimcevic et al. 1998; Little et al. 2015). Some species of Megasphaera have the capability of producing various volatile fatty acids including lactic, formic, acetic, propionic, and butyric acids using sugarcane vinasse (Marx et al. 2011; Sydney et al. 2014). In the raw sugarcane vinasse, high concentrations of propionic acids have been reported as indicating a highly active Propionibacteria community (Júnior et al. 2016). Coriobacterium glomerans has been isolated from the intestinal tract of insects. Glucose, L-arabinose, D-xylose, D-ribose, mannose, sucrose, maltose, cellobiose, mannitol, and salicin are used as a carbon source that are fermented to acetic acid, L-lactic acid, ethanol, CO2, and H(Haas and König 1988).

Ethanol and propionate are mainly transformed into simpler organic acids, CO2, and H2 in the acetogenesis step. Acetobacterium sp. converts H2/CO2 into acetate through acetogenic fermentation (Bainotti and Nishio 2000). Acetobacterium woodii has been the most studied species (Bertsch and Müller 2015; Schuchmann and Müller 2016). At this stage, methylamine is also produced. Tissierella sp. is strictly anaerobic and can produce methylamine (Harms et al. 1998). They were also found to be greatly correlated with the recovered biogas (Chen et al. 2018).

In methanogenesis, the conversion of CO2 and hydrogen to methane is carried out by hydrogenotrophic methanogens (Zabranska and Pokorna 2018). Some genera of Methanobacterium have been associated with this activity (Visser et al. 1991; Harada et al. 1996). The H2 produced in the previous stage must be monitored and eliminated so that the acetate is not reduced. Wolinella succinogenes compete with methanogens microorganisms by consuming H2 (Asanuma et al. 1999). Prevotella sp. utilizes saccharides such as xylan, xylose, pectin, and carboxymethylcellulose, and produces acetate and succinate (Ueki et al. 2007). The acetoclastic microorganism consumes acetate, methanol, and some amines. Pseudomonas sp. can facilitate the extracellular electron transfer and can oxidize various organic electron donors, such as acetate and ethanol (Maruthupandy et al. 2015; Barua et al. 2018).

It has been reported that Thioalkalimicrobium sp. oxizes the sulfur to sulfates (Sorokin et al. 2002). Vinasse obtained from the ethanol distillation has sulfate-rich, liquid substrates (Barrera et al. 2013). Methane production can be affected by alternative hydrogen sinks such as sulfates (Johnson and Johnson 1995), where bacteria could remove sulfate in wastewater before anaerobic treatment for biogas production (Promnuan and Sompong 2017). Desulfovibrio sp. removes the dissolved sulfate and produces small amounts of H2 (Martens and Berner 1974; Guyot and Brauman 1986). Desulfomicrobium aspheronum also removes sulfate (Rozanova et al. 1990), and Halothiobacillaceae sp. utilizes reduced sulfur for energy needs (Quek et al. 2017).


  1. The studies reported in this review focused on microbial composition identification at initial and/or final times. Therefore there is little available information about other stages that are crucial for methane production. Furthermore, there is little information on microbial communities associated with the production of methane from tequila vinasse and null for the mezcal vinasse.
  2. Research of microbial communities that participate in the production of biogas from mezcal vinasses is necessary because each microorganism performs a specific function at each stage of the methane production process. In addition, the quality and performance of methane’s production process are related to the composition and activity of the microbial community. In each reactor subjected to different conditions, the bacterial diversity that develops in the reactor should be monitored. This information should correlate to maximize methane production, and increase knowledge in this field of research and industry.
  3. The molecular tools allow rapid advancement in the knowledge of microbial communities in these habitats. Furthermore, it is time to enrich the functional knowledge of microbial communities, so that cellular metabolism and key functional genes of the microorganisms are better understood.


The support of the Science and Technology National Council (CONACyT) is gratefully appreciated.


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Article submitted: October 4, 2019; Peer review completed: January 23, 2020; Revised version received and accepted: February 16, 2020; Published: February 25, 2020.

DOI: 10.15376/biores.15.2.Diaz