Microbiological Research 173 (2015) 10–17
Contents lists available at ScienceDirect
Microbiological Research journal homepage: www.elsevier.com/locate/micres
Bacterial communities in thermophilic H2 -producing reactors investigated using 16S rRNA 454 pyrosequencing Regiane Priscila Ratti ∗ , Tiago Palladino Delforno, Dagoberto Yukio Okada, Maria Bernadete Amâncio Varesche ∗ University of São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 30 September 2014 Received in revised form 5 January 2015 Accepted 26 January 2015 Available online 7 February 2015 Keywords: Fermentation Firmicutes Heat-shock Thermoanaerobacterium Thermohydrogenium
a b s t r a c t In this study, the composition and diversity of the bacterial community in thermophilic H2 -producing reactors fed with glucose were investigated using pyrosequencing. The H2 -producing experiments in batch were conducted using 0.5 and 2.0 g l−1 glucose at 550 ◦ C. Under the two conditions, the H2 production and yield were 1.3 and 1.6 mol H2 mol glucose−1 , respectively. Acetic, butyric, iso-butyric, lactic and propionic acids were detected in the two reactors. The increase in substrate concentration favored a high H2 yield. In this reactor, a predominance of acetic and iso-butyric acids, 27.7% and 40%, were measured, respectively. By means of pyrosequencing, a total of 323 and 247 operational taxonomic units were obtained, with a predominance of the phylum Firmicutes (68.73–67.61%) for reactors with 0.5 and 2.0 g l−1 glucose, respectively. Approximately 40.55% and 62.34% of sequences were affiliated with Thermoanaerobacterium and Thermohydrogenium, microorganisms that produce H2 under thermophilic conditions. © 2015 Elsevier GmbH. All rights reserved.
Introduction Hydrogen gas (H2 ) is an ideal fuel source for producing bioenergy because of its high-energy yield and lack of greenhouse gas production. Many researchers believe that using H2 as an alternative energy source may replace fossil fuels. H2 can be produced by different physical–chemical methods; however, these methods are more costly when compared to methods that utilize fossil fuels. H2 production using microorganisms is a feasible technology that is more environmentally friendly and less energy intensive (Kapdan and Kargi 2006). H2 can be produced by pure or mixed cultures from various simple substrates (glucose, sucrose and xylose) and from waste generated by industry (Kapdan and Kargi 2006; Ratti et al. 2013). The H2 production from carbohydrates is accompanied by formation of organic acids, CO2 and other intermediates. During the anaerobic acidification of carbohydrates, methanogenic
∗ Corresponding authors at: Department of Hydraulics and Sanitation, School of Engineering of São Carlos, University of São Paulo, Av. João Dagnone, 1100, Jd. Santa Angelina, 13563-120 São Carlos, SP, Brazil. Tel.: +55 1633738357; fax: +55 1633739550. E-mail addresses: [email protected] (R.P. Ratti), [email protected] (M.B.A. Varesche). http://dx.doi.org/10.1016/j.micres.2015.01.010 0944-5013/© 2015 Elsevier GmbH. All rights reserved.
microorganisms consume H2 and have a negative impact on H2 production; therefore, many studies have attempted to inhibit hydrogen consumers by pH control, heat-shock, or aeration (Ratti et al. 2013). Another important factor that affects H2 production is temperature. Mesophilic and thermophilic conditions have been commonly used in studies on H2 production (Datar et al. 2007; Ratti et al. 2013). However, thermophilic fermentation presents a number of advantages in comparison to the mesophilic process, such as higher yields of H2 , more efficient degradation of organics, and more resistance to contamination (Zhang et al. 2003). Clostridium and Thermoanaerobacterium are fermentative microorganisms that are able to produce H2 from various carbohydrates under thermophilic conditions (Lebuhn et al. 2014). There is great interest in investigating the microbial community structure in H2 -producing reactors to create a better understanding of syntrophic interactions in this process. Moreover, the knowledge of the microbial population is essential to develop operating methods aimed at improving the H2 production process performance (Wagner and Loy 2002). Various molecular methods have been applied in research to better understand the interactions that the microbial community present in anaerobic digestion reactors. Development of new technologies, such as 454 pyrosequencing, enables the determination of a larger number of sequences in a shorter time. This method
R.P. Ratti et al. / Microbiological Research 173 (2015) 10–17
11
has been used to investigate the microbial community in various environments (Guo et al. 2014). It should also be noted that, to our knowledge, there have been few studies that have investigated the thermophilic microbial population in H2 production using pyrosequencing. The objectives of this study were to investigate the H2 production using a thermophilic microbial consortium. Glucose is readily found in a wide variety of industrial wastes and, therefore, was used as the substrate. 16S rRNA gene pyrosequencing methodologies were then employed to characterize the microbial communities present in H2 -producing reactors.
Table 1 Parameters obtained by modified Gompertz model for H2 production.
Material and methods
DNA extraction and pyrosequencing
Inoculum, culture medium and substrate
For analysis of bacterial diversity, DNA was extracted using a modified phenol–chloroform protocol (Griffiths et al. 2000). DNA quality was assessed by a standard of 260/280 nm > 1.8 using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA) and agarose gel electrophoresis. The pyrosequencing was performed at the Instituto de Agrobiotecnologia Rosario (INDEAR) (Rosario, Argentina) using a 454 Genome Sequencer FLX (Roche). The DNA of each sample was amplified with a set of primers that flanked the hypervariable V4 region of the 16S rRNA gene. The forward primer was 563F (5 -AYTGGGYDTAAAGNG-3 ), and the reverse primer was 802R (5 -CAGGAAACAGCTATGACC-3 ).
The inoculum used was the granular sludge obtained from a thermophilic up-flow anaerobic sludge blanket reactor (UASB), which is used for the treatment of stillage from sugarcane for biogas production (CH4 ), located at the São Martinho distillery plant (Pradópolis, SP, Brazil). The inoculum was subjected a heat-shock to inhibit H2 -consuming microorganisms (Maintinguer et al. 2011). Modified Del Nery medium was used in the experiments following the composition described by Ratti et al. (2013). Glucose is readily found in a wide variety of industrial wastes and was used as the model substrate in two concentrations: 0.5 and 2.0 g l−1 . Experimental procedures The experiment was carried out in triplicate in 5.0 l batch reactors. The reactors were inoculated with inoculum (10% v/v) and the appropriate amount of substrate in a final working volume of 1.0 l. The initial pH of the solution was adjusted at 7.0 with HCl and NaOH. Nitrogen (N2 ) gas was flushed into the reactors to create anaerobic culture conditions. The reactors were covered with rubber stoppers, closed with plastic stoppers and incubated at 55 ◦ C. During the experiments, the liquid samples were collected for the analysis of pH, carbohydrate consumption, and soluble metabolite composition. Biogas production was simultaneously monitored over time. Analytical methods The hydrogen content in the biogas was determined by gas chromatography (GC) using a GC 2010 Shimadzu system (Maintinguer et al. 2011; Ratti et al. 2013, 2014). The concentrations of organic compounds, such as volatile acids and alcohols, in the liquid phase were determined by high-performance liquid chromatography (HPLC) (Penteado et al. 2013). The concentration of total volatile solids (TVS) was determined according to standard methods for the examination of water and wastewater (APHA 2012). The soluble carbohydrate concentration was determined indirectly by the phenol–sulfuric acid method using glucose as a standard (Dubois et al. 1956). Kinetic analysis The experimental data were fitted to the mean values of the triplicate sets of reactors using the software Statistica® 8.0. The cumulative hydrogen production (H) data were fitted to the modified Gompertz model (Eq. (1)), which is suitable for describing the cumulative biogas production in batch experiments (Ratti et al. 2013, 2014). In this equation, H represents the hydrogen evolution data, t is the period of incubation (h), P is the hydrogen production
Reactors
P (mmol)
Rm (mmol/h)
H2 yield
0.5 g l−1 glucose 2.0 g l−1 glucose
4.2 13.3
0.24 0.04
20 41.5
1.3 1.6
potential (mmol), Rm is the maximum hydrogen production rate (mmol/h), is the length of the lag phase (h) and e is 2.71. H = P · exp
− exp
R · e m P
( − t) + 1
(1)
Sequencing processing and bacterial population analysis The sequences obtained were processed using the Ribosomal Database Project (RDP-Pyrosequencing Pipeline) (http://pyro.cme. msu.edu/index.jsp) (Cole et al. 2009). First, the sequences were trimmed to remove adaptor, barcodes, primers and sequences shorter than 200 bases. Chimeras introduced in the PCR process were removed using the DECIPHER program (http://decipher.cee.wisc.edu/index.html; Wright et al. 2012). The processed and trimmed sequences were aligned using the 210 secondary-structure aware Infernal aligner-RDP. Similar sequences were clustered into Operational Taxonomic Units (OTUs) based on 97% similarity using Hierarchical clustering. The taxonomy assignment was performed by the RDP classifier (Cole et al. 2009) using one representative sequence for each OTU. Rarefaction curves were constructed based on the OTUs phylogenetic domain. The relative abundance of bacterial taxons was analyzed in each sample, according to Chapleur et al. (2014). The diversity (Shannon and Simpson) and richness (Chao 1) indices were generated using Past software. The sequences were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk) under accession numbers ERS553966 (0.5 g l−1 glucose) and ERS553967 (2.0 g l−1 glucose); the project number is PRJEB7362. Results and discussion H2 production and organic acids In reactors fed with 0.5 and 2.0 g l−1 glucose, the generation of H2 after 20 h (2.1 mmol H2 ) and 23 h (0.21 mmol H2 ) was observed, and the maximum value was observed at 60 h (4.7 mmol H2 ) and 162 h (14.3 mmol H2 ) of incubation (Fig. 1). In the reactors, increased H2 production was observed with increased glucose concentration due to increased availability of substrate. Values of potential production of H2 (P), maximum rate of production of H2 (Rm ), lag phase time () and H2 yield are shown in Table 1.
12
R.P. Ratti et al. / Microbiological Research 173 (2015) 10–17
H2 Production (mmol)
16
Table 2 Results of conditions studied.
14 12 10 8 6 4 2 0
0
20
40
60
80 100 120 140 160 180 200 220 240 Time (h)
Fig. 1. H2 production. () A reactor fed with 0.5 g l−1 glucose; (䊉) a reactor fed with 2.0 g l−1 glucose.
The maximum hydrogen production potential (P) and H2 yield increased with increasing glucose concentration. P was 13.36 and 4.22, and H2 yield was 1.6 and 1.3 mol H2 mol glucose−1 for assays fed with 2.0 and 0.5 g l−1 glucose, respectively. The periods of lagphase were similar to others studies that utilized glucose as the substrate (Wang and Wan 2008). In the reactor fed with 0.5 g l−1 glucose, was the lowest (20 h), as the lower amount of substrate favored rapid growth of microorganisms. The lag phase is influenced by initial pH in assays of H2 production (Kapdan and Kargi 2006). In the present study, the initial pH was 7.0 and was verified longer lag phases (Table 1). Longer lag phases (20 h) were detected at low initial pH of 4.0–4.5 (Khanal 2004). According to Zhang et al. (2003), high initial pH (9.0) reduces lag time leading to lower H2 yields. The H2 yield obtained in the present study is comparable to reported values of hydrogen-producing enrichment cultures (1.7 mol H2 mol glucose−1 ) (Lin and Chang 1999). In contrast, higher yields of H2 were observed by Morimoto (2004) and Yokoyama et al. (2009) in thermophilic and extreme thermophilic conditions. The H2 yields were 2.1 mol H2 mol glucose−1 (Morimoto 2004) and 2.63–3.32 mol H2 mol glucose−1 (Yokoyama et al. 2009), respectively. Probably, higher yields were related to the type of inoculum used. Furthermore, Yokoyama et al. (2009) verified the predominance of Caldanaerobacter subterraneus in the inoculum that led to high H2 yields. According to Lin and Chang (Lin 2004), the H2 yield could be less than 1.7 mol H2 mol glucose−1 when more than 95% glucose was degraded. In the present study, glucose was linearly degraded, and at the end of the assay, 100% of the glucose was degraded. After 40 h and 100 h of the assay, the degradation of the substrate was high and the production of H2 stabilized in the reactor fed with 0.5 g l−1 of glucose and 2.0 g l−1 glucose, respectively. Using TVS analysis, it was verified that the increase in the substrate concentration was favorable for microorganism growth. The TVS values were 0.8 and 1.2 mg l−1 for assays with 0.5 and 2.0 g l−1 glucose, respectively (Table 2). Increasing the substrate concentration favored the development of microorganisms and resulted in an increase in H2 production. The pH was not controlled in these batch cultures, and under both conditions, a reduction of pH to 4.7 and 3.8 for assays fed with 0.5 and 2.0 g l−1 glucose, respectively, was observed (Table 2). The decrease in pH was due to production of organic acids during fermentation. The decrease in pH inhibits the H2 production (Dabrock et al. 1992). In the present study, the production of hydrogen ceased at low pH; the assays were started at neutral pH and terminated at acidic pH values. The optimum pH for H2 production was observed
Parameters analyzed
0.5 g l−1 of glucose
2.0 g l−1 of glucose
Degradation of glucose (%) TVS (g l−1 ) Initial pH Final pH Acetic acid (mmol) Butyric acid (mmol) Iso-butyric acid (mmol) Lactic acid (mmol) Propionic acid (mmol) H2 yield (mol H2 mol glucose−1 )
100 0.8 7.0 4.7 1.8 1.08 8.8 0.08 2.4 1.3
100 1.2 7.0 3.8 5.4 1.8 7.8 0.8 2.6 1.6
in the pH range of 5–6 (Ueno et al. 2001). Ratti et al. (2013) reported that the maintenance of an optimal pH range (5.5–6.5) was necessary to avoid the rapid inhibition of fermentation. The intermediate products obtained from the reactors with glucose are illustrated in Fig. 2. The main volatile fatty acid in the fermentation broth was isobutyric acid in all the assays. Low amounts of lactic and propionic acids were produced from glucose. Butyric, isobutyric and acetic acids constituted more than 75% of total end products. The production of these organic acids favors the H2 production, according to Eqs. (2) and (3). Theoretically, 4 mol and 2 mol of H2 are produced from 1 mol of glucose when acetate and butyrate are the main fermentation products, respectively (Ratti et al. 2014; Santos et al. 2014). C6 H12 O6 + 2H2 O → 4H2 + 2CO2 + 2 C2 H4 O2
(2)
C6 H12 O6 → 2H2 + 2CO2 + 2 C4 H8 O2
(3)
At the end of the test of the reactor containing 0.5 g l−1 glucose, the percentages of acetic, butyric, isobutyric, lactic and propionic acids were 12.72%, 7.6%, 62.23%, 0.56% and 16.87%, respectively. For the reactor containing 2.0 g l−1 glucose, the percentages of acetic, butyric, isobutyric, lactic and propionic acids were 27.73%, 9.2%, 40%, 4.6%, and 18.5%, respectively. In both reactors, a predominance of iso-butyric acids was observed. However, in the reactor fed with 2.0 g l−1 glucose, an increase in acetic acid (5.4 mmol) was observed, explaining the higher H2 yield (Table 2). According to Kapdan and Kargi (2006), the production of butyric acid rather than acetic acid may be one of the reasons for lower H2 yields (see Eqs. (2) and (3)). pH may also affect the type of organic acids produced and the H2 yield. pH between 4.0 and 6.0 favored the production of butyric acid (Fang and Liu 2002). In the present study, the production of H2 occurred preferentially via butyric acid (isobutyric acid), which was evident because the concentration of this acid was higher. Several previous studies have indicated a predominance of butyric acid in assays for H2 production using glucose (Fang and Liu 2002; Morimoto 2004). Probably, the substrate concentration also affected the production of organic acids and the H2 yield. In Table 2 was possible verify that the total acetic acid concentration increased in reactors fed with 2.0 g glucose where was observed major H2 yield. Bacterial community compositions and diversities Pyrosequencing produced a total of 7806 (average length = 273 ± 1.6 bp) and 4318 (average length = 272 ± 2.1 bp) bacterial 16S rDNA sequences with high coverage of diversity (98–98.2%). These sequences were assigned to 323 and 247 OTUS for the reactors with 0.5 and 2.0 g l−1 glucose, respectively. According to the RDP-Classifier, 95.7–99.9% of the sequences were classified into a phylum and 76.4–93.6% into a genus. The sequences were affiliated into 5 and 7 phyla and 35 and 30 genera for the reactors fed with 0.5 and 2.0 g l−1 glucose, respectively. In the two conditions, the most prevalent phylum was Firmicutes
R.P. Ratti et al. / Microbiological Research 173 (2015) 10–17
Fig. 2. Production of organic acids ( acetate, glucose.
butyrate,
iso-butyrate,
lactate,
(68.73–67.61%). According to the literature, this phylum encompasses the main microorganisms that produce H2 (Ratti et al. 2013; Santos et al. 2014). The diversity and richness of the microbial consortium were analyzed (Table 3). The microbial community in the reactor with 0.5 g l−1 glucose contained high diversity and richness according to the Simpson index (0.99) and the Shannon index (5.25). The community composition was composed of 323 operational taxonomic units (OTUs) with a predominance of the phylum Firmicutes (68.73%). Members of the Acidobacteria, Proteobacteria and Verrucomicrobia were present at an abundance of
Contents lists available at ScienceDirect
Microbiological Research journal homepage: www.elsevier.com/locate/micres
Bacterial communities in thermophilic H2 -producing reactors investigated using 16S rRNA 454 pyrosequencing Regiane Priscila Ratti ∗ , Tiago Palladino Delforno, Dagoberto Yukio Okada, Maria Bernadete Amâncio Varesche ∗ University of São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 30 September 2014 Received in revised form 5 January 2015 Accepted 26 January 2015 Available online 7 February 2015 Keywords: Fermentation Firmicutes Heat-shock Thermoanaerobacterium Thermohydrogenium
a b s t r a c t In this study, the composition and diversity of the bacterial community in thermophilic H2 -producing reactors fed with glucose were investigated using pyrosequencing. The H2 -producing experiments in batch were conducted using 0.5 and 2.0 g l−1 glucose at 550 ◦ C. Under the two conditions, the H2 production and yield were 1.3 and 1.6 mol H2 mol glucose−1 , respectively. Acetic, butyric, iso-butyric, lactic and propionic acids were detected in the two reactors. The increase in substrate concentration favored a high H2 yield. In this reactor, a predominance of acetic and iso-butyric acids, 27.7% and 40%, were measured, respectively. By means of pyrosequencing, a total of 323 and 247 operational taxonomic units were obtained, with a predominance of the phylum Firmicutes (68.73–67.61%) for reactors with 0.5 and 2.0 g l−1 glucose, respectively. Approximately 40.55% and 62.34% of sequences were affiliated with Thermoanaerobacterium and Thermohydrogenium, microorganisms that produce H2 under thermophilic conditions. © 2015 Elsevier GmbH. All rights reserved.
Introduction Hydrogen gas (H2 ) is an ideal fuel source for producing bioenergy because of its high-energy yield and lack of greenhouse gas production. Many researchers believe that using H2 as an alternative energy source may replace fossil fuels. H2 can be produced by different physical–chemical methods; however, these methods are more costly when compared to methods that utilize fossil fuels. H2 production using microorganisms is a feasible technology that is more environmentally friendly and less energy intensive (Kapdan and Kargi 2006). H2 can be produced by pure or mixed cultures from various simple substrates (glucose, sucrose and xylose) and from waste generated by industry (Kapdan and Kargi 2006; Ratti et al. 2013). The H2 production from carbohydrates is accompanied by formation of organic acids, CO2 and other intermediates. During the anaerobic acidification of carbohydrates, methanogenic
∗ Corresponding authors at: Department of Hydraulics and Sanitation, School of Engineering of São Carlos, University of São Paulo, Av. João Dagnone, 1100, Jd. Santa Angelina, 13563-120 São Carlos, SP, Brazil. Tel.: +55 1633738357; fax: +55 1633739550. E-mail addresses: [email protected] (R.P. Ratti), [email protected] (M.B.A. Varesche). http://dx.doi.org/10.1016/j.micres.2015.01.010 0944-5013/© 2015 Elsevier GmbH. All rights reserved.
microorganisms consume H2 and have a negative impact on H2 production; therefore, many studies have attempted to inhibit hydrogen consumers by pH control, heat-shock, or aeration (Ratti et al. 2013). Another important factor that affects H2 production is temperature. Mesophilic and thermophilic conditions have been commonly used in studies on H2 production (Datar et al. 2007; Ratti et al. 2013). However, thermophilic fermentation presents a number of advantages in comparison to the mesophilic process, such as higher yields of H2 , more efficient degradation of organics, and more resistance to contamination (Zhang et al. 2003). Clostridium and Thermoanaerobacterium are fermentative microorganisms that are able to produce H2 from various carbohydrates under thermophilic conditions (Lebuhn et al. 2014). There is great interest in investigating the microbial community structure in H2 -producing reactors to create a better understanding of syntrophic interactions in this process. Moreover, the knowledge of the microbial population is essential to develop operating methods aimed at improving the H2 production process performance (Wagner and Loy 2002). Various molecular methods have been applied in research to better understand the interactions that the microbial community present in anaerobic digestion reactors. Development of new technologies, such as 454 pyrosequencing, enables the determination of a larger number of sequences in a shorter time. This method
R.P. Ratti et al. / Microbiological Research 173 (2015) 10–17
11
has been used to investigate the microbial community in various environments (Guo et al. 2014). It should also be noted that, to our knowledge, there have been few studies that have investigated the thermophilic microbial population in H2 production using pyrosequencing. The objectives of this study were to investigate the H2 production using a thermophilic microbial consortium. Glucose is readily found in a wide variety of industrial wastes and, therefore, was used as the substrate. 16S rRNA gene pyrosequencing methodologies were then employed to characterize the microbial communities present in H2 -producing reactors.
Table 1 Parameters obtained by modified Gompertz model for H2 production.
Material and methods
DNA extraction and pyrosequencing
Inoculum, culture medium and substrate
For analysis of bacterial diversity, DNA was extracted using a modified phenol–chloroform protocol (Griffiths et al. 2000). DNA quality was assessed by a standard of 260/280 nm > 1.8 using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA) and agarose gel electrophoresis. The pyrosequencing was performed at the Instituto de Agrobiotecnologia Rosario (INDEAR) (Rosario, Argentina) using a 454 Genome Sequencer FLX (Roche). The DNA of each sample was amplified with a set of primers that flanked the hypervariable V4 region of the 16S rRNA gene. The forward primer was 563F (5 -AYTGGGYDTAAAGNG-3 ), and the reverse primer was 802R (5 -CAGGAAACAGCTATGACC-3 ).
The inoculum used was the granular sludge obtained from a thermophilic up-flow anaerobic sludge blanket reactor (UASB), which is used for the treatment of stillage from sugarcane for biogas production (CH4 ), located at the São Martinho distillery plant (Pradópolis, SP, Brazil). The inoculum was subjected a heat-shock to inhibit H2 -consuming microorganisms (Maintinguer et al. 2011). Modified Del Nery medium was used in the experiments following the composition described by Ratti et al. (2013). Glucose is readily found in a wide variety of industrial wastes and was used as the model substrate in two concentrations: 0.5 and 2.0 g l−1 . Experimental procedures The experiment was carried out in triplicate in 5.0 l batch reactors. The reactors were inoculated with inoculum (10% v/v) and the appropriate amount of substrate in a final working volume of 1.0 l. The initial pH of the solution was adjusted at 7.0 with HCl and NaOH. Nitrogen (N2 ) gas was flushed into the reactors to create anaerobic culture conditions. The reactors were covered with rubber stoppers, closed with plastic stoppers and incubated at 55 ◦ C. During the experiments, the liquid samples were collected for the analysis of pH, carbohydrate consumption, and soluble metabolite composition. Biogas production was simultaneously monitored over time. Analytical methods The hydrogen content in the biogas was determined by gas chromatography (GC) using a GC 2010 Shimadzu system (Maintinguer et al. 2011; Ratti et al. 2013, 2014). The concentrations of organic compounds, such as volatile acids and alcohols, in the liquid phase were determined by high-performance liquid chromatography (HPLC) (Penteado et al. 2013). The concentration of total volatile solids (TVS) was determined according to standard methods for the examination of water and wastewater (APHA 2012). The soluble carbohydrate concentration was determined indirectly by the phenol–sulfuric acid method using glucose as a standard (Dubois et al. 1956). Kinetic analysis The experimental data were fitted to the mean values of the triplicate sets of reactors using the software Statistica® 8.0. The cumulative hydrogen production (H) data were fitted to the modified Gompertz model (Eq. (1)), which is suitable for describing the cumulative biogas production in batch experiments (Ratti et al. 2013, 2014). In this equation, H represents the hydrogen evolution data, t is the period of incubation (h), P is the hydrogen production
Reactors
P (mmol)
Rm (mmol/h)
H2 yield
0.5 g l−1 glucose 2.0 g l−1 glucose
4.2 13.3
0.24 0.04
20 41.5
1.3 1.6
potential (mmol), Rm is the maximum hydrogen production rate (mmol/h), is the length of the lag phase (h) and e is 2.71. H = P · exp
− exp
R · e m P
( − t) + 1
(1)
Sequencing processing and bacterial population analysis The sequences obtained were processed using the Ribosomal Database Project (RDP-Pyrosequencing Pipeline) (http://pyro.cme. msu.edu/index.jsp) (Cole et al. 2009). First, the sequences were trimmed to remove adaptor, barcodes, primers and sequences shorter than 200 bases. Chimeras introduced in the PCR process were removed using the DECIPHER program (http://decipher.cee.wisc.edu/index.html; Wright et al. 2012). The processed and trimmed sequences were aligned using the 210 secondary-structure aware Infernal aligner-RDP. Similar sequences were clustered into Operational Taxonomic Units (OTUs) based on 97% similarity using Hierarchical clustering. The taxonomy assignment was performed by the RDP classifier (Cole et al. 2009) using one representative sequence for each OTU. Rarefaction curves were constructed based on the OTUs phylogenetic domain. The relative abundance of bacterial taxons was analyzed in each sample, according to Chapleur et al. (2014). The diversity (Shannon and Simpson) and richness (Chao 1) indices were generated using Past software. The sequences were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk) under accession numbers ERS553966 (0.5 g l−1 glucose) and ERS553967 (2.0 g l−1 glucose); the project number is PRJEB7362. Results and discussion H2 production and organic acids In reactors fed with 0.5 and 2.0 g l−1 glucose, the generation of H2 after 20 h (2.1 mmol H2 ) and 23 h (0.21 mmol H2 ) was observed, and the maximum value was observed at 60 h (4.7 mmol H2 ) and 162 h (14.3 mmol H2 ) of incubation (Fig. 1). In the reactors, increased H2 production was observed with increased glucose concentration due to increased availability of substrate. Values of potential production of H2 (P), maximum rate of production of H2 (Rm ), lag phase time () and H2 yield are shown in Table 1.
12
R.P. Ratti et al. / Microbiological Research 173 (2015) 10–17
H2 Production (mmol)
16
Table 2 Results of conditions studied.
14 12 10 8 6 4 2 0
0
20
40
60
80 100 120 140 160 180 200 220 240 Time (h)
Fig. 1. H2 production. () A reactor fed with 0.5 g l−1 glucose; (䊉) a reactor fed with 2.0 g l−1 glucose.
The maximum hydrogen production potential (P) and H2 yield increased with increasing glucose concentration. P was 13.36 and 4.22, and H2 yield was 1.6 and 1.3 mol H2 mol glucose−1 for assays fed with 2.0 and 0.5 g l−1 glucose, respectively. The periods of lagphase were similar to others studies that utilized glucose as the substrate (Wang and Wan 2008). In the reactor fed with 0.5 g l−1 glucose, was the lowest (20 h), as the lower amount of substrate favored rapid growth of microorganisms. The lag phase is influenced by initial pH in assays of H2 production (Kapdan and Kargi 2006). In the present study, the initial pH was 7.0 and was verified longer lag phases (Table 1). Longer lag phases (20 h) were detected at low initial pH of 4.0–4.5 (Khanal 2004). According to Zhang et al. (2003), high initial pH (9.0) reduces lag time leading to lower H2 yields. The H2 yield obtained in the present study is comparable to reported values of hydrogen-producing enrichment cultures (1.7 mol H2 mol glucose−1 ) (Lin and Chang 1999). In contrast, higher yields of H2 were observed by Morimoto (2004) and Yokoyama et al. (2009) in thermophilic and extreme thermophilic conditions. The H2 yields were 2.1 mol H2 mol glucose−1 (Morimoto 2004) and 2.63–3.32 mol H2 mol glucose−1 (Yokoyama et al. 2009), respectively. Probably, higher yields were related to the type of inoculum used. Furthermore, Yokoyama et al. (2009) verified the predominance of Caldanaerobacter subterraneus in the inoculum that led to high H2 yields. According to Lin and Chang (Lin 2004), the H2 yield could be less than 1.7 mol H2 mol glucose−1 when more than 95% glucose was degraded. In the present study, glucose was linearly degraded, and at the end of the assay, 100% of the glucose was degraded. After 40 h and 100 h of the assay, the degradation of the substrate was high and the production of H2 stabilized in the reactor fed with 0.5 g l−1 of glucose and 2.0 g l−1 glucose, respectively. Using TVS analysis, it was verified that the increase in the substrate concentration was favorable for microorganism growth. The TVS values were 0.8 and 1.2 mg l−1 for assays with 0.5 and 2.0 g l−1 glucose, respectively (Table 2). Increasing the substrate concentration favored the development of microorganisms and resulted in an increase in H2 production. The pH was not controlled in these batch cultures, and under both conditions, a reduction of pH to 4.7 and 3.8 for assays fed with 0.5 and 2.0 g l−1 glucose, respectively, was observed (Table 2). The decrease in pH was due to production of organic acids during fermentation. The decrease in pH inhibits the H2 production (Dabrock et al. 1992). In the present study, the production of hydrogen ceased at low pH; the assays were started at neutral pH and terminated at acidic pH values. The optimum pH for H2 production was observed
Parameters analyzed
0.5 g l−1 of glucose
2.0 g l−1 of glucose
Degradation of glucose (%) TVS (g l−1 ) Initial pH Final pH Acetic acid (mmol) Butyric acid (mmol) Iso-butyric acid (mmol) Lactic acid (mmol) Propionic acid (mmol) H2 yield (mol H2 mol glucose−1 )
100 0.8 7.0 4.7 1.8 1.08 8.8 0.08 2.4 1.3
100 1.2 7.0 3.8 5.4 1.8 7.8 0.8 2.6 1.6
in the pH range of 5–6 (Ueno et al. 2001). Ratti et al. (2013) reported that the maintenance of an optimal pH range (5.5–6.5) was necessary to avoid the rapid inhibition of fermentation. The intermediate products obtained from the reactors with glucose are illustrated in Fig. 2. The main volatile fatty acid in the fermentation broth was isobutyric acid in all the assays. Low amounts of lactic and propionic acids were produced from glucose. Butyric, isobutyric and acetic acids constituted more than 75% of total end products. The production of these organic acids favors the H2 production, according to Eqs. (2) and (3). Theoretically, 4 mol and 2 mol of H2 are produced from 1 mol of glucose when acetate and butyrate are the main fermentation products, respectively (Ratti et al. 2014; Santos et al. 2014). C6 H12 O6 + 2H2 O → 4H2 + 2CO2 + 2 C2 H4 O2
(2)
C6 H12 O6 → 2H2 + 2CO2 + 2 C4 H8 O2
(3)
At the end of the test of the reactor containing 0.5 g l−1 glucose, the percentages of acetic, butyric, isobutyric, lactic and propionic acids were 12.72%, 7.6%, 62.23%, 0.56% and 16.87%, respectively. For the reactor containing 2.0 g l−1 glucose, the percentages of acetic, butyric, isobutyric, lactic and propionic acids were 27.73%, 9.2%, 40%, 4.6%, and 18.5%, respectively. In both reactors, a predominance of iso-butyric acids was observed. However, in the reactor fed with 2.0 g l−1 glucose, an increase in acetic acid (5.4 mmol) was observed, explaining the higher H2 yield (Table 2). According to Kapdan and Kargi (2006), the production of butyric acid rather than acetic acid may be one of the reasons for lower H2 yields (see Eqs. (2) and (3)). pH may also affect the type of organic acids produced and the H2 yield. pH between 4.0 and 6.0 favored the production of butyric acid (Fang and Liu 2002). In the present study, the production of H2 occurred preferentially via butyric acid (isobutyric acid), which was evident because the concentration of this acid was higher. Several previous studies have indicated a predominance of butyric acid in assays for H2 production using glucose (Fang and Liu 2002; Morimoto 2004). Probably, the substrate concentration also affected the production of organic acids and the H2 yield. In Table 2 was possible verify that the total acetic acid concentration increased in reactors fed with 2.0 g glucose where was observed major H2 yield. Bacterial community compositions and diversities Pyrosequencing produced a total of 7806 (average length = 273 ± 1.6 bp) and 4318 (average length = 272 ± 2.1 bp) bacterial 16S rDNA sequences with high coverage of diversity (98–98.2%). These sequences were assigned to 323 and 247 OTUS for the reactors with 0.5 and 2.0 g l−1 glucose, respectively. According to the RDP-Classifier, 95.7–99.9% of the sequences were classified into a phylum and 76.4–93.6% into a genus. The sequences were affiliated into 5 and 7 phyla and 35 and 30 genera for the reactors fed with 0.5 and 2.0 g l−1 glucose, respectively. In the two conditions, the most prevalent phylum was Firmicutes
R.P. Ratti et al. / Microbiological Research 173 (2015) 10–17
Fig. 2. Production of organic acids ( acetate, glucose.
butyrate,
iso-butyrate,
lactate,
(68.73–67.61%). According to the literature, this phylum encompasses the main microorganisms that produce H2 (Ratti et al. 2013; Santos et al. 2014). The diversity and richness of the microbial consortium were analyzed (Table 3). The microbial community in the reactor with 0.5 g l−1 glucose contained high diversity and richness according to the Simpson index (0.99) and the Shannon index (5.25). The community composition was composed of 323 operational taxonomic units (OTUs) with a predominance of the phylum Firmicutes (68.73%). Members of the Acidobacteria, Proteobacteria and Verrucomicrobia were present at an abundance of
