Advanced
Identification and Characterization of an Anaerobic Ethanol-Producing Cellulolytic Bacterial Consortium from Great Basin Hot Springs with Agricultural Residues and Energy CropsS
Identification and Characterization of an Anaerobic Ethanol-Producing Cellulolytic Bacterial Consortium from Great Basin Hot Springs with Agricultural Residues and Energy CropsS
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1280-1290
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : January 13, 2014
  • Accepted : May 06, 2014
  • Published : September 30, 2014
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Chao Zhao
National Engineering Research Center of Juncao, Fuzhou, Fujian 350002, P. R. China
Yunjin Deng
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P. R. China
Xingna Wang
Sanya Institute of Deep-Sea Science and Engineering, Chinese Academy of Sciences, Sanya, Hainan 572000, P. R. China
Qiuzhe Li
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P. R. China
Yifan Huang
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P. R. China
zjhyfang@163.com
Bin Liu
National Engineering Research Center of Juncao, Fuzhou, Fujian 350002, P. R. China
zjhyfang@163.com

Abstract
In order to obtain the cellulolytic bacterial consortia, sediments from Great Basin hot springs (Nevada, USA) were sampled and enriched with cellulosic biomass as the sole carbon source. The bacterial composition of the resulting anaerobic ethanol-producing celluloytic bacterial consortium, named SV79, was analyzed. With methods of the full-length 16S rRNA library-based analysis and denaturing gradient gel electrophoresis, 21 bacteria belonging to eight genera were detected from this consortium. Clones with closest relation to the genera Acetivibrio , Clostridium , Cellulosilyticum , Ruminococcus , and Sporomusa were predominant. The cellulase activities and ethanol productions of consortium SV79 using different agricultural residues (sugarcane bagasse and spent mushroom substrate) and energy crops ( Spartina anglica , Miscanthus floridulus , and Pennisetum sinese Roxb) were studied. During cultivation, consortium SV79 produced the maximum filter paper activity (FPase, 9.41 U/ml), carboxymethylcellulase activity (CMCase, 6.35 U/ml), and xylanase activity (4.28 U/ml) with sugarcane bagasse, spent mushroom substrate, and S. anglica , respectively. The ethanol production using M. floridulus as substrate was up to 2.63 mM ethanol/g using gas chromatography analysis. It has high potential to be a new candidate for producing ethanol with cellulosic biomass under anoxic conditions in natural environments.
Keywords
Introduction
The world economy has been largely depending on fossil energy resources such as oil, coal, and natural gas. There has been significant research on renewable sources of liquid fuels to replace fossil fuels, to improve energy security and reduce greenhouse emissions. Nowadays, bioethanol is gaining worldwide acceptance, essentially to overcome problems associated with exploitation and depletion of fossil fuels and environmental pollution [23] . Bioethanol has been receiving widespread interest and the global bioethanol market has entered the rapid growth phase [30] . Bioethanol produced from renewable biomass, such as agricultural residues, energy crops, or cellulosic materials, is one of the alternative energy resources that are both renewable and environmentally friendly [20 , 39] . It is indigenous and can therefore contribute to reducing dependency on oil imports and increasing the security of supply.
Ethanol production using easily available substrates and microbes that could convert them to ethanol could be very useful. The debate of potential conflict between food and fuel will continue unless the feedstock for the biofuel shifts from major crops to production of second generation biofuels, which uses waste materials and nonfood crops that can be grown on marginal land as feedstocks [52] . Agricultural processes yield by-products and waste streams collectively known as residues, such as sugarcane bagasse and spent mushroom substrate, which yield significant energy potential and are relatively conducive to utilization because they have already been collected [13] . Moreover, perennial herbaceous crops are a huge potential resource for producing fuel ethanol. The term energy crop is used to principally describe a crop grown primarily to provide a natural cellulosic feedstock for biofuels such as ethanol.
Cellulosic biomass, available in enormous quantities, is the feasible replacement and a renewable resource with great potential for bioconversion to value-added byproducts [10 , 17] . However, the biorefining process remains economically unfeasible owing to a lack of biocatalysts that can overcome costly hurdles, such as cooling from high temperature, pumping of oxygen/stirring, and neutralization from acidic or basic pH [33] . The bottleneck of cellulosic ethanol production is how to convert cellulose into glucose effectively [3] . Highly efficient cellulase is a key factor for cellulosic biomass conversion into biofuel and for biorefinery. Screening and isolation of microbes from the thermophilic environment help to overcome these challenges [7 , 36] . Many cellulose-decomposing strains were isolated based on cultivation and pure culture. However, the isolated bacterium achieved by high dilution principle cannot represent the natural bacterial consortium, and most of the microbial species in nature cannot be cultured. The functions of uncultured bacteria would be neglected. Therefore, the bacterial consortia rather than cultured bacteria have provided more information about how bacteria play their roles. The exploitation of a bacterial consortium in the search for improved strategies provides a means to upgrade the feasibility for biomass conversion [33] .
In this study, an anaerobic ethanol-producing cellulolytic bacterial consortium, named SV79, was isolated from Great Basin hot spring (Nevada, USA). Its morphological characterization and microbial structure were studied. Structure dynamics of the consortium was analyzed by denaturing gradient gel electrophoresis (DGGE) to assess its stability during the cellulose degradation procedure, and the microbial diversity was checked by construction of a 16S rRNA gene library. The enzyme fermentation characteristics of SV79 and the ability of the consortium in using different agricultural residues and energy crops were also determined.
Materials and Methods
- Samples and Medium
The water and sludge sediment samples were collected from Great Basin hot springs in Nevada, USA (41D 31’57.2”N, 120D 04’15.7”W- 41D 32’03.4”N, 120D 04’23.9”W). The water temperatures of the hot springs were between 40℃ and 78℃. The Z-AECC medium for enrichment and subcultivation of the anaerobic cellulolytic consortium was composed of the following (per liter): KH 2 PO 4 0.75 g, K 2 HPO 4 1.5 g, MgCl 2 ·6H 2 O 0.4 g, NH 4 Cl 0.9 g, NaCl 0.9 g, yeast extract 0.6 g, peptone 2.0 g, L-cysteine 0.5 g, carbon source 10.0 g, 10% FeSO 4 0.3 ml, 1% Resazurin 1 ml, Wolfe's mineral solution 9 ml, and Wolfe’s vitamin solution 1 ml [51] . The agricultural residues (sugarcane bagasse and spent mushroom substrate) and energy crops ( Spartina anglica , Miscanthus floridulus and Pennisetum sinese Roxb) were used as the carbon sources. The whole operation for enrichment and subcultivation of microbial consortia was performed under anoxic conditions in a Forma 1029 Anaerobic Chamber (Thermo Scientific, Marietta, USA) with the gas phase N 2 /CO 2 /H 2 = 85:5:10. All culturing manipulations were performed as described by Hungate [24] .
- Consortium Enrichment and Characteristics of the Bacterial Consortium
For isolation, approximately 5.0 g of the environmental sample at each site was transferred to fresh 100 ml of the Z-AECC medium containing filter paper as the sole carbon source in 125 ml serum bottles, for incubation at 40-78℃ for 24-72 h without shaking. Five milliliters of the suspension was inoculated into 100 ml of Z-AECC medium and incubated statically for one week. A volume of 0.5 ml of each positive culture, in which filter paper was completely decomposed, was subinoculated into 5 ml of Z-AECC medium in Hungate’s tubes. Positive samples were continuously subinoculated using fresh Z-AECC medium in order to construct stable cellulolytic consortia. A stable cellulolytic bacterial consortium, SV79, was enriched after subcultivation for seven times. For evaluation of the cellulose degradation efficiency of the consortium, the culture of SV79 was inoculated into seven Hungate’s tubes with Z-AECC medium. Time-course degradation dynamics of filter paper were observed on each day. The reducing sugars were liberated in the reaction mixture. Fermentation characteristics of the reducing sugar production of the microbial consortium were determined. The effect of carbon source, time, temperature, and initial pH values on the reducing sugar production of the bacterial consortium was studied. The temperature ranges were determined from 30℃ to 70℃ with 5℃ interval. The pH dependence was tested from pH 4.0 to 11.0. The reducing sugar yield in the cultural process was determined with the 3,5-dinitrosalicylic acid colorimetric method [35] . Reducing sugar yield (%) was represented as the gram amount of reducing sugars per 10.0 g of carbon substrates. Furthermore, cell morphologies of the species from consortium SV79 were also examined by transmission electron microscopy (JEM-1230, JEOL, Peabody, MA, USA) after cultivation under the optimal temperature and pH value [39] .
- Structure Dynamics of the Microbial Consortium
Total genomic DNA of the consortium from the one-week-incubation cultures was extracted by the SDS-CTAB method as previously described [50] . PCR primers GC341F (5’-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCGCCTACGGG AGGCAGCAG-3’; containing 40 bp of GC clamp) and 534R (5’-ATTACCGCGGCTGCTGG-3’) were used to amplify the variable V3 region of bacterial 16S rRNA [47] . The PCR procedure included an initial cycle of 5 min at 94℃, followed by 20 cycles of 1 min at 94℃, 45 sec at 65℃, and 1 min at 72℃, with a touchdown of 0.5℃ per cycle, followed by 16 cycles of 1 min at 94℃, 45 sec at 55℃, and 1 min at 72℃; for the final extension, the reaction was held at 72℃ for 10 min. The PCR products of the consortium were loaded on the same gel for DGGE analysis with a DCode Universal Mutation Detection System (Bio-Rad, USA). Electrophoresis was performed at 60℃ in an 8% (w/v) polyacrylamide gel with a denaturant gradient ranging from 30% to 60% for 15 min at 30 V, and then 4.5 h at 130 V in 1× Tris-acetate-EDTA buffer. After electrophoresis, the gel was stained with ethidium bromide for 15 min, and patterns were visualized and photographed using the Gel Doc XR system (Bio-Rad, USA). The band patterns on gels were checked to analyze the dynamics of the microbial consortium structures in the incubation process of one week and in the procedure of subcultivation. The dynamics of the microbial structure of consortium SV79 in different biomass substrates were also analyzed by DGGE gels.
- Diversity Analysis of the Microbial Consortium
The 16S rRNA library of the microbial consortium was constructed [34] . Nearly complete 16S rRNA gene sequences were amplified by PCR from genomic DNA of the microbial consortium using universal primers 27F and 1492R [48] . They were purified and ligated into pMD18-T (TaKaRa, Japan). The ligation mixture was transformed into E. coli DH5α (TaKaRa, Japan) to generate a gene library. More than 300 clones were selected randomly and checked for correct insert size via PCR and agarose gel electrophoresis, and then the inserted fragments were sequenced. Pairwise alignments were carried out using the EzTaxon-e program [26] . To test the evolutionary relationships, phylogenetic analysis was performed with the program MEGA 4.0 [43] . Multiple alignments of the sequences were performed using CLUSTAL W [44] . Distance matrices were calculated according to the Kimura’s two-parameter correction model [27] . Phylogenetic trees were inferred using neighbor joining. Bootstrap values were determined based on 1,000 replications.
- Cellulase and Ethanol Production Abilities of the Microbial Consortium
The consortium SV79 was grown in the Z-AECC medium under the optimum fermentation condition, in which the carbon source was replaced by agricultural residues (sugarcane bagasse and spent mushroom substrate) and energy crops ( S. anglica , M. floridulus , and P. sinese Roxb). The enzymatic activities were assayed by filter paper cellulase (FPase) and carboxymethylcellulase (CMCase) methods and determined using the IUPAC standard procedure [21] . Hemicellulose is the second most abundant renewable polysaccharide after cellulose. Xylan is the main component of hemicellulose. The assay to measure xylanase activity was also performed according to the method of Liu et al . [31] . One unit (U) of enzyme activity was defined as the amount of enzyme releasing 1 μg reducing sugar per 1 min under the assay conditions using glucose as a standard. Ethanol production was analyzed with gas chromatography (Agilent 6280) equipped with a flame ionization detector (FID) and HP-INNOWAX columns (0.25 mm × 30 m × 0.25 m; Agilent).
Results
- Isolation and Analysis of the Anaerobic Cellulolytic Consortium SV79
After incubation in Z-AECC medium for one week under anaerobic static conditions, five cultures showed the capability of decomposing filter paper. These positive cultures were chosen for successive subcultivation using the same medium. After subcultivation for seven times, consortium SV79, which exhibited the highest efficiency on filter paper degradation, was chosen for further study. Time-course dynamics of cellulose degradation by consortium SV79 was determined using filter paper as the substrate (Z-AECC medium) ( Fig. 1 A). After a 2-3 day incubation period in the medium, it was observed that small visible spots started to appear on the filter paper and the whole filter paper shattered. The filter paper was obviously decomposed after incubation for 4 days, and almost completely degraded after 7 days, which implied SV79’s high efficiency for cellulose degradation. Filter paper could be degraded efficiently even after successive subcultivation for seven times, but more than one week was needed for complete decomposition. Moreover, the different strains that developed in the medium were observed. The bacteria group consisted of both gram-positive and gram-negative bacteria. Differences in shapes were observed. The electron microscopy results revealed three forms of bacteria, including rod, spherical, and arc shapes, in consortium SV79 ( Fig. 1 B). The flagella were also seen on the electron micrographs of the cells a , b , and d .
PPT Slide
Lager Image
Characteristics of the anaerobic cellulolytic consortium SV79. (A) Time-course degradation of filter paper by consortium SV79. ck, the control Z-AECC medium that was not inoculated with consortium SV79; 1 -7 d, the Z-AECC media incubated for 1-7 days after being inoculated with consortium SV79. (B) Scanning electron micrographs of consortium SV79. B-a and B-d show an arc with the flagellum; B-b had a flagellum, of length about 5 μm; B-c was about 0.5 μm in diameter size, of globular shape; B-e and B-f show rod bacteria, with no flagellum. The black line for length indicates 1 μm for reference.
Four factors (carbon source, time, temperature, and initial pH) that are known to influence fermentation processes were assayed by measuring the amount of reducing sugars released by the bacterial consortium. The fermentation conditions of the conversion from cellulose to sugar were determined. The optimal fermentation time, fermentation temperature, and pH were 7 days, 42.5℃, and 8.0, respectively ( Fig. 2 ). The growth curve of bacterial consortium SV79 was not identical to the standard growth curve, in which no obvious death phase of bacterial consortium SV79 was observed (data not shown).
PPT Slide
Lager Image
Effects of carbon substrates (A), time (B), temperature (C), and initial pH values (D) on the fermentation of consortium SV79. Reducing sugar yields (%) from 1.0% carbon substrates were tested at pH 7.0, 40℃ for 7 days (A). FP-filter paper, ScB-sugarcane bagasse, SMS-spent mushroom substrate, SA-Spartina anglica, MF-Miscanthus floridulus, and PSR-Pennisetum sinese Roxb. Reducing sugar yields (%) from 1.0% filter paper were tested at pH 7.0, 40℃ for varying lengths of time (B), at various temperatures ranging from 30℃ to 70℃ at pH 7.0 for 7 days (C), and in different pH buffers at 40℃ for 7 days (D).
- Microbial Diversity of Consortium SV79
The time-course dynamics of the microbial structure of consortium SV79 were analyzed by DGGE ( Fig. 3 ). More than 20 bands were found on the gels through the whole cultivation procedure, indicating a complex microbial consortium. Moreover, the DGGE patterns of each gel lane corresponding to each-day samples of the 7th subcultivation were not similar and differences were found. Several weak bands among these samples were changing. However, the main bands of each sample, which stood for the dominant members of the consortium, were almost the same. The dynamics of the microbial structure of consortium SV79 in five different biomass substrates were analyzed by DGGE. The proportion of bacteria in the consortium was a little different by each biomass substrate. However, the main bands were bands 1-5 and remained stable (Fig. S1). Even for a stable community, the proportion of bacteria was also changed in different stages and medium composition. The 16S rRNA clone library of bacterial consortium SV79 was constructed. Subsequently, the different isolates of the 16S rRNA library were subjected to DNA sequencing. The nucleotide sequence data reported in this work will appear in the GenBank nucleotide database under accession numbers JQ316682 to JQ316694 and JQ694703 to JQ694710 ( Table 1 ). As their closest taxonomic relatives were almost <97%, all the clones likely represented new bacterial species. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates belonged to four different families: Ruminococcaceae, Clostridiaceae, Lachnospiraceaea, and Veillonellaceae. Among the detected bacteria in the consortium SV79, the following genera occurred: Acetanaerobacterium , Acetivibrio , Anaerotruncus , Bacteroides , Cellulosilyticum , Clostridium , Ruminococcus , and Sporomusa ( Fig. 4 ). Among the 21 clones, six clones belonged to Clostridium , and five clones belonged to Acetivibrio , while Cellulosilyticum and Ruminococcus had four and two clones, respectively. At the genus rank, Clostridium (70% of the consortium) was prevalent in this community, containing the abundant bacterial species C. straminisolvens . In addition, there were also the following genera: Ruminococcus (12%), Acetivibrio (10%), and Cellulosilyticum (4%).
PPT Slide
Lager Image
Time-course dynamics of the microbial structures of consortium SV79 within 7 days, performed by DGGE using the V3 regions of the 16S rRNA gene. 1, 3, 5, and 7 d, the samples of consortium SV79 incubated for 1, 3, 5, and 7 days.
Bacterial isolates from consortium SV79 enriched with cellulosic biomass.
PPT Slide
Lager Image
Bacterial isolates from consortium SV79 enriched with cellulosic biomass.
PPT Slide
Lager Image
Phylogenetic analysis of the isolates from consortium SV79. Total genomic DNA of the consortium from the one-week-incubation cultures with filter paper at pH 7.0, 42.5℃ was extracted. The phylogenetic tree was constructed based on 16S rRNA gene sequences. Reference strains used in the phylogenetic tree were retrieved by the database accession number in parentheses. The numbers at the branch nodes are bootstrap values based on 1,000 resamplings for maximum likelihood. The scale bar equals approximately 10% nucleotide divergence.
- Cellulase and Ethanol Production Ability of the Microbial Consortium
The abilities of consortium SV79 to utilize agricultural residues (sugarcane bagasse and spent mushroom substrate) and energy crops ( S. anglica , M. floridulus , and P. sinese Roxb) were researched ( Fig. 5 A). The highest FPase activity, CMCase activity, and xylanase activity were 9.41, 6.35, and 4.28 U/ml, using sugarcane bagasse, spent mushroom substrate, and S. anglica as substrate, respectively. Meanwhile, the ethanol production by consortium SV79 was also analyzed in Z-AECC medium by gas chromatography ( Fig. 6 ), in which different natural cellulose biomasses were the sole carbon source. As shown in Fig. 6 , the retention time for ethanol in all tested materials was 1.70 min, approximately. The peaks at about 6.5, 8.0, and 8.7 min in the chromatogram corresponded to the peaks of acetic acid, propionic acid, and butyric acid (data not shown). Ethanol yields were 2.63 mM ethanol/g M. floridulus , which was the highest, and 2.13 mM ethanol/g spent mushroom substrate, which was the lowest ethanol yield ( Fig. 5 B).
PPT Slide
Lager Image
Effects of different cellulosic substrates on the cellulase production (A) and ethanol production (B) of consortium SV79. FP-filter paper, ScB-sugarcane bagasse, SMS-spent mushroom substrate, SA-Spartina anglica, MF-Miscanthus floridulus, and PSR-Pennisetum sinese Roxb.
PPT Slide
Lager Image
GC chromatograms of ethanol production by consortium SV79 with different cellulosic substrates. FP-filter paper, ScB-sugarcane bagasse, SMS-spent mushroom substrate, SA-Spartina anglica, MF-Miscanthus floridulus, and PSR-Pennisetum sinese Roxb.
Discussion
A bacterial consortium usually has advantage over a single bacterium in the biodegradation of carbohydrate and crude fiber. Enrichment of a microbial consortium in natural environments provides an approach to simulate and investigate cellulose degradation. Several cellulolytic consortia have been enriched from composts, soils, decayed straws, etc . [42 , 46 , 54] . These studies mainly focused on microbial communities in terrestrial ecosystems. However, in an actual production process, the high unit cost of cellulases has limited their application to ethanol production, stimulating a demand for more cost-effective enzymes that would make bioethanol an economically feasible alternative to fossil fuels [1] . Thermostable cellulases offer several potential benefits to mitigate the high costs of enzymatic saccharification of lignocellulose. A number of studies have focused on the isolation and characterization of cellulolytic thermophiles as possible sources of cellulases for the biofuels industry [7] . Therefore, hot springs are a potential source for thermophilic cellulases and ethanolproducing microorganisms [28 , 40] .
In this report, hot spring samples were collected from Great Basin in Nevada, USA, and a stable anaerobic cellulolytic consortium, SV79, was enriched. The consortium showed high efficiency for cellulose degradation and maintained the cellulolytic ability after seven successive subcultivations. 16S rRNA sequencing was a preferable method, which can be used to detect each kind of bacterium in a consortium and directly show the proportion of different bacteria in the consortium [56] . Sequence alignments and phylogenetic analyses performed with type species of validly published genera showed that most of the 16S rRNA sequences of bacterium consortium SV79 had a lower similarity with uncultured microorganisms. A large number of novel microbial strains were contained in the bacterium consortium SV79. The phylogenetic tree revealed that the bacterium consortium SV79 consisted of the Ruminococcaceae, Clostridiaceae, Lachnospiraceae, and Veillonellaceae families. These four families had a relationship with lignocellulosic biomass degradation and ethanol production. Moreover, PCR-DGGE analysis further revealed the changes of its microbial community structure in the incubation process of 7 days. The result implied that the strains of consortium SV79 performed different functions at different stages and coexisted and took effect in degrading cellulose.
So far, a variety of microorganisms have been found to produce biofuels from fermentation of an organic complex, including obligate anaerobes such as Acetanaerobacterium , Acetivibrio , Bacteroides , Clostridium , Cellulosilyticum , Ruminococcus , and Sporomusa species. The strains from Acetanaerobacterium produced ethanol, H 2 gas, and acetic acid from glucose fermentation [12] . Cellulolytic strains of Bacteroides were relatively slow in attacking easily degraded cellulose [6] . However, the strains of Anaerotruncus colihominis would be unable to degrade cellulose [29] . Acetivibrio cellulolyticus demonstrated its cellulose-degrading activities [16] . Another related genus, Ruminococcus , was the dominant rumen bacteria [38 , 55] . Bacteria in the genus Clostridium were important members of cellulolytic microorganisms. Clostridium was well known as a typical anaerobic cellulolytic species. Meanwhile, Clostridium was accompanied by the formation of cellulosic ethanol in the process of degradation [25] and became the predominant bacteria with the extension of fermentation. At least two species, Clostridium thermocellum and C. acetobutylicum , have demonstrated the ability of fermenting sugars. C. thermocellum made use of crystalline cellulose to produce sugar and then as its carbon source [22] . A thermophilic anaerobic cellulose-degrading bacterium, C. straminisolvens was also isolated and had the ability of converting cellulose to produce ethanol under high temperature conditions [41] . Cellulosilyticum ruminicola was a novel strain and able to take advantage of a variety of lignocellulosic growth, with the degradation of hemicellulose and lignin [8] . Among consortium SV79 bacteria, the Sporomusa also could contribute to produce ethanol through degrading by-products of cellulose conversion. Balk et al . [5] isolated Sporomusa sp. An4 from the underground gas storage reservoir, which was later identified as a mesophilic bacterium and had the ability to convert various alcohols. Hence, the various strains of consortium SV79 were suitable for the production of hydrolytic enzymes for biomass material degradation.
The negative consequences of existing biofuel technologies have stimulated interest in the development of so-called “second-generation” biofuel technologies, which derive fermentable sugars from crops or lignocellulosic waste produced by agriculture, forestry, and other industries. However, only limited information is available concerning agricultural residues and energy crops degradation [9] . Agricultural residues, which are primarily composed of stalks, leaves, and straws, contain approximately 10-20% lignin, 40-50% glucan, and 15-35% xylan, making them good candidates for bioethanol production through proper pretreatment, hydrolysis, and fermentation. The celluloserich sugarcane bagasse and spent mushroom substrate have been widely investigated for their potential for producing commercial quantities of cellulosic ethanol [2 , 10 , 18] .
The energy crops are the types of plants expected to be cultivated as raw materials for the production of the biofuels such as ethanol. Plants with a fast growing rate, such as grasses or shrubs, should represent the appropriate energy crops for the future. Grasses are targeted as potential energy crops because of their high productivity per hectare, abundance, availability, and utilization of the whole plant. Fibers and storage carbohydrates within some species of grass can be used as substrates to produce ethanol, whereas the species of grasses that contain high amounts of proteins can be used as nitrogenous waste for biorefineries [15] . Three species of energy crops ( S. anglica , M. floridulus , and P. sinese Roxb) with potential value to produce biofuels were selected [32] . They were the most suitable energy utilization index for fuel ethanol. So far, several Miscanthus species have been studied for possible production of second-generation biofuels in a number of laboratories [14 , 53] . These plants are perennial crops, considered suitable as feedstock for lignocellulosic ethanol production because of high yields, low costs, fit for infertile land, and less environmental impacts [4] .
Some studies showed FPase and CMCase played the major roles in crude fiber biodegradation [19] . From analyzing the enzymatic activity of FPase, it was found that the FPase activity obtained with the agricultural residues and energy crops was higher than the CMCase and xylanase activities obtained. The Z-AECC cultures with sugarcane bagasse had greater FPase activity than the other results had reported [9] . The FPase activities were most obvious in this study. Ethanol-producing bacteria have gained much attention in recent years because of increased interest in renewable energy sources. The highest ethanol yield reported is by the thermophilic bacterium T. ethanolicus , with 1.9 mol-EtOH mol-glucose -1 , but several strains are capable of yields reaching 1.5 mol-EtOH mol-glucose -1 [49] . Several investigations on ethanol production by thermophilic bacteria from cellulosic biomass have been recently published. Clostridium thermocellum produced between 4.6 and 8.1 mM ethanol/g alkali-pretreated paddy straw, sorghum stover, and corn stubs [37] . The ethanol production using M. floridulus as substrate was up to 2.63 mM ethanol/g. The alkali treatments could be recommended for paddy straw for microcommunity-based conversions. After treatment, the net yields of reducing sugars were greatly enhanced [57] . Under pretreatment, the ethanol production of consortium SV79 would greatly increase.
Apart from ethanol, anaerobic ethanol-producing cellulolytic bacterial consortia may excrete a variety of metabolic products. These include polyalcohols, and monocarboxylic, dicarboxylic, and tricarboxylic acids [11] . In real applications, the analysis of such variety of reaction products by gas chromatography was difficult. During anaerobic fermentation by Clostridium , the production of ethanol or acetic acid was the major end products [45] . Detection of ethanol and acetic acid showed that Clostridium was the prevalent genus in this community. Although the productivity of ethanol by bacteria is still not better than fungus, bacteria possess other advantages, such as endurance of high temperature during the fermentation process, fast growth, and easy survival. The bacterial consortium SV79 has a good ability in a variety of cellulosic materials degradation and ethanol production with the cooperation of the four families.
In conclusion, a stable ethanol-producing cellulolytic anaerobic microbial consortium, SV79, was isolated using the anaerobic screening medium containing filter paper as the sole carbon source. The consortium could degrade cellulosic biomass efficiently under anaerobic condition. The novel new strains from consortium SV79 also provided a new insight into the microbial diversity of anaerobes in hot spring ecosystems. Acetivibrio , Cellulosilyticum , and Clostridium were detected as key degraders of agricultural residues and energy crops in the sediments of Great Basin hot springs. They and other potential degrading bacteria coexisted and took effect in degrading cellulosic biomass and reducing ethanol. This work would be useful for those involved in developing fuel ethanol production from lignocellulosic biomass. Consortium SV79 provides a candidate for investigating the mechanism of anaerobic cellulose degradation.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (31370146, 41306181 & 31071639), China Postdoctoral Science Foundation (2013M530303 & 2014T70602), Special Fund for Trial-Manufacture of Sanya City, China (2013KS08) and China National Engineering Research Center of Juncao Technology (JCGG1406).
References
Alizadeh H , Teymouri F , Gilbert T , Dale B. 2005 Pretreatment of switchgrass by ammonia fiber explosion (AFEX). Appl. Biochem. Biotechnol. 124 1133 - 1141    DOI : 10.1385/ABAB:124:1-3:1133
Balan V , da Costa Sousa L , Chundawat SP , Vismeh R , Jones AD , Dale BE. 2008 Mushroom spent straw: a potential substrate for an ethanol-based biorefinery. J. Ind. Microbiol. Biotechnol. 35 293 - 301    DOI : 10.1007/s10295-007-0294-5
Balat M. 2011 Production of bioethanol from lignocellulosic materials via the biochemical pathway: a review. Energy Convers. Manage. 52 858 - 875    DOI : 10.1016/j.enconman.2010.08.013
Balat M , Balat H , Öz C. 2008 Progress in bioethanol processing. Prog. Energ. Combust. Sci. 34 551 - 573    DOI : 10.1016/j.pecs.2007.11.001
Balk M , Mehboob F , van Gelder AH , Rijpstra WI , Damsté JS , Stams AJ. 2010 (Per)chlorate reduction by an acetogenic bacterium, Sporomusa sp., isolated from an underground gas storage. Appl. Microbiol. Biotechnol. 88 595 - 603    DOI : 10.1007/s00253-010-2788-8
Betian HG , Linehan BA , Bryant MP , Holdeman LV 1977 Isolation of a cellulolytic Bacteroides sp. from human feces. Appl. Environ. Microbiol. 33 1009 - 1010
Blumer-Schuette SE , Kataeva I , Westpheling J , Adams MW , Kelly RM. 2008 Extremely thermophilic microorganisms for biomass conversion: status and prospects. Curr. Opin. Biotechnol. 19 210 - 217    DOI : 10.1016/j.copbio.2008.04.007
Cai S , Li J , Hu FZ , Zhang K , Luo Y , Janto B 2010 Cellulosilyticum ruminicola, a newly described rumen bacterium that possesses redundant fibrolytic-protein-encoding genes and degrades lignocellulose with multiple carbohydrateborne fibrolytic enzymes. Appl. Environ. Microbiol. 76 3818 - 3824    DOI : 10.1128/AEM.03124-09
Camassola M , Dillon AJP. 2009 Biological pretreatment of sugar cane bagasse for the production of cellulases and xylanases by Penicillium echinulatum. Ind. Crops Prod. 29 642 - 647    DOI : 10.1016/j.indcrop.2008.09.008
Cardona CA , Quintero JA , Paz IC. 2010 Production of bioethanol from sugarcane bagasse: status and perspectives. Bioresour. Technol. 101 4754 - 4766    DOI : 10.1016/j.biortech.2009.10.097
Chaudhary N , Qazi JI. 2011 Lignocellulose for ethanol production: a review of issues relating to bagasse as a source material. Afr. J. Biotechnol. 10 1270 - 1274
Chen SY , Dong XZ. 2004 Acetanaerobacterium elongatum gen. nov., sp. nov., from paper mill waste water. Int. J. Syst. Evol. Microbiol. 54 2257 - 2262    DOI : 10.1099/ijs.0.63212-0
Chen Y , Sharma-Shivappa RR , Keshwani D , Chen C. 2007 Potential of agricultural residues and hay for bioethanol production. Appl. Biochem. Biotechnol. 142 276 - 290    DOI : 10.1007/s12010-007-0026-3
Chung JH , Kim DS. 2012 Miscanthus as a potential bioenergy crop in East Asia. J. Crop Sci. Biotechnol. 15 65 - 77    DOI : 10.1007/s12892-012-0023-0
Dale BE , Allen MS , Laser M , Lynd LR. 2009 Protein feeds coproduction in biomass conversion to fuels and chemicals. Biofuel. Bioprod. Bioref. 3 219 - 230    DOI : 10.1002/bbb.132
Dassa B , Borovok I , Lamed R , Henrissat B , Coutinho P , Hemme CL 2012 Genome-wide analysis of Acetivibrio cellulolyticus provides a blueprint of an elaborate cellulosome system. BMC Genomics 13 210 -    DOI : 10.1186/1471-2164-13-210
French CE. 2009 Synthetic biology and biomass conversion: a match made in heaven. J. R. Soc. Interface 64 547 - 558    DOI : 10.1098/rsif.2008.0527.focus
Geddes CC , Mullinnix MT , Nieves IU , Peterson JJ , Hoffman RW , York SW 2011 Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160. Bioresour. Technol. 102 2702 - 2711    DOI : 10.1016/j.biortech.2010.10.143
Geng A , Zou G , Yan X , Wang Q , Zhang J , Liu F 2012 Expression and characterization of a novel metagenomederived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei. Appl. Microbiol. Biotechnol. 96 951 - 962    DOI : 10.1007/s00253-012-3873-y
Ge X , Burner DM , Xu J , Phillips GC , Sivakumar G. 2011 Bioethanol production from dedicated energy crops and residues in Arkansas. USA Biotechnol. J. 6 66 - 73    DOI : 10.1002/biot.201000240
Ghose TK. 1987 Measurement of cellulase activities. Pure Appl. Chem. 59 257 - 268
Hernandez PE , Ordonez JA , Sanz B. 1985 Utilization of cellobiose and D-glucose by Clostridium thermocellum ATCC-27405. Rev. Esp. Fisiol. 41 195 - 199
Hill J , Nelson E , Tilman D , Polasky S , Tiffany D. 2006 Environmental, economic and energetic costs and benefits of biodiesel and ethanol biofuels. PNAS 103 11206 - 11210    DOI : 10.1073/pnas.0604600103
Hungate RE. 1969 A roll tube method for cultivation of strict anaerobes. Methods Microbiol. 3B 117 - 132
Kato S , Haruta S , Cui ZJ. 2004 Clostridium straminisolvens sp. nov., a moderately thermophilic, aerotolerant and cellulolytic bacterium isolated from a cellulose-degrading bacterial community. Int. J. Syst. Evol. Microbiol. 54 2043 - 2047    DOI : 10.1099/ijs.0.63148-0
Kim OS , Cho YJ , Lee K , Yoon SH , Kim M , Na H 2012 Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62 716 - 721    DOI : 10.1099/ijs.0.038075-0
Kimura M. 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16 111 - 120    DOI : 10.1007/BF01731581
Koskinen PEP , Lay CH , Beck SR , Tolvanen KE , Koksonen AH , Orlygsson J 2008 Bioprospecting thermophilic microorganisms from Icelandic hot springs for hydrogen and ethanol production. Energy Fuels 22 134 - 140    DOI : 10.1021/ef700275w
Lawson PA , Song Y , Liu C , Molitoris DR , Vaisanen ML , Collins MD , Finegold SM 2004 Anaerotruncus colihominis gen. nov., sp. nov., from human faeces. Int. J. Syst. Evol. Microbiol. 54 413 - 417    DOI : 10.1099/ijs.0.02653-0
Limayem A , Ricke SC. 2012 Lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects. Prog. Energ. Combust. Sci. 38 449 - 467    DOI : 10.1016/j.pecs.2012.03.002
Liu B , Zhang N , Zhao C , Lin BX , Xie LH , Huang YF. 2012 Characterization of a recombinant thermostable xylanase from hot spring thermophilic Geobacillus sp. TC-W7. J. Microbiol. Biotechnol. 22 1391 - 1397
Liu CC , Xiao L , Jiang JX , Wang WX , Gu F , Song DL 2013 Biomass properties from different Miscanthus species. Food Energy Security    DOI : 10.1002/fes3.19.
Maki M , Leung KT , Qin W. 2009 The prospects of cellulaseproducing bacteria for the bioconversion of lignocellulosic biomass. Int. J. Biol. Sci. 5 500 - 516    DOI : 10.7150/ijbs.5.500
McKew BA , Coulon F , Osborn AM , Timmis KN , McGenity TJ. 2007 Determining the identity and roles of oil-metabolizing marine bacteria from the Thames estuary. UK Environ. Microbiol. 9 165 - 176    DOI : 10.1111/j.1462-2920.2006.01125.x
Miller GL. 1959 Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31 426 - 428    DOI : 10.1021/ac60147a030
Peacock JP , Cole JK , Murugapiran SK , Dodsworth JA , Fisher JC , Moser DP , Hedlund BP. 2013 Pyrosequencing reveals high-temperature cellulolytic microbial consortia in Great Boiling Spring after in situ lignocellulose enrichment. PLoS One 8 e59927 -    DOI : 10.1371/journal.pone.0059927
Rani KS , Swamy MV , Seenayya G. 1997 Increased ethanol production by metabolic modulation of cellulose fermentation in Clostridium thermocellum. Biotechnol. Lett. 19 819 - 823    DOI : 10.1023/A:1018312931542
Rincon MT , Dassa B , Flint HJ , Travis AR , Jindou S , Borovok I 2010 Abundance and diversity of dockerin-containing proteins in the fiber-degrading rumen bacterium, Ruminococcus flavefaciens FD1. PLoS One 5 e12476 -    DOI : 10.1371/journal.pone.0012476
Sarkar N , Ghosh SK , Bannerjee S , Aikat K. 2012 Bioethanol production from agricultural wastes: an overview. Renew. Energ. 37 19 - 27    DOI : 10.1016/j.renene.2011.06.045
Sigurbjornsdottir M-A , Orlygsson J. 2012 Combined hydrogen and ethanol production from sugars and lignocellulosic biomass by Thermoanaerobacterium AK54, isolated from hot spring. Appl. Energ. 97 785 - 791    DOI : 10.1016/j.apenergy.2011.11.035
Sizova MV , Izquierdo JA , Panikov NS , Lynd LR. 2011 Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost. Appl. Environ. Microbiol. 77 2282 - 2291    DOI : 10.1128/AEM.01219-10
Syutsubo K , Nagaya Y , Sakai S , Miya A. 2005 Behavior of cellulose-degrading bacteria in thermophilic anaerobic digestion process. Water Sci. Technol. 52 79 - 84
Tamura K , Dudley J , Nei M , Kuma S. 2007 MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24 1596 - 1599    DOI : 10.1093/molbev/msm092
Thompson JD , Gibson TJ , Plewniak F , Jeanmougin F , Higgins DG. 1997 The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24 4876 - 4882    DOI : 10.1093/nar/25.24.4876
Tsuey LS , Ariff AB , Mohamad R , Rahim RA. 2006 Improvements of GC and HPLC analyses in solvent (acetonebutanol-ethanol) fermentation by Clostridium saccharobutylicum using a mixture of starch and glycerol as carbon source Biotechnol. Bioproc. E 11 293 - 298    DOI : 10.1007/BF03026243
Warnick TA , Methe BA , Leschine SB. 2002 Clostridium phytofermentans sp. nov. a cellulolytic mesophile from forest soil. Int. J. Syst. Evol. Microbiol. 52 1155 - 1160    DOI : 10.1099/ijs.0.02125-0
Watanabe K , Teramoto M , Futamata H , Harayama S. 1998 Molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria in activated sludge. Appl. Environ. Microbiol. 64 4396 - 4402
Weisburg WG , Barns SM , Pelletier DA , Lane DJ. 1991 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173 697 - 703
Wiegel J , Ljungdahl LG. 1981 Thermoanaerobacter ethanolicus gen. nov., spec. nov., a new, extreme thermophilic, anaerobic bacterium. Arch. Microbiol. 128 343 - 348    DOI : 10.1007/BF00405910
Wilson K. 1990 Preparation of genomic DNA from bacteria, In Ausubel FM, Brent R (eds.). Current Protocols in Molecular Biology. Greene Publ. Assoc. and Wiley Interscience New York. 241 - 245
Wolin EA , Wolin MJ , Wolfe RS. 1963 Formation of methane by bacterial extracts. J. Biol. Chem. 238 2882 - 2886
Wongwatanapaiboon J , Kangvansaichol K , Burapatana V , Inochanon R , Winayanuwattikun P , Yongvanich T , Chulalaksananukul W. 2012 The potential of cellulosic ethanol production from grasses in Thailand. J. Biomed. Biotechnol. 303748 -
Xi Q , Jezowski S. 2004 Plant resources of Triarrhena and Miscanthus species in China and its meaning for Europe. Plant Breed. Seed Sci. 49 63 - 77
Yoon MH , Ten LN , Im WT , Lee ST. 2008 Cellulomonas chitinilytica sp. nov. a chitinolytic bacterium isolated from cattle-farm compost. Int. J. Syst. Evol. Microbiol. 58 1878 - 1884    DOI : 10.1099/ijs.0.64768-0
Zhang K , Dong X. 2009 Selenomonas bovis sp. nov., isolated from yak rumen contents. Int. J. Syst. Evol. Microbiol. 59 2080 - 2083    DOI : 10.1099/ijs.0.007641-0
Zhao C , Ruan LW. 2011 Biodegradation of Enteromorpha prolifera by mangrove degrading micro-consortium with physical-chemical pretreatment. Appl. Microbiol. Biotechnol. 92 709 - 916    DOI : 10.1007/s00253-011-3384-2
Zhao Y , Wang Y , Zhu JY , Ragauskas A , Deang Y. 2008 Enhanced enzymatic hydrolysis of spruce by alkaline pretreatment at low temperature. Biotechnol. Bioeng. 99 1320 - 1328    DOI : 10.1002/bit.21712