Control of Lactobacillus plantarum Contamination in Bioethanol Fermentation by Adding Plantaricins.
Bacteriocin is considered as a potential biological method for controlling bacterial contamination. Plantaricins can inhibit the growth of Lactobacillus species closely related to the producer. In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with Saccharomyces cerevisiae S288C to mimic the bacterial contamination in industrial bioethanol fermentation. Plantaricins produced by L. plantarum ATCC BAA-793 was added into the co-cultivation system to control L. plantarum 8014 contamination. The final ethanol content and cell number of S. cerevisiae and L. plantarum 8014 were determined to assess the controlling effect. Results showed that plantaricins could effectively control L. plantarum contamination and remarkably reduce the inhibition effect on S. cerevisiae. Furthermore, plantaricins did no harm to the S. cerevisiae growth and bioethanol yield.
These results suggested the potential of plantaricins as a novel antibacterial agent for controlling L. plantarum contamination during the bioethanol fermentation.
Keywords: Plantaricins; Saccharomyces cerevisiae; Bioethanol; Lactobacillus plantarum; Bacterial contamination
With the continuous development of economy, the world falls into the midst of energy crisis and environmental pollution (Giampietro et al., 2012). Therefore, environment-friendly and sustainable alternative energy resources, such as bioenergy are urgently needed (Fukuda et al., 2009). As a renewable clean bioenergy, bioethanol would facilitate the reform of energy proportion, relieve energy crisis, and lighten global warming to a certain extent (Katakura et al., 2011).
Bioethanol can be produced by many kinds of microorganisms, of which Saccharomyces cerevisiae is the most employed species for industrial production (Widiastuti et al., 2011). However, industrial-scale bioethanol fermentation is frequently stressed by bacterial contaminants (Muthaiyan et al., 2011). Bacterial contamination can inhibit the growth of S. cerevisiae and result in decreased bioethanol yield, then eventually lead to economic losses (Thomas et al., 2001; Narendranath and Power, 2005). Lactobacillus is the major bacteria contaminant in bioethanol fermentation because of its rapid proliferation and tolerance to ethanol and low pH (Narendranath et al., 1997). Skinner's study on bacterial contaminants of three fuel ethanol facilities has also shown that Lactobacillus species were the most abundant isolates, averaging 51, 38, and 77% of total isolates, respectively (Skinner and Leathers, 2004).
In addition, it has been reported while the final ethanol concentrations were approximately 100 g/L (12.7%, vol/vol), the presence of lactobacilli at various concentrations would cause the loss in produced ethanol ranged from 0 to 7.5% (Narendranath et al., 1997).
Lactobacillus inhibits the growth of S. cerevisiae mainly through generating lactate and competing for nutrients and living space. Firstly, Lactobacillus can compete against S. cerevisiae for saccharides and other micronutrients in fermentation broth (Narendranath and Power, 2005). Secondly, lactate generated by Lactobacillus can decrease the fermentation pH value, thus inhibiting yeast biomass and bioethanol yield (Watanabe et al., 2008; Katakura et al., 2011). Addition of 4% (w/v) exogenous lactic acid can significantly decrease the bioethanol yield (Graves et al., 2006). Thirdly, Lactobacillus would also contend with S. cerevisiae for subsisting space. This kind of competition is common among microorganisms living in an enclosed environment.
Various methods have been attempted to prevent the adverse effects of bacterial contamination, such as adding antibiotics (Narendranath and Power, 2005; Bischoff et al., 2009), exogenous ethanol (Katakura et al., 2011), lactate (Watanabe et al., 2008), and acetate (Saithong et al., 2009). Antibiotics are widely used to eliminate bacterial contamination. However, overuse of antibiotics can result in increased antibiotic-resistant bacteria, which might make antibiotics ineffective, and drug residue, which might be fed to livestock and eventually threaten the safety of foodstuff (Narendranath et al., 2000). Moreover, overuse of antibiotics would also lead to emerging and spreading of antibiotic-resistant genes (Zhu et al., 2013). Exogenous lactate, which is safer and more acceptable for public, could also inhibit Lactobacillus effectively; but the exogenous lactate can also inhibit the fermentation capability of S. cerevisiae.
In addition, high temperature can reduce the incidence of bacterial contamination. However, most industrial-scale S. cerevisiae strains cannot grow or ferment at temperature higher than 35degC (Limtong et al., 2007; Watanabe et al., 2010). Moreover, other methods have also been attempted to control Lactobacillus contamination, such as subjoining sulfite and hydrogen peroxide (Chang et al., 1997), adding peptides derived from bovine lactoferrin (Enrique et al., 2009), chitosan (Gil et al., 2004), and sulfuric acid (Pant and Adholeya, 2007; Tang et al., 2010). Sulfuric acid would cause high levels of sulfate ions in the wastewater and heighten the osmotic stress and change pH value of fermentation broth (Narendranath and Power, 2005). Consequently it is meaningful to find alternative and applicable antibacterial methods to manage bacterial contamination in industrial-scale bioethanol production.
Bacterial contamination can be inhibited by biological control, among which bacteriocins produced by lactic acid bacteria (LAB) might be a potential method to safely and economically control Lactobacillus contamination occurred in bioethanol fermentation. Over the past decades, LAB- bacteriocins including nisin, pediocin produced by Pediococcus acidilactici, and plantaricin produced by Lactobacillus plantarum have gained comprehensive attention for their antibacterial activity and have been widely used in food preservation and pharmaceutical industries (Nishie et al., 2012; Balciunas et al., 2013). Plantaricins produced by L. plantarum ATCC BAA-793, are proved heat-stable, degradable by proteases and exhibit strain-specific antimicrobial activity. They could inhibit the growth of species closely related to the producing strain, and do not harm to other organisms (Daeschel et al., 1990).
This character provides the possible use of plantaricins as novel anti-microbial agent to control LAB contamination occurred in bioethanol fermentation.
In this study, L. plantarum ATCC 8014 was co-cultivated with S. cerevisiae in YPD broth at the beginning of the culture to simulate the bacterial contamination in industrial bioethanol fermentation. Plantaricins produced by L. plantarum ATCC BAA-793 were added into the co-cultivation system to evaluate the controlling effect of bacteriocins on the L. plantarum ATCC 8014 contamination, and the side effect of plantaricins on bioethanol fermentation was also examined.
Materials and Methods
Strains, Media and Culture Conditions
The S. cerevisiae strain used in this study was S288C and the ATCC No. for this strain was 204508 (http://www.atcc.org/Products/All/204508.aspx). It was cultured in YPD broth (2% glucose, 1% yeast extract, and 2% peptone) at 30degC and shaken at 150 rpm. Both the bacteriocins producer L. plantarum ATCC BAA-793 (http://www.atcc.org/Products/All/BAA-793.aspx) and the contaminating L. plantarum was ATCC 8014 (http://www.atcc.org/Products/All/8014.aspx) were grown at 30degC in MRS medium (1% peptone, 1% beef extract, 1% yeast extract, 2% glucose, 0.5% sodium acetate, 0.2% diammonium hydrogen citrate, 0.2% dipotassium hydrogen phosphate, 0.058% magnesium sulfate, 0.025% manganese sulfate, and 0.1% (v/v) tween 80, pH 6.8).
The plantaricins were prepared as described by Nissen-Meyer et al. (1993). After growing at 30degC for 12~16 h to early stationary phase, L. plantarum ATCC BAA-793 culture was centrifugalized at 8,000 x g for 20 min, and the supernatant was collected. With addition of ammonium sulfate (75% w/v, final concentration), the plantaricins were precipitated. After separating by centrifugation (12,000 g, 4degC, 15 min), the precipitated plantaricins were resuspended in 20 mM-sodium phosphate buffer (pH 7.0). The pellet was stored (4degC) or to be quantified by Bradford method (Bradford, 1976).
Plantaricins Antimicrobial Activity Optimization
The antimicrobial activity of plantaricins produced by L. plantarum ATCC BAA-793 was evaluated by the Oxford cup method with L. plantarum ATCC 8014 used as indicator strain and S. cerevisiae S288C as negative control; penicillin and clindamycin (1 mg/mL) were used as contrasting agents (Vincent et al., 1944). Two hundred microlitres of prepared plantaricins, penicillin solution, and clindamycin solution were respectively added into Oxford cup, on MRS agar plate (1.5% w/v agar) seeded with for L. plantarum ATCC 8014 in exponential phase or YPD agar plate for S. cerevisiae S288C in exponential phase, respectively. Plates were incubated at 30degC for 16 h, and then inhibitory zone was measured.
To further optimize the concentration of plantaricins for inhibiting L. plantarum ATCC 8014, different concentrations (i.e., 0, 2, 5, 10, 20 and 50 ug/mL) of plantaricins were added into L. plantarum ATCC 8014 incubation system at the beginning of culture. L. plantarum cell number was determined by serial dilution and plate counting method. The experiment was conducted in triplicate.
Inhibition of L. plantarum Contamination using Plantaricins during S. cerevisiae Fermentation L. plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288C in YPD broth at the beginning of fermentation to mimic the L. plantarum contamination in industrial bioethanol fermentation. The final cell densities of inoculated S. cerevisiae and L. plantarum were 4x105 and 1.6-2.3x106 cells/mL, respectively. To assess the effect of plantaricins against L. plantarum contamination, 50 ug/mL (final concentration) plantaricins was added into the S. cerevisiae and L. plantarum co-cultivation system.
Altogether, three groups including control group (only S. cerevisiae in YPD mediumbroth), contamination group (S. cerevisiae and L. plantarum were co-cultured in YPD mediumbroth), and plantaricins treatment group (plantaricins were added into S. cerevisiae and L. plantarum co-cultivation system), were cultured at 30degC and shaken at 170 rpm. S. cerevisiae cell number was counted with a haemocytometer, while L. plantarum cell number was determined by serial dilution and plate counting method.
The final ethanol content was measured by gas chromatography (GC) analysis. The experiment was conducted in triplicate.
Side Effect Evaluation of Plantaricins on S. cerevisiae Fermentation
To monitor the side effect of plantaricins on S. cerevisiae fermentation, two groups, that were control group (only S. cerevisiae in YPD broth), and side effect evaluation group (plantaricins were added into S. cerevisiae fermentation system, the final concentration of plantaricins was 50 ug/mL), were cultivated at 30degC and shaken at 170 rpm. S. cerevisiae cell number was counted with a haemocytometer. The final ethanol content was measured by GC analysis. The experiment was conducted in triplicate.
Data were analyzed by IBM SPSS statistics 20 and differences exhibiting P<0.05 were considered statistically significant.
Plantaricins Antimicrobial Activity Optimization
It could be clearly observed that Oxford cup with penicillin or clindamycin showed a clear and distinct zone of sterilization, while Oxford cup with plantaricins showed a cloudy zone of inhibition. Although the inhibitory effect was <that of penicillin or clindamycin, plantaricins still showed antimicrobial activity against L. plantarum ATCC 8014 (Fig. 1a). Moreover, both plantaricins and tested antibiotics showed no antimicrobial activity against S. cerevisiae S288C (Fig. 1b).
In the L. plantarum ATCC 8014 cultures, the addition of different concentrations of plantaricins inhibited cell growth. The growth of L. plantarum ATCC 8014 was suppressed from 9 h after culture under low (2 and 5 ug/mL) and intermediate (10 and 20 ug/mL) plantaricins concentrations, while the growth was inhibited from 3 h after culture under high (50 ug/mL) plantaricins concentration (P<0.05) (Fig. 2, Supplementary Table S1). According to these results, 50 ug/mL was chosen as the optimized concentration of plantaricins for subsequent experiments.
Inhibition of L. plantarum Contamination using Plantaricins during S. cerevisiae Fermentation
To mimic the bacterial contamination in S. cerevisiae fermentation, L. plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288C in YPD medium, and plantaricins were added to assess the efficacy of plantaricins treatment. Compared to the control group, the growth of S. cerevisiae in the contamination group was inhibited from 3 h after co-culture (P<0.05) (Fig. 3a).
However, addition of plantaricins reversed the inhibitory effect of L. plantarum on the growth of S. cerevisiae. The yeast cell number in the plantaricins treatment group increased remarkably from 3 h to 18 h after co-culture in comparison with the contamination group (P<0.05) (Fig. 3a).
The final ethanol production in the presence of L. plantarum also decreased about 31% in comparison with the control group (P<0.01) (Fig. 3b). However, the addition of plantaricins reversed the inhibition of L. plantarum on the final ethanol production (P0.05) (Fig. 3b).
Moreover, the cell number of L. plantarum ATCC 8014 was determined to evaluate the antimicrobial activity of plantaricins on L. plantarum. Compared to the contamination group, the cell number of L. plantarum in the plantaricins treatment group decreased remarkably during early exponential phase (P0.05) (Fig. 4a). Interestingly, the final ethanol production in the plantaricins treatment group increased 4.6% than that in the control group (P<0.01) (Fig. 4b). These results suggested that addition of plantaricins into S. cerevisiae ferementation system is harmless.
With the continuous development of economy, energy crisis and environmental pollution are increasingly prominent. Bioethanol is considered as an alternative fuel for reducing consumption of crude oil and then facilitate alleviating environmental pollution (Balat and Balat, 2009). During the bioethanol fermentation, the ethanol yield and the growth of S. cerevisiae cells are often inhibited by bacterial contamination, among which LAB such as L. plantarum, are the major bacterial contaminants (Skinner and Leathers, 2004). LAB is tolerant to ethanol and low pH and can quickly outnumber the S. cerevisiae cells (Bayrock et al., 2003). Therefore, they can greatly influence yeast growth.
Results shown here also indicated the inhibitory effect of L. plantarum on the growth of S. cerevisiae and the final ethanol production (P<0.05) (Fig. 3).
Supplementary Table S1: The statistical P values about L. plantarum ATCC 8014 cell number of plantaricins treated groups in comparison with the control group (no plantaricins)
2###0.311 0.287 0.006 0.085 0.037 0.003
5###0.175 0.190 0.003 0.029 0.015 0.001
10###0.116 0.153 0.000 0.018 0.005 0.000
20###0.081 0.100 0.000 0.021 0.002 0.000
50###0.032 0.049 0.001 0.001 0.002 0.000
Bacterial contaminants controlling method should be taken seriously for the purpose of enhancing the bioethanol yield and simultaneously decreasing the cost of production. Biological control has gained much attention for its non-hazardous characteristic and is considered alternative to traditional chemical agents for inhibiting bacterial contamination (Santos et al., 2011). Among the biological controlling methods, plantaricins offer an environment- friendly treatment of L. plantarum contamination with no harm to S. cerevisiae. In the present study, the addition of plantaricins reversed the inhibition of L. plantarum on the growth of S. cerevisiae to the control group level and removed the negative impact of L. plantarum on final bioethanol production (Fig. 3a, b). Moreover, the addition of plantaricins did markedly decrease the cell number of L. plantarum at early exponential phase (Fig. 3c). At the beginning of co-culture, resources are relatively abundant.
So compared with other phases, hyper proliferative of L. plantarum at early exponential phase makes use of the resources originally belonging to S. cerevisiae and makes great damage to S. cerevisiae. Thus, inhibition of L. plantarum contamination at early fermentation is more effective. This laid the foundation for L. plantarum control during the whole ethanol fermentation process. These results suggested that plantaricins were effective to control L. plantarum contamination during S. cerevisiae fermentation. Although these results were acquired from laboratory scale experiments, which containing ethanol at low concentrations, they were still significant for potential application of plantaricins for controlling of L. plantarum contamination in industrial bioethanol production containing higher ethanol yield and in which L. plantarum contamination was still serious (Skinner and Leathers, 2004).
Compared with antibiotics, natural bacteriocins including plantaricins are safe to environment. In particular, antimicrobial activity of plantaricins has strain-specificity (Daeschel et al., 1990), which means they might have no side effect on S. cerevisiae growth and fermentation. Additionally, plantaricins not only did no harm to the growth of S. cerevisiae (Fig. 4a), but also slightly promoted the production of bioethanol (Fig. 4b). S. cerevisiae can secrete several kinds of proteases (Ogrydziak, 1993). As L. plantarum contamination was controlled, plantaricins might be degraded by proteases secreted by S. cerevisiae. The degraded plantaricins might be utilized by S. cerevisiae as nitrogen source. Moreover, it has been reported that supplementation of exogenous tryptophan and proline into culture medium could increase the ethanol tolerance of S. cerevisiae (Takagi et al., 2005; Ma and Liu, 2010).
Some free amino acids might be produced through degradation of plantarcins and conferred ethanol tolerance to S. cerevisiae to some extent. Of course, such hypothesis needs further verification. Overall, these results illustrated the biosafety of plantaricins for controlling of L. plantarum contamination occurred during bioethanol fermentation.
In summary, the present work illustrated the utility of plantaricins to control L. plantarum contamination occurred in bioethanol fermentation. Results showed that plantaricins treatment can effectively control L. plantarum contamination in bioethanol fermentation. Moreover, plantaricins treatment did no harm to the growth of S. cerevisiae, and to some extent, enhanced bioethanol production. This research implied the potential of plantaricins as a novel bactericide for controlling of bacterial contamination in bioethanol production. Although these results were acquired from laboratory scale experiments, results shown here can still lay the theoretical basis for utilization of plantaricins to control contaminants in industrial bioethanol production, thereby lowering the consumption of antibiotics. In the future, purified plantaricins might be produced as liquid or powder or other forms for application.
Finally, there are still other contaminating microbes in the bioethanol fermentation, and use of bacteriocins acting on these microbes will make bacteriocins treatment more efficient.
This work was supported by the National Nature Science Foundation of China (No. 31201413), the Fundamental Research Funds for the Central Universities (No. YS1407) and the Higher Education and High-quality and World-class Universities (No. PY201617).
Balat, M. and H. Balat, 2009. Recent trends in global production and utilization of bio-ethanol fuel. Appl. Energy, 86: 2273-2282
Balciunas, E.M., F.A. Castillo Martinez, S.D. Todorov, B.D.G.D.M. Franco, A. Converti and R.P.D.S. Oliveira, 2013. Novel biotechnological applications of bacteriocins: a review. Food Cont., 32: 134-142
Bayrock, D.P., K.C. Thomas and W.M. Ingledew, 2003. Control of Lactobacillus contaminants in continuous fuel ethanol fermentations by constant or pulsed addition of penicillin G. Appl. Microbiol. Biotechnol., 62: 498-502
Bischoff, K.M., S. Liu, T.D. Leathers, R.E. Worthington and J.O. Rich, 2009. Modeling bacterial contamination of fuel ethanol fermentation. Biotechnol. Bioeng., 103: 117-122
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254
Chang, I.S., B.H. Kim and P.K. Shin, 1997. Use of sulfite and hydrogen peroxide to control bacterial contamination in ethanol fermentation. Appl. Environ. Microbiol., 63: 1-6
Daeschel, M.A., M.C. McKenney and L.C. McDonald, 1990. Bacteriocidal activity of Lactobacillus plantarum C-11. Food Microbiol., 7: 91-98
Enrique, M., P. Manzanares, M. Yuste, M. Martinez, S. Valles and J.F. Marcos, 2009. Selectivity and antimicrobial action of bovine lactoferrin derived peptides against wine lactic acid bacteria. Food Microbiol., 26: 340-346
Fukuda, H., A. Kondo and S. Tamalampudi, 2009. Bioenergy: Sustainable fuels from biomass by yeast and fungal whole-cell biocatalysts. Biochem. Eng. J., 44: 2-12
Giampietro, M., J.R. Martin and S. Ulgiati, 2012. Can we break the addiction to fossil energy?. Energy, 37: 2-4
Gil, G., S. del Monaco, P. Cerrutti and M. Galvagno, 2004. Selective antimicrobial activity of chitosan on beer spoilage bacteria and brewing yeasts. Biotechnol. Lett., 26: 569-574
Graves, T., N.V. Narendranath, K. Dawson and R. Power, 2006. Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash. J. Ind. Microbiol. Biotechnol., 33: 469-474
Katakura, Y., C. Moukamnerd, S. Harashima and M. Kino-oka, 2011. Strategy for preventing bacterial contamination by adding exogenous ethanol in solid-state semi-continuous bioethanol production. J. Biosci. Bioeng., 111: 343-345
Limtong, S., C. Sringiew and W. Yongmanitchai, 2007. Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus. Bioresour. Technol., 98: 3367-3374
Ma, M. and Z.L. Liu, 2010. Mechanisms of ethanol tolerance in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 87: 829-845
Muthaiyan, A., A. Limayem and S.C. Ricke, 2011. Antimicrobial strategies for limiting bacterial contaminants in fuel bioethanol fermentations. Prog. Energy Combust. Sci., 37: 351-370
Narendranath, N.V., S.H. Hynes, K.C. Thomas and W.M. Ingledew, 1997. Effects of lactobacilli on yeast-catalyzed ethanol fermentations. Appl. Environ. Microbiol., 63: 4158-4163
Narendranath, N.V. and R. Power, 2005. Relationship between pH and medium dissolved solids in terms of growth and metabolism of Lactobacilli and Saccharomyces cerevisiae during ethanol production. Appl. Environ. Microbiol., 71: 2239-2243
Narendranath, N.V., K.C. Thomas and W.M. Ingledew, 2000. Urea hydrogen peroxide reduces the numbers of lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation. Appl. Environ. Microbiol., 66: 4187-4192
Nishie, M., J. Nagao and K. Sonomoto, 2012. Antibacterial peptides" bacteriocins": an overview of their diverse characteristics and applications. Biocont. Sci., 17: 1-16
Nissen-Meyer, J., A.G. Larsen, K. Sletten, M. Daeschel and I.F. Nes, 1993. Purification and characterization of plantaricin A, a Lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides. J. Gen. Microbiol., 139: 1973-1978
Ogrydziak, D.M., 1993. Yeast extracellular proteases. Crit. Rev. Biotechnol., 13: 1-55
Pant, D. and A. Adholeya, 2007. Biological approaches for treatment of distillery wastewater: a review. Bioresour. Technol., 98: 2321-2334
Saithong, P., T. Nakamura and J. Shima, 2009. Prevention of bacterial contamination using acetate-tolerant Schizosaccharomyces pombe during bioethanol production from molasses. J. Biosci. Bioeng., 108: 216-219
Santos, A., E. Navascues, E. Bravo and D. Marquina, 2011. Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis. Int. J. Food Microbiol., 145: 147-154
Skinner, K.A. and T.D. Leathers, 2004. Bacterial contaminants of fuel ethanol production. J. Ind. Microbiol. Biotechnol., 31: 401-408
Takagi, H., M. Takaoka, A. Kawaguchi and Y. Kubo, 2005. Effect of L-proline on sake brewing and ethanol stress in Saccharomyces cerevisiae. Appl. Environ. Microbiol., 71: 8656-8662
Tang, Y.Q., M.Z. An, Y.L. Zhong, M. Shigeru, X.L. Wu and K. Kida, 2010. Continuous ethanol fermentation from non-sulfuric acid-washed molasses using traditional stirred tank reactors and the flocculating yeast strain KF-7. J. Biosci. Bioeng., 109: 41-46
Thomas, K.C., S.H. Hynes and W.M. Ingledew, 2001. Effect of lactobacilli on yeast growth, viability and batch and semi-continuous alcoholic fermentation of corn mash. J. Appl. Microbiol., 90: 819-828
Vincent, J.G., H.W. Vincent and J. Morton, 1944. Filter paper disc modification of the Oxford cup penicillin determination. Exp. Biol. Med. (Maywood), 55: 162-164
Watanabe, I., T. Nakamura and J. Shima, 2008. A strategy to prevent the occurrence of Lactobacillus strains using lactate-tolerant yeast Candida glabrata in bioethanol production. J. Ind. Microbiol. Biotechnol., 35: 1117-1122
Watanabe, T., S. Srichuwong, M. Arakane, S. Tamiya, M. Yoshinaga, I.Watanabe, M. Yamamoto, A. Ando, K. Tokuyasu and T. Nakamura, 2010. Selection of stress-tolerant yeasts for simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash to ethanol. Bioresour. Technol., 101: 9710-9714
Widiastuti, H., J.Y. Kim, S. Selvarasu, I.A. Karimi, H. Kim, J.S. Seo and D.Y.Lee, 2011. Genome-scale modeling and in silico analysis of ethanologenic bacteria Zymomonas mobilis. Biotechnol. Bioeng., 108:655-665
Zhu, Y.G., T.A. Johnson, J.Q. Su, M. Qiao, G.X. Guo, R.D. Stedtfeld, S.A.Hashsham and J.M. Tiedje, 2013. Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc. Natl. Acad. Sci. U S A,110: 3435-3440
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|Author:||Liu Bo; Liu Huaqing; Zhang, Yunxiao; Li, Hao|
|Publication:||International Journal of Agriculture and Biology|
|Date:||Feb 28, 2017|
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