Abstract
The dynamics of bacterial communities play an important role in solid-state fermentation (SSF). Poly-γ-glutamic acid (γ-PGA) was produced by Bacillus amyloliquefaciens C1 in SSF using dairy manure compost and monosodium glutamate production residuals as basic substrates. The production of γ-PGA reached a maximum of 0.6% after 20 days fermentation. Real-time polymerase chain reaction showed the amount of total bacteria reached 3.95 × 109 16S rDNA copies/g sample after 30 days, which was in good accordance with the 4.80 × 109 CFU/g obtained by plate counting. Denaturing gradient gel electrophoresis profile showed a reduction of microbial diversity during fermentation, while the inoculum, B. amyloliquefaciens C1, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbial, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce γ-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.
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References
Ahn J-h, Y-j K, Kim T, H-g S, Kang C, J-o K (2009) Quantitative improvement of 16S rDNA DGGE analysis for soil bacterial community using real-time PCR. J Microbiol Methods 78:216–222
Amann RI, Ludwig W, Schleifer K (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169
Asano R, Otawa K, Ozutsumi Y, Yamamoto N (2010) Development and analysis of microbial characteristics of an acidulocomposting system for the treatment of garbage and cattle manure. J Biosci Bioeng 110:419–425
Ashiuchi M (2010) Occurrence and biosynthetic mechanism of poly-γ-glutamic acid. In: Hamano Y (ed) Amino-acid homopolymers occurring in nature (microbiology monographs), vol 15. Springer, Heidelberg, pp 77–94
Bassam BJ, Caetano-Anollés G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196:80–83
Bustin S, Benes V, Ja G, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Chen X (2005) High yield of poly-γ-glutamic acid from Bacillus subtilis by solid-state fermentation using swine manure as the basis of a solid substrate. Bioresour Technol 96:1872–1879
Daniel R (2005) The metagenomics of soil. Nat Rev Microbiol 3:470–478
Danon M, Franke-whittle IH, Insam H, Chen Y, Hadar Y (2008) Molecular analysis of bacterial community succession during prolonged compost curing. FEMS Microbiol Ecol 65:133–144
Filion M, Novinscak A, Decoste NJ (2009) Characterization of bacterial and fungal communities in composted biosolids over a 2 year period using denaturing gradient gel electrophoresis. Can J Microbiol 387:375–387
Finstein MS, Morris ML (1975) Microbiology of municipal solid waste composting. Adv Appl Microbiol 19:113–151
Fracchia L, Dohrmann AB, Martinotti MG, Tebbe CC (2006) Bacterial diversity in a finished compost and vermicompost: differences revealed by cultivation-independent analyses of PCR-amplified 16S rRNA genes. Appl Microbiol Biotechnol 71:942–952
Giraffa G (2004) Studying the dynamics of microbial populations during food fermentation. FEMS Microbiol Rev 28:251–260
Haruta S, Ueno S, Egawa I, Hashiguchi K, Fujii A, Nagano M, Ishii M, Igarashi Y (2006) Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis. Int J Food Microbiol 109:79–87
Head IM, Saunders JR, Pickup RW (1998) Microbial evolution, diversity, and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol 35:1–21
Innerebner G, Knapp B, Vasara T, Romantschuk M, Insam H (2006) Traceability of ammonia-oxidizing bacteria in compost-treated soils. Soil Biol Biochem 38:1092–1100
Ivánovics G, Erdos L (1937) Ein beitrag zum wesen der kapselsubstanz des milzbrandbazillus. Z Immunitatsforsch 90:5–19
Konstantinov SR, W-y Z, Williams BA, Tamminga S (2003) Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA. FEMS Microbiol Ecol 43:225–235
LaMontagne MG, Michel FC Jr, Holden PA, Reddy CA (2002) Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis. J Microbiol Methods 49:255–264
Lauber CL, Hamady M, Knight R, Fierer N (2009) Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl Environ Microbiol 75(15):5111–5120. doi:https://doi.org/10.1128/aem.00335-09
Maeda K, Morioka R, Hanajima D (2010) The impact of using mature compost on nitrous oxide emission and the denitrifier community in the cattle manure composting process. Microb Ecol 59:25–36
Mieszkin S, Furet J-P, Gr C, Gourmelon Ml (2009) Estimation of pig fecal contamination in a river catchment by real-time PCR using two pig-specific Bacteroidales 16S rRNA genetic markers. Appl Environ Microbiol 75(10):3045–3054
Muyzer G, Waal ECD, Uitierlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700
Nakasaki K, Sasaki M, Shoda M, Kubota H (1985) Characteristics of mesophilic bacteria isolated during thermophilic composting of sewage-sludge. Appl Environ Microbiol 49:42–45
Pandey A (2003) Solid-state fermentation. Biochem Eng J 13:81–84
Peters S, Koschinsky S, Schwieger F, Tebbe CC (2000) Succession of microbial communities during hot composting as detected by PCR–single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes. Appl Environ Microbiol 66:930–936
Petrosino JF, Highlander S, Luna RA, Gibbs RA, Versalovic J (2009) Metagenomic pyrosequencing and microbial identification. Clin Chem 55(5):856–866. doi:https://doi.org/10.1373/clinchem.2008.107565
Rai SK, Konwarh R, Mukherjee AK (2009) Purification, characterization and biotechnological application of an alkaline β-keratinase produced by Bacillus subtilis RM-01 in solid-state fermentation using chicken-feather as substrate. Biochem Eng J 45:218–225
Sasaki H, Yano H, Sasaki T, Nakai Y (2005) A survey of ammonia-assimilating micro-organisms in cattle manure composting. J Appl Microbiol 99:1356–1363
Schloss PD, Hay AG, Wilson DB, Walker LP (2003) Tracking temporal changes of bacterial community fingerprints during the initial stages of composting. FEMS Microbiol Ecol 46:1–9
Shih IL, Van YT (2001) The production of poly-(γ-glutamic acid) from microorganisms and its various applications. Bioresour Technol 79:207–225
Strom PF (1985a) Effect of temperature on bacterial species diversity in thermophilic solid-waste composting. Appl Environ Microbiol 50:899–905
Strom PF (1985b) Identification of thermophilic bacteria in solid-waste composting. Appl Environ Microbiol 50:906–913
Tang J, Maie N, Tada Y, Katayama A (2006) Characterization of the maturing process of cattle manure compost. Process Biochem 41:380–389
Tang J-c, Shibata A, Zhou Q, Katayama A (2007) Effect of temperature on reaction rate and microbial community in composting of cattle manure with rice straw. J Biosci Bioeng 104:321–328
Wang H-Y, Zhang X-J, Zhao L-P, Xu Y (2008a) Analysis and comparison of the bacterial community in fermented grains during the fermentation for two different styles of Chinese liquor. J Ind Microbiol Biotechnol 35:603–609
Wang Q, Shouwen C, Jibin Z, Ming S, Ziduo L, Ziniu Y (2008b) Co-producing lipopeptides and poly-γ-glutamic acid by solid-state fermentation of Bacillus subtilis using soybean and sweet potato residues and its biocontrol and fertilizer synergistic effects. Bioresour Technol 99:3318–3323
Xu J, Shouwen C, Ziniu Y (2005) Optimization of process parameters for poly-γ-glutamate production under solid state fermentation from Bacillus subtilis CCTCC202048. Process Biochem 40:3075–3081
Yu Z, Dong B, Lu W (2009) Dynamics of bacterial community in solid-state fermented feed revealed by 16S rRNA. Lett Appl Microbiol 49:166–172
Zhang YC, Ronimus RS, Turner N, Zhang Y, Morgan HW (2002) Enumeration of thermophilic species in composts and identification with a random amplification polymorphic DNA (RAPD) protocol. Syst Appl Microbiol 25:618–626
Zhu W-y, Williams BA, Konstantinov SR, Tamminga S (2003) Analysis of 16S rDNA reveals bacterial shift during in vitro fermentation of fermentable carbohydrate using piglet faeces as inoculum. Anaerobe 9:175–180
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The study was financially supported by Chinese Ministry of Science and Technology (2011CB100503) and by Jiangsu Bureau of Science and Technology (BE2010722 and BA2008027).
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Yong, X., Cui, Y., Chen, L. et al. Dynamics of bacterial communities during solid-state fermentation using agro-industrial wastes to produce poly-γ-glutamic acid, revealed by real-time PCR and denaturing gradient gel electrophoresis (DGGE). Appl Microbiol Biotechnol 92, 717–725 (2011). https://doi.org/10.1007/s00253-011-3375-3
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DOI: https://doi.org/10.1007/s00253-011-3375-3