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ABSTRACT: Ketogulonigenium vulgare has long been used in industry to produce 2-keto-L-gulonic acid (2KGA), the precursor of vitamin C. This fermentation process involves co-culture of K. vulgare and a Bacillus species. Early studies demonstrated that the presence of the Bacillus strain can enhance the cellular growth and 2KGA production of K. vulgare. However, the molecular mechanism behind how Bacillus affects the growth of K. vulgare and 2KGA production remains unclear. In addition, the inclusion of Bacillus in the fermentation process presents difficulties for the post-separation and purification of 2KGA. To address these issues, efforts have been made to replace the Bacillus strain with chemical compounds. In this study, we found that adding thiol compounds such as reduced glutathione (GSH) and dithiothreitol (DTT) to the K. vulgare mono-culture system can increase the growth of K. vulgare about twofold, and increase 2KGA production by about fivefold. The effects of thiols on the concentrations of some cellular metabolites were determined using gas chromatography coupled to time-of-flight mass spectrometry. The results showed that the levels of intracellular amino acids and intermediates in the pentose phosphate pathway increased significantly after thiol addition. Interestingly, when GSH was added, the levels of key intracellular metabolites in primary metabolic pathways and the cell biomass both reached their maximum in the first 36 h, and then decreased when the thiol was exhausted. These findings indicate that cell growth needs the assistance of a high concentration of thiols. This study is the first report that chemically defined compounds were used to enhance the growth of K. vulgare and 2KGA production. Furthermore, it also provides new insights into the possible cellular interaction between Bacillus species and K. vulgare.
Omics: a journal of integrative biology 06/2012; 16(7-8):387-96. · 2.29 Impact Factor
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ABSTRACT: The metabolic cooperation in the ecosystem of Bacillus megaterium and Ketogulonicigenium vulgare was investigated by cultivating them spatially on a soft agar plate. We found that B. megaterium swarmed in a direction along the trace of K. vulgare on the agar plate. Metabolomics based on gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) was employed to analyze the interaction mechanism between the two microorganisms. We found that the microorganisms interact by exchanging a number of metabolites. Both intracellular metabolism and cell-cell communication via metabolic cooperation were essential in determining the population dynamics of the ecosystem. The contents of amino acids and other nutritional compounds in K. vulgare were rather low in comparison to those in B. megaterium, but the levels of these compounds in the medium surrounding K. vulgare were fairly high, even higher than in fresh medium. Erythrose, erythritol, guanine, and inositol accumulated around B. megaterium were consumed by K. vulgare upon its migration. The oxidization products of K. vulgare, including 2-keto-gulonic acids (2KGA), were sharply increased. Upon coculturing of B. megaterium and K. vulgare, 2,6-dipicolinic acid (the biomarker of sporulation of B. megaterium), was remarkably increased compared with those in the monocultures. Therefore, the interactions between B. megaterium and K. vulgare were a synergistic combination of mutualism and antagonism. This paper is the first to systematically identify a symbiotic interaction mechanism via metabolites in the ecosystem established by two isolated colonies of B. megaterium and K. vulgare.
Applied and environmental microbiology 07/2011; 77(19):7023-30. · 3.69 Impact Factor
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ABSTRACT: An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus megaterium has been used in industry to produce 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. During the mix culture fermentation process, sporulation and cell lysis of B. megaterium can be observed. In order to investigate how these phenomena correlate with 2-KGA production, and to explore how two species interact with each other during the fermentation process, an integrated time-series proteomic and metabolomic analysis was applied to the system. The study quantitatively identified approximate 100 metabolites and 258 proteins. Principal Component Analysis of all the metabolites identified showed that glutamic acid, 5-oxo-proline, L-sorbose, 2-KGA, 2, 6-dipicolinic acid and tyrosine were potential biomarkers to distinguish the different time-series samples. Interestingly, most of these metabolites were closely correlated with the sporulation process of B. megaterium. Together with several sporulation-relevant proteins identified, the results pointed to the possibility that Bacillus sporulation process might be important part of the microbial interaction. After sporulation, cell lysis of B. megaterium was observed in the co-culture system. The proteomic results showed that proteins combating against intracellular reactive oxygen stress (ROS), and proteins involved in pentose phosphate pathway, L-sorbose pathway, tricarboxylic acid cycle and amino acids metabolism were up-regulated when the cell lysis of B. megaterium occurred. The cell lysis might supply purine substrates needed for K. vulgare growth. These discoveries showed B. megaterium provided key elements necessary for K. vulgare to grow better and produce more 2-KGA. The study represents the first attempt to decipher 2-KGA-producing microbial communities using quantitative systems biology analysis.
PLoS ONE 01/2011; 6(10):e26108. · 4.09 Impact Factor
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ABSTRACT: A high cell density strategy has been used in bioethanol production to shorten the fermentation period. To reveal the molecular basis of fermentative behavior in high cell density, the profiling of the phospholipids and sterols of Saccharomyces cerevisiae during fermentation at five different pitching rates (1, 5, 10, 20, and 40 g/L) was investigated. Using LC/ESI/MS(n) technology, 148 phospholipid species were detected, of which 91 species were quantified, and using the gas chromatography-time-of-flight mass spectrometry procedure, a total of 11 sterols were quantified. Phospholipid samples from different pitching rates were discriminated into three groups using principal component analysis (1, 5 g/L, and the others). The main changes in the lipid profile of yeast cells with higher pitching rates were as follows: (a) the relative contents of phosphatidylglycerol and phosphatidylserine were higher while phosphatidylinositol was lower compared with lower pitching rates, (b) the saturated and the relatively shorter fatty acyl chains of phospholipids decreased, and (c) the content of ergosterol was higher. These findings suggested a regulation of the property of the membrane at the situation of high cell density and a possible approach of self-protection of the yeast cells against the high density stresses.
Applied Microbiology and Biotechnology 05/2010; 87(4):1507-16. · 3.42 Impact Factor
