Qian Ma

Metabolome Profiling Reveals Metabolic Cooperation between Bacillus megaterium and Ketogulonicigenium vulgare during Induced Swarm Motility

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.

Proteomic Analysis of Ketogulonicigenium vulgare under Glutathione Reveals High Demand for Thiamin Transport and Antioxidant Protection

Ketogulonicigenium vulgare, though grows poorly when mono-cultured, has been widely used in the industrial production of the precursor of vitamin C with the coculture of Bacillus megaterium. Various efforts have been made to clarify the synergic pattern of this artificial microbial community and to improve the growth and production ability of K. vulgare, but there is still no sound explanation. In previous research, we found that the addition of reduced glutathione into K. vulgare monoculture could significantly improve its growth and productivity. By performing SEM and TEM, we observed that after adding GSH into K. vulgare monoculture, cells became about 4–6 folds elongated, and formed intracytoplasmic membranes (ICM). To explore the molecular mechanism and provide insights into the investigation of the synergic pattern of the co-culture system, we conducted a comparative iTRAQ-2-D-LC-MS/MS-based proteomic analysis of K. vulgare grown under reduced glutathione. Principal component analysis of proteomic data showed that after the addition of glutathione, proteins for thiamin/thiamin pyrophosphate (TPP) transport, glutathione transport and the maintenance of membrane integrity, together with several membrane-bound dehydrogenases had significant up-regulation. Besides, several proteins participating in the pentose phosphate pathway and tricarboxylic acid cycle were also up-regulated. Additionally, proteins combating intracellular reactive oxygen species were also up-regulated, which similarly occurred in K. vulgare when the co-cultured B. megaterium cells lysed from our former research results. This study reveals the demand for transmembrane transport of substrates, especially thiamin, and the demand for antioxidant protection of K. vulgare.

Design, analysis and application of synthetic microbial consortia

The rapid development of synthetic biology has conferred almost perfect modification on single cells, and provided methodological support for synthesizing microbial consortia, which have a much wider application potential than synthetic single cells. Co-cultivating multiple cell populations with rational strategies based on interacting relationships within natural microbial consortia provides theoretical as well as experimental support for the successful obtaining of synthetic microbial consortia, promoting it into extensive research on both industrial applications in plenty of areas and also better understanding of natural microbial consortia. According to their composition complexity, synthetic microbial consortia are summarized in three aspects in this review and are discussed in principles of design and construction, insights and methods for analysis, and applications in energy, healthcare, etc.

Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana.

High density cultivation is essential to industrial production of biodiesel from microalgae, which involves in variations of micro‐environment around individual cells, including light intensity, nutrition distribution, other abiotic stress and so on. To figure out the main limit factor in high inoculum cultivation, a quantitative proteomic analysis (iTRAQ‐on‐line 2‐D nano‐LC/MS) in a non‐model green microalga, Chlorella sorokiniana, under different inoculum sizes was conducted. The resulting high‐quality proteomic dataset consisted of 695 proteins. Using a cutoff of P < 0.05, 241 unique proteins with differential expression levels were identified between control and different inoculum sizes. Functional analysis showed that proteins participating in photosynthesis (light reaction) and Calvin cycle (carbon reaction pathway) had highest expression levels under inoculum size of 1 × 106 cells mL−1, and lowest levels under 1 × 107 cells mL−1. Canonical correlation analysis of the photosynthesis related proteins and metabolites biomarkers showed that a good correlation existed between them (canonical coefficient was 0.987), suggesting photosynthesis process greatly affected microalgae biodiesel productivity and quality. Proteomic study of C. sorokiniana under different illuminations was also conducted to confirm light intensity as a potential limit factor of high inoculum size. Nearly two thirds of proteins showed up‐regulation under the illumination of 70–110 µmol m−2 s−1, compared to those of 40 µmol m−2 s−1. This result suggested that by elegantly adjusting light conditions, high cell density cultivation and high biodiesel production might be achieved. Biotechnol. Bioeng. 2013; 110: 773–784.

Comparative Proteomic Analysis of Experimental Evolution of the Bacillus cereus-Ketogulonicigenium vulgare Co-Culture

The microbial co-culture system composing of Ketogulonicigenium vulgare and Bacillus cereus was widely adopted in industry for the production of 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. We found serial subcultivation of the co-culture could enhance the yield of 2-KGA by 16% in comparison to that of the ancestral co-culture. To elucidate the evolutionary dynamics and interaction mechanisms of the two microbes, we performed iTRAQ-based quantitative proteomic analyses of the pure cultures of K. vulgare, B. cereus and their co-culture during serial subcultivation. Hierarchy cluster analyses of the proteomic data showed that the expression level of a number of crucial proteins associated with sorbose conversion and oligopeptide transport was significantly enhanced by the experimental evolution. In particular, the expression level of sorbose/sorbosone dehydrogenase was enhanced in the evolved K. vulgare, while the expression level of InhA and the transport efficiency of oligopeptides were increased in the evolved B. cereus. The decreased sporulating protein expression and increased peptide transporter expression observed in evolved B. cereus, together with the increased amino acids synthesis in evolved K. vulgare suggested that serial subcultivation result in enhanced synergistic cooperation between K. vulgare and B. cereus, enabling an increased production of 2-KGA.

Phospholipid Metabolism in an Industry Microalga Chlorella sorokiniana: The Impact of Inoculum Sizes

Chlorella sorokiniana is an important industry microalga potential for biofuel production. Inoculum size is one of the important factors in algal large-scale culture, and has great effects on the growth, lipid accumulation and metabolism of microalgae. As the first barrier of cell contents, membrane plays a vital role in algal inoculum-related metabolism. The knowledge of phospholipids, the main membrane component and high accumulation of phospholipids as the major content of total lipids mass in some microalgae, is necessary to understand the role of membrane in cell growth and metabolism under different inoculum density. Profiling of C. sorokiniana phospholipids with LC-MS led to the identification of 119 phospholipid species. To discover the phospholipid molecules most related to change of inoculum sizes, Partial Least Squares Discriminant Analysis (PLS-DA) was employed and the results revealed that inoculum sizes significantly affected phospholipid profiling. Phosphatidylglycerol (PG), phosphatidyl- ethanolamine (PE) and several phosphatidylcholine (PC) species might play an important role under our experimental conditions. Further analysis of these biomarkers indicated that cell membrane status of C. sorokiniana might play an important role in the adaption to the inoculum sizes. And the culture with inoculum size of 1×106 cells mL−1 presented the best membrane status with the highest content of PC and PG, and the lowest content of PE. We discovered that the inoculum size of 1×106 cells mL−1 might provide the best growth condition for C. sorokiniana. Also we proposed that PG, PE and several PC may play an important role in inoculum-related metabolism in C. sorokiniana, which may work through thylakoid membrane and photosynthetic pathway. Thus this study would provide more potential targets for metabolic engineering to improve biofuel production and productivity in microalgae.