Engineering an Artificial Membrane Vesicle Trafficking System (AMVTS) for the Excretion of ß-Carotene in Escherichia coli.

Large hydrophobic molecules, such as carotenoids, cannot be effectively excreted from cells by natural transportation systems. These products accumulate inside the cells and affect normal cellular physiological functions, which hinders further improvement of carotenoid production by microbial cell factories. In this study, we proposed to construct a novel artificial transport system utilizing membrane lipids to carry and transport hydrophobic molecules. Membrane lipids allow the physiological mechanism of membrane dispersion to be reconstructed and amplified to establish a novel artificial membrane vesicle transport system (AMVTS). Specifically, a few proteins in E. coli were reported or proposed to be related to the formation mechanism of outer membrane vesicles, and were individually knocked out or overexpressed to test their physiological functions. The effects on tolR and nlpI were the most significant. Knocking out both tolR and nlpI resulted in a 13.7% increase of secreted ß-carotene with a 35.6% increase of specific production. To supplement the loss of membrane components of the cells due to the increased membrane vesicle dispersion, the synthesis pathway of phosphatidylethanolamine was engineered. While overexpression of AccABCD and PlsBC in TW-013 led to 15% and 17% increases of secreted ß-carotene, respectively, the overexpression of both had a synergistic effect and caused a 53-fold increase of secreted ß-carotene, from 0.2 to 10.7 mg/g dry cell weight (DCW). At the same time, the specific production of ß-carotene increased from 6.9 to 21.9 mg/g DCW, a 3.2-fold increase. The AMVTS was also applied to a ß-carotene hyperproducing strain, CAR025, which led to a 24-fold increase of secreted ß-carotene, from 0.5 to 12.7 mg/g DCW, and a 61% increase of the specific production, from 27.7 to 44.8 mg/g DCW in shake flask fermentation. The AMVTS built in this study establishes a novel artificial transport mechanism different from natural protein-based cellular transport systems, which has great potential to be applied to various cell factories for the excretion of a wide range of hydrophobic compounds.

Characterization of microbial community structure and metabolic potential using Illumina MiSeq platform during the black garlic processing.

Black garlic is a distinctive garlic deep-processed product made from fresh garlic at high temperature and controlled humidity. To explore microbial community structure, diversity and metabolic potential during the 12days of the black garlic processing, Illumina MiSeq sequencing technology was performed to sequence the 16S rRNA V3-V4 hypervariable region of bacteria. A total of 677,917 high quality reads were yielded with an average read length of 416bp. Operational taxonomic units (OTU) clustering analysis showed that the number of species OTUs ranged from 148 to 1974, with alpha diversity increasing remarkably, indicating the high microbial community abundance and diversity. Taxonomic analysis indicated that bacterial community was classified into 45 phyla and 1125 distinct genera, and the microbiome of black garlic samples based on phylogenetic analysis was dominated by distinct populations of four genera: Thermus, Corynebacterium, Streptococcus and Brevundimonas. The metabolic pathways were predicted for 16S rRNA marker gene sequences based on Kyoto Encyclopedia of Genes and Genomes (KEGG), indicating that amino acid metabolism, carbohydrate metabolism and membrane transport were important for the black garlic fermentation process. Overall, the study was the first to reveal microbial community structure and speculate the composition of functional genes in black garlic samples. The results contributed to further analysis of the interaction between microbial community and black garlic components at different stages, which was of great significance to study the formation mechanism and quality improvement of black garlic in the future.

Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria

ABSTRACT Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.

Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria.

Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.

Corynebacterium accolens Releases Antipneumococcal Free Fatty Acids from Human Nostril and Skin Surface Triacylglycerols.

UNLABELLED: Bacterial interspecies interactions play clinically important roles in shaping microbial community composition. We observed that Corynebacterium spp. are overrepresented in children free of Streptococcus pneumoniae (pneumococcus), a common pediatric nasal colonizer and an important infectious agent. Corynebacterium accolens, a benign lipid-requiring species, inhibits pneumococcal growth during in vitro cocultivation on medium supplemented with human skin surface triacylglycerols (TAGs) that are likely present in the nostrils. This inhibition depends on LipS1, a TAG lipase necessary for C. accolens growth on TAGs such as triolein. We determined that C. accolens hydrolysis of triolein releases oleic acid, which inhibits pneumococcus, as do other free fatty acids (FFAs) that might be released by LipS1 from human skin surface TAGs. Our results support a model in which C. accolens hydrolyzes skin surface TAGS in vivo releasing antipneumococcal FFAs. These data indicate that C. accolens may play a beneficial role in sculpting the human microbiome. IMPORTANCE: Little is known about how harmless Corynebacterium species that colonize the human nose and skin might impact pathogen colonization and proliferation at these sites. We show that Corynebacterium accolens, a common benign nasal bacterium, modifies its local habitat in vitro as it inhibits growth of Streptococcus pneumoniae by releasing antibacterial free fatty acids from host skin surface triacylglycerols. We further identify the primary C. accolens lipase required for this activity. We postulate a model in which higher numbers of C. accolens cells deter/limit S. pneumoniae nostril colonization, which might partly explain why children without S. pneumoniae colonization have higher levels of nasal Corynebacterium. This work narrows the gap between descriptive studies and the needed in-depth understanding of the molecular mechanisms of microbe-microbe interactions that help shape the human microbiome. It also lays the foundation for future in vivo studies to determine whether habitat modification by C. accolens could be promoted to control pathogen colonization.

Engineering Corynebacterium crenatum to produce higher alcohols for biofuel using hydrolysates of duckweed (Landoltia punctata) as feedstock.

Early trials have demonstrated great potential for the use of duckweed (family Lemnaceae) as the next generation of energy plants for the production of biofuels. Achieving this technological advance demands research to develop novel bioengineering microorganisms that can ferment duckweed feedstock to produce higher alcohols. In this study, we used relevant genes to transfer five metabolic pathways of isoleucine, leucine and valine from the yeast Saccharomyces cerevisiae into the bioengineered microorganism Corynebacterium crenatum. Experimental results showed that the bioengineered strain was able to produce 1026.61 mg/L of 2-methyl-1-butanol by fermenting glucose, compared to 981.79 mg/L from the acid hydrolysates of duckweed. The highest isobutanol yields achieved were 1264.63 mg/L from glucose and 1154.83 mg/L from duckweed, and the corresponding highest yields of 3-methyl-1-butanol were 748.35 and 684.79 mg/L. Our findings demonstrate the feasibility of using bioengineered C. crenatum as a platform to construct a bacterial strain that is capable of producing higher alcohols. We have also shown the promise of using duckweed as the basis for developing higher alcohols, illustrating that this group of plants represents an ideal fermentation substrate that can be considered the next generation of alternative energy feedstocks.

Isolation and characterization of crude oil degrading bacteria from the Persian Gulf (Khorramshahr provenance).

Fifteen crude oil degrading bacteria were isolated from oil contaminated sites in the Persian Gulf at Khorramshahr provenance. These bacteria were screened with two important factors such as growth rate on crude oil and hydrocarbon biodegradation, and then three strains were selected from 15 isolated strains for further study. One strain (PG-Z) that show the best crude oil biodegradation was selected between all isolates. Nucleotides sequencing of the gene encoding for 16S rRNA show that strain PG-Z belong to Corynebacterium variabile genus. This strain was efficient in degrading of crude oil. This strain was capable to degraded 82% of crude-oil after one week incubation in ONR7a medium. The PG-Z strain had high emulsification activity and biosurfactant production between all isolates. GC-MS analysis shows that C. variabile strain PG-Z can degrade different alkanes in crude oil.

Biodegradation of petroleum hydrocarbons in the presence of nickel and cobalt.

Bioremediation of environments co-contaminated with hydrocarbons and heavy metals often pose a challenge as heavy metals exert toxicity to existing communities of hydrocarbon degraders. Multi-resistant bacterial strains were studied for ability to degrade hydrocarbons in chemically defined media amended with 5.0 mM Ni(2+), and Co(2+). The bacteria, Pseudomonas aeruginosa CA207Ni, Burkholderia cepacia AL96Co, and Corynebacterium kutscheri FL108Hg, utilized crude oil and anthracene without lag phase at specific growth rate spanning 0.3848-0.8259 per day. The bacterial populations grew in hydrocarbon media amended with nickel (Ni) and cobalt (Co) at 0.8393-1.801 days generation time (period of exponential growth, t = 15 days). The bacteria degraded 96.24-98.97, and 92.94-96.24% of crude oil, and anthracene, respectively, within 30 days without any impedance due to metal toxicity (at 5.0 mM). Rather, there was reduction of Ni and Co concentrations in the axenic culture 30 days post-inoculation to 0.08-0.12 and 0.11-0.15 mM, respectively. The metabolic functions of the bacteria are active in the presence of toxic metals (Ni and Co) while utilizing petroleum hydrocarbons for increase in biomass. These findings are useful to other baseline studies on decommissioning of sites co-contaminated with hydrocarbons and toxic metals.

A silicon cell cycle in a bacterial model of calcium phosphate mineralogenesis.

The prokaryote Corynebacterium matruchotii produces calcium phosphate (bone salt) and may serve as a convenient model for examining individual factors relevant to vertebrate calcification. A factor of current clinical uncertainty is silicon. To investigate its possible role in biomineralisation advanced optical (digital deconvolution and 3D fluorescent image rendering) and electron microscopy (EDX microanalysis and elemental mapping) were applied to calcifying microbial colonies grown in graded Si concentrations (0-60mM). Cell viability was confirmed throughout by TO-PRO-3-iodide and SYTO-9 nucleic acid staining. It was observed that calcium accumulated in dense intracellular microspherical objects (types i-iii) as nanoparticles (5 nm, type i), nanospheres (30-50 nm, type ii) and filamentous clusters (0.1-0.5 µm, type iii), with a regular transitory Si content evident. With bacterial colony development (7-28 days) the P content increased from 5 to 60%, while Si was displaced from 60 to 5%, distinguishing the phenomenon from random contamination, and with a significant relationship (p<0.001) found between calcified object number and Si supplementation (optimum 0.01mM). The Si-containing, intracellular calcified objects (also positive for Mg and negative with Lysensor blue DND-167 for acidocalcisomes) were extruded naturally in bubble-like chains to complete the cycle by coating the cell surface with discrete mineral particles. These could be harvested by lysis, French press and density fractionation when Si was confirmed in a proportion. It was concluded that the unexplained orthopaedic activity of Si may derive from its special property to facilitate calcium phosphorylation in biological systems, thereby recapitulating an ancient and conserved bacterial cycle of calcification via silicification.

Identification of McbR as transcription regulator of aecD and genes involved in methionine and cysteine biosynthesis in Corynebacterium jeikeium K411.

The C-S lyase aecD (MetC) from skin corynebacteria plays an important role in body odour formation by releasing odoriferous sulfanylalkanols from cysteine conjugates in human axilla secretions. The expression of the aecD gene from Corynebacterium jeikeium K411, a strain originally isolated from the human axilla, was down-regulated in cells grown in minimal medium supplemented with methionine. A candidate transcription regulator binding in front of the aecD coding region was detected by DNA affinity chromatography and identified as McbR by peptide mass fingerprinting. A 16-bp McbR-binding site was localized in the mapped promoter region of the aecD gene. The binding of purified McbR protein to the 16-bp sequence motif was demonstrated by DNA band shift assays. Comparative DNA microarray hybridizations and bioinformatic motif searches revealed the gene composition of the McbR regulon from C. jeikeium, including 28 genes that are organized in 16 transcription units. The McbR protein from C. jeikeium K411 directly regulates genes involved in methionine uptake and biosynthesis, in cysteine biosynthesis and sulfate reduction, and in the biosynthesis of amino acids belonging to the aspartate family.

Polyprenylated benzoylphloroglucinol-type derivatives including novel cage compounds from Hypericum erectum.

Hyperici erecti herba (Hypericum erectum THUNB.) showed a suppressive effect on generation of isovaleric acid by Corynebacterium xerosis. An ethyl acetate (AcOEt) soluble fraction of methanol extract of H. erectum showed the activity. The AcOEt fraction was separated by various successive choromatographical methods to give seven new compounds 1-7 along with some known compounds. The structures of the new compounds were elucidated to be polyprenylated benzoylphloroglucinol derivatives by means of HR-MS and NMR spectra including 2D-NMR. Some of these compounds had novel cage structures having benzoyltricyclo[3,3,1,1(3,7)]decane and benzoyltricyclo[4,3,1,1(3,8)]undecane skeletons arising from a polyprenylated phloroglucinol precursor by a transannular cyclization reaction. The isolated compounds were tested for suppressive activity, but they showed only weak activity.

Antioxidative and antibacterial effects of seeds and fruit rind of nutraceutical plants belonging to the Fabaceae family.

In the present study, the seeds and fruit rind of six plants of the Fabaceae family were selected to evaluate their potential as antioxidant and antibacterial agents. The dried powders were individually extracted with various organic solvents by the cold percolation method, were evaluated for antibacterial activity and methanol extracts used for antioxidant activities. Total phenol, protein and sugar contents were also measured. Antioxidant activities were measured by DPPH free radical scavenging activity, superoxide anion radical scavenging activity and reducing capacity assessment. Antibacterial activity was measured by the agar well diffusion method against four Gram positive and four Gram negative bacteria. The methanol extract of the fruit rind of C. indica showed the maximum DPPH free radical scavenging activity, superoxide anion radical scavenging activity, a high reducing capacity assessment and also had the highest total phenol content. There was a direct correlation between the phenol content and the antioxidant activity. The antibacterial activity of all the extracts was more pronounced on Gram positive bacteria than on Gram negative bacteria. Thus, the fruit rind of C. indica showed the best antioxidant and antibacterial activities.

Biosurfactant production by Corynebacterium kutscheri from waste motor lubricant oil and peanut oil cake.

AIM: Production and characterization of biosurfactant from renewable sources. METHODS AND RESULTS: Biosurfactant production was carried out in 3-l fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (9.8 mg ml(-l)) and biosurfactant production (6.4 mg ml(-l)) occurred with peanut oil cake at 120 and 132 h, respectively. Chemical characterization of the biosurfactant revealed that it is a glycolipopeptide with chemical composition of carbohydrate (40%), lipid (27%) and protein (29%). The biosurfactant is able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene; the emulsification activity was comparatively higher than the activity found with Triton X-100. CONCLUSION: This study indicates the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources like waste motor lubricant oil and peanut oil cake. Emulsification activity found with the biosurfactant against different hydrocarbons showed the possibility of the application of biosurfactants against diverse hydrocarbon pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained from the study could be useful for large-scale biosurfactant production using economically cheaper substrates. Information obtained in emulsification activity and laboratory-scale experiment on bioremediation inferred that bioremediation of hydrocarbon-polluted sites may be treated with biosurfactants or the bacteria that produces it.

L-lysine fermentation.

Amino acids are the basic bioelements of proteins, which are the most important macromolecules for the functions of humans and animals. Out of the 20 L-amino acids, ecumenically found in most of living organisms, L-lysine is one of the 9 amino acids which are essential for human and animal nutrition. L-lysine is useful as medicament, chemical agent, food material (food industry) and feed additive (animal food). Its demand has been steadily increasing in recent years and several hundred thousands tones of L-lysine (about 800,000 tones/year) are annually produced worldwide almost by microbial fermentation. The stereospecificity of amino acids (the L isomer) makes the fermentation advantageous compared with synthetic processes. Mutant auxotrophic or resistant to certain chemicals strains of so-called gram positive coryneform bacteria are generally used, including the genera Brevibacterium and Corynebacterium, united to the genus. The significance of Research and Development increased rapidly since the discovery of fermentative amino acid production in the fifties (S. Kinoshita et al., Proceedings of the International Symposium on Enzyme Chemistry 2:464-468 (1957)), leading to innovative fermentation processes which replaced the classical manufacturing methods of L-lysine like acid hydrolysis. L-Lysine is separated and purified by suitable downstream processes involving classical separation or extraction methods (ultrafiltration or centrifugation, separation or ion exchange extraction, crystallization, drying) and is sold as a powder. Alternatively, spray dried pellets or liquid fermentation broth can be used as animal feed supplement. On behalf of today's strong competition in amino acid industry, Biotechnology companies are continuously aiming in innovative research developments and use complex management concepts and business strategies, towards gaining market leadership in the field of amino acid production.

Evaluation of bioremediation effectiveness on crude oil-contaminated sand.

A treatability study was conducted using sea sand spiked with 3% or 6% (w/w) of Arabian light crude oil to determine the most effective bioremediation strategies for different levels of contamination. The sea sand used in the study was composed of gravel (0.1%), sand (89.0%), and silt and clay (10.9%). The water content of the sea sand was adjusted to 12.6% (w/w) for the study. Different combinations of the following treatments were applied to the sand in biometer flasks: the concentration of oil (3% or 6%), the concentration of a mixture of three oil-degrading microorganisms (Corynebacterium sp. IC-10, Sphingomonas sp. KH3-2 and Yarrowia sp. 180, 1x10(6) or 1x10(8) cells g-1 sand), the concentration of the surfactant Tween 80 (1 or 10 times the critical micelle concentration), and the addition of SRIF in a C:N:P ratio of 100:10:3. Three biometer flasks per combination of experimental conditions were incubated, and the performance of each treatment was examined by monitoring CO2 evolution, microbial activity, and oil degradation rate. The results suggest that the addition of inorganic nutrients accelerated the rate of CO2 evolution by a factor of 10. The application of oil-degrading microorganisms in a concentration greater than that of the indigenous population clearly increased biodegradation efficiency. The application of surfactant slightly enhanced the oil degradation rate in the contaminated sand treated with the higher concentration of oil-degrading microorganisms. The initial CO2 evolution rate was shown to efficiently evaluate the treatability test by providing significant data within a short period, which is critical for the rapid determination of the appropriate bioremediation approach. The measurements of microbial activity and crude oil degradation also confirmed the validity of the CO2 evolution rate as an appropriate criterion.

Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium.

The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l(-1) of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l(-1)h(-1)) every 2 h to the production medium increased the production of methionine to 3.39 g l(-1). A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers.

Effects of crude oil on phospholipid fatty acid compositions of marine hydrocarbon degraders: estimation of the bacterial membrane fluidity.

In this study, we investigated, in vitro, the effects of petroleum hydrocarbons on the phospholipid ester-linked fatty acid composition of Corynebacterium sp. Strain 8. The usual ratio of monounsaturated fatty acids E/Z (or trans/cis) was calculated. This ratio led to unexpected results because we found similar values for growths on either a hydrophobic substrate (crude oil) or a soluble carbon source (rich medium). The use of such an indicator seemed limited for monitoring an environmental stress, so we proposed an index based on the homeoviscous adaptation theory. A membrane viscosity index was defined and applied to Corynebacterium sp. Strain 8 (in vitro growth) and to a sedimentary community (in situ experiment). The results allowed us to estimate the membrane fluidity of both an isolated strain and a bacterial community in accordance with the medium hydrophobicity.

A microbial consortium isolated from a crude oil sample that uses asphaltenes as a carbon and energy source.

A microbial consortium capable of mineralizing asphaltenes was obtained from the Maya crude oil. The enrichment system was built with a glass column reactor containing mineral medium supplied with asphaltenes as energy and carbon source. The consortium growth was evaluated in Casoy agar during 40 weeks. The steady-state phase of the enriched bacterial community was observed after 10 weeks when the culture reach 10(5) to 10(6) CFU ml(-1). The isolates belong to bacterial genus reported for degradation of other hydrocarbons and they were identified as Corynebacterium sp., Bacillus sp., Brevibacillus sp. and Staphylococcus sp. The bacterial consortium growth was evaluated by a viable counts during 14 days exposed to different aeration, temperature, salinity, and pH conditions. The ability of the consortium to mineralize asphaltenes was evaluated using the method of ISO 9439 in glass column reactors of 20 x 3.2 cm during 13 days. Temperatures of 55 degrees C and salinity of 1.8% were growth limiting. The respiration of the microbial consortium using asphaltenes as a sole carbon source (800 micromoles CO2 in 13 days) was significantly higher than those of the samples containing only the microbial consortium (200 micromoles CO2) or only asphaltenes (300 micromoles CO2). These results indicated the existence of asphaltenes-degradating microbes in the crude oil and confirmed that the consortium could mineralize asphaltenes in conditions of room temperature, salinity of 100 ppm, aeration of 1 l min(-1) and pH of 7.4.

[Effect of photosynthetic bacteria and compost on degradation of petroleum products in soil]. / Vliianie fotosinteziruiushchikh bakterii i komposta na degradatsiiu nefteproduktov v pochve.

Addition of diesel fuel and waste engine oil to soil was found to cause biostimulation of hydrocarbon-oxidizing microorganisms. Corynebacteria constitute a large group of hydrocarbon-oxidizing microorganisms. Addition of a liquid culture of photosynthetic bacteria to soil not only facilitates degradation of petroleum products, but also stimulates growth of hydrocarbon-oxidizing microorganisms. Combined addition of photosynthetic bacteria and compost to soil polluted with petroleum products causes even a more significant increase in the count of hydrocarbon-oxidizing bacteria and substantially increases the rate of pollutant degradation.

Production of a novel polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum.

The production of a novel polygalacturonic acid bioflocculant REA-11 from a newly isolated strain, Corynebacterium glutamicum CCTCC M201005, was investigated. Sucrose was chosen as a carbon source for REA-11 production. Complex nitrogen sources containing urea and an organic nitrogen compound enhanced both bacterial growth and REA-11 production, among which urea plus corn steep liquor was shown to be the most efficient combination. A cost-effective medium for REA-11 production mainly comprised 17 g/l sucrose, 0.45 g/l urea, and 5 ml/l corn steep liquor, under which conditions the flocculating activity reached 390 U/ml. The molar ratio of carbon to nitrogen (C/N) significantly affected REA-11 production, where a C/N ratio of 20:1 was shown to be the best. Interestingly, by simultaneously feeding sucrose and urea at a C/N ratio of 20:1 at 24 h of fermentation, REA-11 production (458 U/ml) was enhanced by 17% compared to the control. In a 10 l jar fermentor, lower dissolved oxygen tension was favorable for REA-11 production: a flocculating activity of 520 U/ml was achieved at a kappaLa of 100 h(-1). REA-11 raw product is relatively thermo-stable at acidic pH ranges of 3.0-6.5. Preliminary application studies showed that REA-11 had stronger flocculating activity to Kaolin clay suspension compared to chemical flocculants. In addition, the capability of decolorizing molasses wastewater indicates the industrial potential of this novel bioflocculant.