Earticle

Currently, the most promising microorganism used for the bio-production of butyric acid is Clostridium tyrobutyricum ATCC 25755; however, it is unable to use sucrose as a sole carbon source. Consequently, a newly isolated strain, Bacillus sp. SGP1, that was found to produce a levansucrase enzyme, which hydrolyzes sucrose into fructose and glucose, was used in a co-culture with this strain, permitting C. tyrobutyricum ATCC 25755 to ferment sucrose to butyric acid. B. sp. SGP1 alone did not show any butyric acid production and the main metabolite produced was lactic acid. This allowed C. tyrobutyricum ATCC 25755 to utilize the monosaccharides resulting from the activity of levansucrase together with the lactic acid produced by B. sp. SGP1to generate butyric acid, which was the main fermentative product within the co-culture. Furthermore, the final acetic acid concentration in the co-culture was significantly lower when compared with pure C. tyrobutyricum ATCC 25755 cultures grown on glucose. In fed-batch fermentations, the optimum conditions for the production of butyric acid were around pH 5.50 and a temperature of 37°C. Under these conditions, the final butyrate concentration was 34.2±1.8 g/L with yields of 0.35±0.03 g butyrate/g sucrose and maximum productivity of 0.3±0.04 g/L/h.

In a medium containing sodium propionate, pentanoic acid was produced by Megasphaera sp. BS-4 during anaerobic fermentation of fructose. Statistical optimization of medium was carried out in order to find a medium composition for the highest production of pentanoic acid by Megasphaera sp. BS-4. According to one-factor-at-a-time (OFAT) experiments, pH and two substrates, acetate and propionate, were found to have prominent effects on the pentanoic acid production. Especially, the concentration of propionate significantly influenced the production of pentanoic acid. As the initial concentration of sodium propionate increased, pentanoic acid production also increased. Meanwhile, acetate concentration influenced cell growth of Megasphaera sp. BS-4. But high concentration of acetate also stimulated the production of hexanoic acid, not intended to produce in this study. The production of pentanoic acid increased to 10 gL-1 in a medium containing 20 gL-1 fructose and additional two substrates, 3 gL-1 of sodium acetate and 15 gL-1 of sodium propionate. Further statistical experiments followed by Response Surface Methodology (RSM) were conducted for the understanding of optimum medium composition.

Megasphaera elsdenii NCIMB 702410 is a gram-negative strictly anaerobic bacterium isolated in the rumen of cattle and sheep. This strain is able to utilize sucrose to produce 4.53 g/L of hexanoic acid in a batch culture. However, high titer of hexanoic acid is toxic to the cellular growth. Therefore, the objective of this study is to improve hexanoic acid production of Megasphaera elsdenii by using reactive extraction method. The extraction of hexanoic acid was performed with 10 % (v/v) Alamine 336 in Oleyl alcohol as a solvent and this method led the selective removal of hexanoic acid from the fermentation broth with high recovery rate. Compared to conventional fermentation, in situ extractive fermentation showed a 5.7-folds higher production of hexanoic acid (25.83 g/L) due to immediate extraction of hexanoic acid. In addition, this extraction method was applied to fed-batch fermentation, resulting that the titer of hexanoic acid was increased up to about 70.04 g/L (15.5-folds higher). In conclusion, the efficient extraction process is crucial for fermentative production of hexanoic acid by Megasphaera elsdenii NCIMB 702410 using sucrose as a sole carbon.

The draft genome of a hexanoic acid-producing bacterium Megasphaera sp. BS-4 was obtained using GS-FLX titanium and 441,216,354 total base pairs data was generated from 1,213,411 reads. Both fragment library and paired-end library were used as sequencing templates and one-fouth plate was used to generate sequence reads for each library. Assembly was carried out using Newbler and the assembled genome was annotated using RAST. The assembled genome was consisted with 7 scaffolds and 82 contigs. The approximate size and coverage of the genome was 2.8 Mb and 155X respectively. The G+C content of the genome was 48.99%. The genome contained 2,713 ORFs, 5 rRNA genes and 53 tRNA genes. The longest contig was 384,514 bp. A total of 1966 genes were categorized based on COG. Among these genes, 240 genes were assigned to ‘mino acid transport and metabolism’(E). ‘arbohydrate transport and metabolism’(G) and ‘nergy production and conversion genes’(C) were 111 genes and 152 genes.

Butanol and ethanol could be good alternative biofuels for transportation. The bio- butanol and ethanol can be produced simultaneously in cultures of solventogenic Clostridium species through the acetonebutanol-ethanol (ABE) fermentation. For the isolation of microorganism capable of producing only butanol and ethanol without the acetone production, tidal flat samples were collected from seashore area of Tae-An, Korea. The isolated bacterium was designated Clostridium sp. nov. BS-5 and its 16S rDNA revealed 96% similarity to Clostirdium algidixylanolyticum SPL73T. Clostridium sp. nov. BS-5 produced acetone, ethanol, butanol, acetic acid, and butyric acid using glucose as a carbon source. To improve alcohol production, P2 medium modified with 3% sea salt was used and 0~50 g/L of yeast extract was initially added to investigate the effects of trace nutrients on two alcohol production. Ethanol and butanol concentration increased by the addition of yeast extract, up to 20 g/L. Clostridium sp. nov. BS-5 produced 14.71 g/L of alcohols with the yield of 0.54 g/g (0.20 g/L/hr). This result shows that Clostridium sp. nov. BS-5 is a promising new microorganism for two alcohol production.

Hexanoic acid, six carbon and saturated fatty acid could be produced by bacteria like Clostridium kluyveri, Megsphaera elsdenii, and Clostridium sp. BS-1. In this study, we investigated optimization of hexanoic acid production by Clostridium sp. BS-1. To determine the effective factors for hexanoic acid production, fractional factorial design was used and concentration range of the factors was determined via steepest ascent method (SAM). Response surface methodology (RSM) was used for investigation of interactions between the factors and optimized hexaonoic acid production. The proposed solutions from RSM were verified experimentally. Yeast extract, FeSO4•7H2O, and butyric acid were determined as the effective factors.

In a previous study, we co-cultured the novel strain Bacillus licheniformis SGP1 with Clostridium tyrobutyricum ATCC 25755 for butyric acid production using sucrose as a carbon source in RCM medium. In this co-culture, B. licheniformis produces levansucrase enzyme in the medium which degrades sucrose into monosaccharides allowing C. tyrobutyricum to utilize them along with the lactic acid produced by B. licheniformis for butyric acid production. In this study, we wanted to determine the effect of different factors on the growth of this novel Bacillus strain and its levansucrase production and activity. Optimum pH was around 7.0. Optimum temperature for growth is 37⁰C. Enzyme production is decreased dramatically as the temperature rises from 30⁰C to 43⁰C and the reverse happens for enzyme activity. This enzyme has a molecular weight of 55 KDa. In fed-batch fermentation at pH 5.50 and temperature of 37⁰C, the co-culture could give a final butyrate concentration of 36 g/L with productivity equals 0.24 g/L/hr, while at 40⁰C, we could get 28.6 g/L and 0.34 g/L/hr respectively. Real time PCR experiment was done finally to determine the growth pattern of the two strains in the co-culture.

Covalent attachment, aggregate coat ing or entrapment of enzymes with nanof ibers yields highly act ive and stable systems for the applicat ions in the various f ields such ac bioconversion, biomedicals, and biosensors. In this study, we compared the stabilit ies of enzymes, which were immobilized by several nano-based biosilif icat ion. Glutathione Stransferase (GST) as the model enzyme are used, and GST and R5 tagged GST gene was amplif ied by PCR and inserted into the pET-28 a(+) vector (Novagen). It was expressed in E.coli BL21, and then purif ied with aff inity chromatography. GST was immobilized into the nanof iber consisted of polystyrene (PS) and poly (styrene-co-maleic anhydride) (PSMA). R5 catalyzed the format ion of silica nanopart icles (about 200 nm in diameter) with GST retained its act ivity.