Mulumebet Worku

Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes

The main objective of this research is to develop, by adaptive evolution, mutant strains of Enterobacter aerogenes ATCC 13048 that are capable of withstanding high glycerol concentration as well as resisting ethanol-inhibition. The mutant will be used for high ethanol fermentation from glycerol feedstock. Ethanol production from pure (P-) and recovered (R-) glycerol using the stock was evaluated. A six-tube-subculture-generations method was used for developing the mutant. This involved subculturing the organism six consecutive times in tubes containing the same glycerol and ethanol concentrations at the same culture conditions. Then, the glycerol and/or ethanol concentration was increased and the six subculture generations were repeated. A strain capable of growing in 200 g/L glycerol and 30 g/L ethanol was obtained. The ability of this mutant, vis-à-vis the original strain, in utilizing glycerol in a high glycerol containing medium, with the concomitant ethanol yield, was assessed. Tryptic soy broth without dextrose (TSB) was used as the fermentation medium. Fermentation products were analyzed using HPLC.In a 20 g/L glycerol TSB, E. aerogenes ATCC 13048 converted 18.5 g/L P-glycerol and 17.8 g/L R-glycerol into 12 and 12.8 g/L ethanol, respectively. In a 50 g/L P-glycerol TSB, it utilized only 15.6 g/L glycerol; but the new strain used up 39 g/L, yielding 20 g/L ethanol after 120 h, an equivalence of 1.02 mol ethanol/mol-glycerol. This is the highest ethanol yield reported from glycerol bioconversion. The result of this P-glycerol fermentation can be duplicated using the R-glycerol from biodiesel production.

Enzymatic activity of Lactobacillus reuteri grown in a sweet potato based medium with the addition of metal ions

The effect of metal ions on the enzymatic activity of Lactobacillus reuteri was studied. The enzymatic activity was determined spectrophotometrically using the corresponding substrate. In the control group, L. reuteri MF14-C, MM2-3, SD2112, and DSM20016 produced the highest α-glucosidase (40.06 ± 2.80 Glu U/mL), β-glucosidase (17.82 ± 1.45 Glu U/mL), acid phosphatase (20.55 ± 0.74 Ph U/mL), and phytase (0.90 ± 0.05 Ph U/mL) respectively. The addition of Mg2+ and Mn2+ led to enhance α-glucosidase produced by L. reuteri MM2-3 by 113.6% and 100.6% respectively. α-Glucosidase produced by MF14-C and CF2-7F was decrease in the presence of K+ by 65.8 and 69.4% respectively. β-Glucosidase activity of MM7 and SD2112 increased in the presence of Ca2+ (by 121.8 and 129.8%) and Fe2+ (by 143.9 and 126.7%) respectively. Acid phosphatase produced by L. reuteri CF2-7F and MM2-3 was enhanced in the presence of Mg2+, Ca2+ or Mn2+ by (94.7, 43.2, and 70.1%) and (63.1, 67.8, and 45.6%) respectively. On the other hand, Fe2+, K+, and Na+ caused only slight increase or decrease in acid phosphatase activity. Phytase produced by L. reuteri MM2-3 was increase in the presence of Mg2+ and Mn2+ by 51.0 and 74.5% respectively. Ca2+ enhanced phytase activity of MM2-3 and DSM20016 by 27.5 and 28.9% respectively. The addition of Na+ or Fe2+ decreased phytase activity of L. reuteri. On average, Mg2+ and Mn2+ followed by Ca2+ led to the highest enhancement of the tested enzymes. However, the effect of each metal ion on the enzymatic activity of L. reuteri was found to be a strain dependent. Therefore, a maximized level of a target enzyme could be achieved by selecting a combination of specific strain and specific metal ion.

Optimization of cultural conditions for conversion of glycerol to ethanol by Enterobacter aerogenes S012

The aim of this research is to optimize the cultural conditions for the conversion of glycerol to ethanol by Enterobacter aerogenes S012. Taguchi method was used to screen the cultural conditions based on their signal to noise ratio (SN). Temperature (°C), agitation speed (rpm) and time (h) were found to have the highest influence on both glycerol utilization and ethanol production by the organism while pH had the lowest. Full factorial design, statistical analysis, and regression model equation were used to optimize the selected cultural parameters for maximum ethanol production. The result showed that fermentation at 38°C and 200 rpm for 48 h would be ideal for the bacteria to produce maximum amount of ethanol from glycerol. At these optimum conditions, ethanol production, yield and productivity were 25.4 g/l, 0.53 g/l/h, and 1.12 mol/mol-glycerol, repectively. Ethanol production increased to 26.5 g/l while yield and productivity decreased to 1.04 mol/mol-glycerol and 0.37 g/l/h, respectively, after 72 h. Analysis of the fermentation products was performed using HPLC, while anaerobic condition was created by purging the fermentation vessel with nitrogen gas.

Effect of probiotic supplementation on growth and global gene expression in dairy cows

ABSTRACT Use of probiotic supplements as a non-chemical approach to promote health has increased in animal production. The present study evaluated the effect of oral probiotic administration on growth and global gene expression profile in dairy cows. Lactating Holstein-Friesian cows received a daily dose (50 ml) of a commercial probiotic (containing Lactobacillus acidophilus, Saccharomyces cerevisiae, Enterococcus faecium, Aspergillus oryza and Bacillus subtilis) for 60 days. A microarray experiment was performed with blood collected at day-0 and day-60. Although probiotic supplementation had no effect on body weight, PCV and total protein concentration in plasma (P > 0.05), per cent lymphocyte count increased (P < 0.05), and per cent neutrophil count decreased (P < 0.05) in probiotic-treated animals. Gene expression analysis identified 10,859 differentially expressed genes, 1168 up-regulated and 9691 down-regulated genes, respectively, following probiotic treatment. Single experiment pathway analysis identified 87 bovine pathways impacted by probiotic treatment. These pathways included the Toll-like receptor (TLR), inflammation response and Wingless signalling pathways. Oral administration of probiotics to dairy cows had a systemic effect on global gene expression, such as on genes involved in immunity and homeostasis. The results of this study show that the utilization of probiotics in animal agriculture impacts genes important to dairy cow health and production.

Enzymatic Activity of Lactobacillus Grown in a Sweet Potato Base Medium

The objective of this work was to study the enzymatic activity of Lactobacillus in a sweet potato-based medium (SPM). SPM was formed using an extract from baked sweet potatoes and supplemented with 4 g/L of each nitrogen source (beef extract, yeast extract, and proteose peptone #3). Lactobacillus strains were grown in SPM and MRS for 16 h at 37 °C and then plated to determine the final bacterial populations. The strains were screened spectrophotometrically for α-glucosidase, β-glucosidase, acid phosphatase, and phytase using the corresponding substrate. Our results showed no significant (p > 0.05) differences in the final bacterial populations of Lactobacillus strains grown in SPM and MRS. All Lactobacillus strains, except L. reuteri, showed similar α-glucosidase and β-glucosidase activity in SPM and MRS. L. reuteri showed lower α-glucosidase and higher β-glucosidase activity in SPM compared to MRS. Acid phosphatase activity of Lactobacillus in SPM was similar to that in MRS except for L. reuteri SD2112 having higher acid phosphatase in SPM than MRS. In regard to phytase, all strains showed higher activity in SPM than MRS. Strains of L. reuteri showed the highest enzymatic activity of α-glucosidase, acid phosphatase, and phytase whereas L. delbrueckii subsp. bulgaricus SR35 showed the highest β-glucosidase activity. Thus, the growth of Lactobacillus in a SPM could result in enhanced or comparable level of enzymatic activity while showing similar growth compared to MRS.

Transcriptional profiling of the effect of lipopolysaccharide (LPS) pretreatment in blood from probiotics-treated dairy cows

Probiotic supplements are beneficial for animal health and rumen function; and lipopolysaccharides (LPS) from gram negative bacteria have been associated with inflammatory diseases. In this study the transcriptional profile in whole blood collected from probiotics-treated cows was investigated in response to stimulation with lipopolysaccharides (LPS) in vitro. Microarray experiment was performed between LPS-treated and control samples using the Agilent one-color bovine v2 bovine (v2) 4x44K array slides. Global gene expression analysis identified 13,658 differentially expressed genes (fold change cutoff ≥ 2, P < 0.05), 3816 upregulated genes and 9842 downregulated genes in blood in response to LPS. Treatment with LPS resulted in increased expression of TLR4 (Fold change (FC) = 3.16) and transcription factor NFkB (FC = 5.4) and decreased the expression of genes including TLR1 (FC = − 2.54), TLR3 (FC = − 2.43), TLR10 (FC = − 3.88), NOD2 (FC = − 2.4), NOD1 (FC = − 2.45) and pro-inflammatory cytokine IL1B (− 3.27). The regulation of the genes involved in inflammation signaling pathway suggests that probiotics may stimulate the innate immune response of animal against parasitic and bacterial infections. We have provided a detailed description of the experimental design, microarray experiment and normalization and analysis of data which have been deposited into NCBI Gene Expression Omnibus (GEO): GSE75240.

Microarray analysis of the effect of Cowpea (Vigna unguiculata) phenolic extract in bovine peripheral blood

ABSTRACT In this study, the effect of polyphenolic extracts from cowpea (Vigna unguiculata) on global gene expression in bovine peripheral blood was investigated. Blood collected from Holstein-Friesian cows (n = 10) was treated with 10 µg/mL of cowpea phenolic extract (CPE) and subsequently used for transcriptional profiling using the Agilent bovine (v2) 4 × 44 K array. Calculation of fold change in gene expression and pathway analysis was conducted using the GeneSpring GX software 13.0. Real-time quantitative PCR was performed to validate the microarray data. Phenolic extracts of cowpea impacted global gene expression and resulted in 3170 differentially expressed genes (p < .05); 1716 genes were upregulated and 1454 genes were downregulated. Exposure to CPE impacted 66 pathways (p < .05) including the Wnt signalling pathway, Toll-like receptor pathway, inflammation response pathway, MAPK cascade pathway, prostaglandin synthesis and regulation pathway, cell cycle pathway, insulin signalling pathway, and the adipogenesis pathway. Expression of immune markers such as CD40, CD68, Toll-like receptors, and Wnt signalling changed. Exposure to CPE modulated expression of genes associated with immunity and homeostasis. Transcriptional profiles of the response to polyphenols may aid in the design of targeted diets to influence animal production and health and thus requires further study.