Rapid Screening of Cultural Parameters for Extracellular Halophilic Glutaminase Production from Tetragenococcus Muriaticus FF5302 using the Plackett-Burman’s Experimental Design

The Plackett-Burman’s experiemental design was used to efficiently select the key cultural parameters for the production of halophilic glutaminase by moderating halophilic bacterium Tetragenococcus muriaticus FF5302. Eleven variables were selected, which involved glutamine, peptone, yeast extract, glucose, fructose, KCl, MgSO4, NaCl, temperature, pH, and inoculum size, to identify the most significant variables affecting halophilic glutaminase production in 12 experimental trials. The results of the statistical analyses demonstrated that glutamine, pH, and temperature had significant effects on halophilic glutaminase production. The maximum halophilic glutaminase activity of 199.27 U mL-1 was observed after 120 h of fermentation. After Plackett-Burman’s design experiments, the glutaminase activity was found to be 2.28 folds increase compared to basal conditions. Thus, the cultivation of T. muriaticus FF5302 under the optimal condition could enhance the production of halophilic glutaminase enzyme effectively.

Experimental Design to Improve Cell Growth and Ethanol Production in Syngas Fermentation by Clostridium carboxidivorans

Fermentation of gases from biomass gasification, named syngas, is an important alternative process to obtain biofuels. Sequential experimental designs were used to increase cell growth and ethanol production during syngas fermentation by Clostridium carboxidivorans. Based on ATCC (American Type Culture Collection) 2713 medium composition, it was possible to propose a best medium composition for cell growth, herein called TYA (Tryptone-Yeast extract-Arginine) medium and another one for ethanol production herein called TPYGarg (Tryptone-Peptone-Yeast extract-Glucose-Arginine) medium. In comparison to ATCC® 2713 medium, TYA increased cell growth by 77%, reducing 47% in cost and TPYGarg increased ethanol production more than four-times, and the cost was reduced by 31%. In 72 h of syngas fermentation in TPYGarg medium, 1.75-g/L of cells, 2.28 g/L of ethanol, and 0.74 g/L of butanol were achieved, increasing productivity for syngas fermentation.

Proteolytic Bacillus spp. associated with tannery industries: Conventional and molecular identification

Samples collected from different stages of the tannery industry were found to be alkaline (pH 7.52 to 12.11). A good number of bacteria were found to be associated with the samples. The bacterial count ranged in between 1.34×105 to 3.44×105 cfu/ml and 1.04×105 to 6×105 cfu/ml on nutrient agar (NA) and peptone yeast extract glucose agar (PYG) medium, respectively. The maximum bacterial count was observed in bating stage while the minimum count was in the deliming stage. Primarily, 70 isolates were selected based on their different colonial morphology. After heat shock test 27 isolates were finally selected for identification and proteolytic potential. All the selected isolates were the members of different species of the genus Bacillus. The conventionally identified species were B. stearothermophilus (9), B. subtilis (4), B. brevis (3), B. pumilus (3), B. alcalophilus (2), B. badius (2), B. firmus (2) and B. lentus (2). Four important proteolytic isolates of Bacillus were selected for molecular identification. The isolates were confirmed as Bacillus subtilis strain B20 (L/P/2/1), B. subtilis strain PB18 (D/P/3/1), Bacillus sp. strain BVC38 (D/P/3′/2) and B. amyloliqefaciens strain Egy25 (B/N/3′/1). Except B/N/3′/1 all the conventional identification was in accordance with the molecular identification as the isolate B/N/3′/1 was conventionally identified as B. pumilus (B/N/3′/1). All the isolates showed positive proteolytic activities on skim milk agar and the zone ratio was in between 2.61 ± 0.44 and 6.42 ± 0.95. These isolates could be commercially utilized in the tannery and detergent industries for their proteolytic activity.

Fretibacterium fastidiosum gen. nov., sp. nov., isolated from the human oral cavity

SGP1T, a strain belonging to a lineage of the phylum Synergistetes with no previously cultivated representatives was subjected to a comprehensive range of phenotypic and genotypic tests. For good growth the strain was dependent on co-culture with, or extracts from, selected other oral bacteria. Cells of strain SGP1T were asaccharolytic and major amounts of acetic acid and moderate amounts of propionic acid were produced as end products of metabolism in peptone-yeast extract-glucose broth supplemented with a filtered cell sonicate of Fusobacterium nucleatum subsp. nucleatum ATCC 25586T (25 %, v/v). Hydrogen sulphide was produced and gelatin was weakly hydrolysed. The major cellular fatty acids were C14 : 0, C18 : 0 and C16 : 0. The DNA G+C content of strain SGP1T was 63 mol%. Phylogenetic analysis of the full-length 16S rRNA gene showed that strain SGP1T represented a novel group within the phylum Synergistetes . A novel species in a new genus, Fretibacterium fastidiosum gen. nov., sp. nov., is proposed. The type strain of Fretibacterium fastidiosum is SGP1T ( = DSM 25557T = JCM 16858T).

Effects of Temperature and pH on the Growth of Bacteria Isolated from Blown Packs of Vacuum-Packaged Beef

Bacteria recovered from the microflora of blown packs of vacuum-packaged beef were identified as Leuconostoc mesenteroides, Lactococcus lactis, Carnobacterium maltaromaticum, and Clostridium estertheticum, with L. mesenteroides predominant. Isolates of these lactic acid bacteria all grew in peptone yeast extract glucose starch broth (PYGSB) at temperatures between −2 and 30°C but generally grew more slowly and over a more restricted temperature range in meat juice medium (MJM). A C. estertheticum isolate and the type strain of C. estertheticum subsp. estertheticum (ATCC 51377) both grew in PYGSB and MJM at similar rates at temperatures between −2 and 17°C and grew at 20°C in MJM but not in PYGSB. Square root models of the variation of the growth rate with temperature indicated that the C. maltaromaticum isolate and the C. estertheticum strains grew at similar rates that were faster than those of the other isolates at temperatures between 22 and 0°C. The L. mesenteroides and L. lactis isolates grew in PYGSB at pH 5.0, but the C. maltaromaticum isolate and both strains of C. estertheticum did not grow in PYGSB at pH ≤ 5.3. C. estertheticum stopped growing in MJM buffered at pH 6.5 when glucose was exhausted, although these bacteria then utilized lactate. The findings suggest that, like carnobacteria, C. estertheticum may predominate during the early stages of development of the spoilage microflora of vacuum-packaged beef but that C. estertheticum will likely be inhibited by a falling pH and so may be only a minor part of the spoilage microflora when maximum numbers are attained.

Brooklawnia cerclae gen. nov., sp. nov., a propionate-forming bacterium isolated from chlorosolvent-contaminated groundwater

Two novel facultatively anaerobic bacterial strains, BL-34T and BL-35, isolated from groundwater contaminated by a mixture of chlorosolvents were characterized using a polyphasic approach. The two strains exhibited essentially identical taxonomic features except for a vitamin B12 requirement by strain BL-35 for optimal growth. Phylogenetically, the isolates were affiliated with members of the family Propionibacteriaceae and were placed in a phylogenetic branch adjacent to, but distinct from, those of the genera Propionimicrobium, Propionibacterium, Luteococcus, Propioniferax and Tessaracoccus. The cells of the novel strains were Gram-positive, non-motile, non-spore-forming pleomorphic rods. They produced catalase but not oxidase, and nitrate reduction did not occur in peptone/yeast extract/glucose medium. Propionate and acetate were the predominant products of glucose fermentation. Fermentation occurred in the presence of 1,2-dichloroethane and 1,1,2-trichloroethane at concentrations up to at least 9.8 mM. The genomic DNA G+C content was 67.5–67.9 mol%. Menaquinone MK-9(H4) was the predominant respiratory quinone and meso-diaminopimelic acid was present in the cell-wall peptidoglycan layer. The major cellular fatty acids were C15 : 0 and anteiso-C15 : 0. On the basis of the results obtained in this study, strains BL-34T and BL-35 should be classified within a novel taxon, for which the name Brooklawnia cerclae gen. nov., sp. nov. is proposed. The type strain of Brooklawnia cerclae is BL-34T (=LMG 23248T=NRRL B-41418T). An additional strain, BL-35 (=LMG 23249=NRRL B-41419), was also characterized.

Differential Sensitivity of Clostridium botulinum Strains to Nisin is Not Biotype-Associated

The sensitivities of proteolytic and nonproteolytic Clostridium botulinum strains to nisin and other bacteriocins were investigated. Although there were statistically different nisin sensitivities among vegetative cells from 18 C. botulinum strains, these differences were not biotype-associated. When inoculated into tryptose peptone yeast extract glucose broth containing nisin at various levels, spores from strain 56 A were not inhibited at all by 100 IU/ml of nisin. About 2,500 IU/ml was required to inhibit growth for 30 d. In contrast, only 10 IU/ml was required to inhibit strain 169 for 30 d. Both strains were completely inhibited at the 10,000 IU/ml limit allowed in processed cheeses. Spores from strains having limited (strain 56A), moderate (strain 25675), or extreme (strain 169) nisin sensitivity were examined for bacteriocin-mediated inhibition by Lactococcus lactis 11454, Pediococcus pentosaceus 43200, P. pentosaceus 43201, and Lactobacillus plantarum BN using the spot-on-the-lawn method. While the differences in nisin sensitivity were confirmed, there was no statistically significant difference in their sensitivities to the bacteriocins produced by other lactic acid bacteria.

Inhibitory Effects of Various Salts and/or Ionic Strengths on Growth from Clostridium botulinum 52A Spores or Vegetative Cells

Growth response from spores and vegetative cells of Clostridium botulinum strain 52A in peptone-yeast extract-glucose (PYEG) broth at two pH levels (5.55 or 5.85) containing sodium acid pyrophosphate (SAPP) (0, 0.2, 0.4%), NaCl (0, 1.25, 2.50%) and/or potassium sorbate (KS) (0, 0.13, 0.26%) was measured as the mean A630 nm of 20 tubes at 37°C. Additional treatments contained KC1 and MgCl2 (0, 1.25, 2.50%) without SAPP or KS. Growth ratios (GR = treatment/control) based on time to reach A630 = 0.35 were calculated to compare effects of additives on strain 52A. Growth from spores was affected significantly (p≤0.01) by pH level. KS and KS/pH interactions were also significant factors in growth from both spores and vegetative cells; SAPP/pH interactions were significant for cell growth, only. Combinations of SAPP (0.2, 0.4%) NaCl (0%) and KS (0.26%) were the most favorable treatments for delaying growth from spores or vegetative cells. NaCl (1.25, 2.50%) decreased antibotulinal effects produced by combinations of SAPP and KS. Elimination of NaCl enhanced antibotulinal effects. Formulations containing KC1 or MgCl2 (without SAPP and KS) at the same molarity as the NaCl in earlier treatments (0.21, 0.43) resulted in inhibition of growth from vegetative cells greater than growth from spores in the presence of MgCl2 at M = 0.43 (ionic strength = 1.29). This inhibition was more evident at pH 5.55 than pH 5.85. This study in a model system suggests ionic strength and/or chloride salt may be important considerations when manipulating formulations of additives designed to control C. botulinum growth.

Inhibition of Clostridium botulinum Okra B by N-Acyl Amino Acid Ester (Nα-Cocoil Arginine Ethylester•DL-Pyrrolidone Carbonate)

An amino acid ester, Nα-cocoil-L-arginine ethylester·DL-pyrrolidone carbonate (CAE), was inhibitory to growth and toxin production of Clostridium botulinum okra in peptone-yeast extract-glucose (PYG) medium, pH 7.0, at 30°C. Addition of 10 mg of CAE/L to PYG medium delayed toxin production and 25 mg of CAE/L inhibited growth and toxin production, whereas 5 mg of CAE/L had no effect on both growth and toxin production.

Acetoin and Diacetyl Production by Homo- and Heterofermentative Lactic Acid Bacteria

Eleven strains of homofermentative and heterofermentative lactic acid bacteria were screened for acetoin (A) and diacetyl (D) production from pyruvate and citrate in a peptone-yeast extract-glucose broth. The homofermenters, except Streptococcus faecalis subsp. liquefaciens, produced much more AD from pyruvate than from citrate; the opposite was true for the heterofermenters. Acetoin and diacetyl were produced from pyruvate as soon as growth was initiated. The production was exponential up to 24 h. Destruction of the accumulated AD coincided with entry into the stationary phase. Production of AD from citrate did not begin until 6 h of the logarithmic phase of growth. Formation of gas from citrate by Lactobacillus plantarum did not implicate greater ability to form AD from citrate than from pyruvate. Fifty μmoles ml−1 citrate caused about 50% inhibition of growth of Streptococcus lactis subsp. diacetylactis. All strains examined for ability to use pyruvate as a sole source of carbon were able to do so. Acetate (50 μmoles ml−1 generally stimulated AD formation from pyruvate. With the exception of a Pediococcus sp. and S. faecalis subsp. liquefaciens, acetaldehyde (100 μg ml−1) enhanced AD production but not growth. Concentrations higher than 100 μg ml−1 had different effects.