Determination of poly-β-hydroxybutyrate (PHB) production by some <Emphasis Type="Italic">Bacillus</Emphasis> spp.

In this study, 29 strains of the genus Bacillus were isolated from different soil samples which were taken from grasslands of Ankara, Turkey and were identified as B. brevis, B. sphaericus, B. cereus, B. megaterium, B. circulans, B. subtilis, B. licheniformis and B. coagulans. Two strains, B. sphaericus ATCC 14577 and B. subtilis ATCC 6633 were also included in this study. Poly-β-hydroxybutyrate (PHB) production by these strains was determined by the spectrophotometric method, and it was found that PHB production ranged from 1.06–41.67% (w/v) depending on the dry cell weight. The highest PHB production and productivity percentage was found in B. brevis M6 (41.67% w/v).     

Technological characteristics of yeast strains and their potential as starter adjuncts in Greek-style black olive fermentation

The technological properties of Debaryomyces hansenii (15 strains) and Torulaspora delbrueckii (32 strains) isolated from Greek-style black olives under conditions typical for black olive fermentation were studied. Furthermore, the killer character of the strains was assessed as well as their antimicrobial action against food-borne pathogens. All strains could grow at 15°C and low pH (2.5), whereas the majority of the strains were able to grow at 10% (w/v) NaCl, assimilated d-galacturonic acid, and showed lipolytic activity. Only 33% of D. hansenii and 9% of T. delbrueckii strains could hydrolyse 1% (w/v) oleuropein. A large majority of the strains tolerated 0.3% (w/v) bile salts, which in correlation with acid resistance indicates probiotic potential. Cross-reactions between culture filtrates of D. hansenii and T. delbrueckii and 56 yeast strains isolated during spontaneous Greek-style black olive fermentation were conducted. Focusing on their lytic activity, 17 mycogenic strains were selected. Culture filtrates of the mycogenic strains inhibited strains of L. monocytogenes, B. cereus and S. typhimurium. The active substance was heat resistant (stable after heating at 100°C for 10 min) as well as stable over a pH range from 4.0 to 6.5. The possible inhibition of undesirable yeast contaminants and food-borne pathogens in situ on fermented olives as well as the probiotic potential of strains used as starter adjuncts would contribute to the improvement of quality of the fermented product.     

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Summary Self-compatible Brassica napus var Westar was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the Westar cultivar.     

Combined Application of the Biological Product LS213 with Bacillus, Pseudomonas or Chryseobacterium for Growth Promotion and Biological Control of Soil-Borne Diseases in Pepper and Tomato

Recent works suggest that the combination of several PGPRs could be more effective than individual strains as a horticultural product. LS213 is a product formed by a combination of two PGPRs, Bacillus subtilis strain GB03 (a growth-promoting agent), B. amyloliquefaciens strain IN937a (an inducer of systemic resistance) and chitosan. The aim of this work is to establish if the combination of three PGPR, B. licheniformis CECT 5106, Pseudomonas fluorescens CECT 5398 and Chryseobacterium balustinum CECT 5399 with LS213 would have a synergistic effect on growth promotion and biocontrol on tomato and pepper against Fusarium wilt and Rhizoctonia damping off. When individual rhizobacterium and the LS213 were put together, the biometric parameters were higher than with individual rhizobacterium both in tomato and pepper, revealing a synergistic effect on growth promotion, being the most effective combination that of B. licheniformis and LS213. When P. fluorescens CECT 5398 was applied alone, it gave good results, which could be due to the production of siderophores by this strain. Biocontrol results also indicate that those treatments that combined LS213 and each of the bacteria (Treatments: T7 and T8) gave significantly higher percentages of healthy plants for both tomato (T7: 65%) and pepper (T7: 75% and T8: 70%) than the LS213 alone (45% of healthy plants for tomato and 60% for pepper) three weeks after pathogen attack. The effects in pepper were more marked than in tomato. The best treatment in biocontrol was the combination of P. fluorescens and LS213. In summary, the combination of microorganisms gives better results probably due to the different mechanisms used.     

Fermentation pattern ofZymomonas mobilis strains on different substrates—a comparative study

The optimum conditions (pH and initial sugar concentration) of fermentation for the production of ethanol by 4 strains ofZymomonas mobilis (ATCC 10988, ATCC 12526, NRRL B 4286 and IFO 13756) were studied. An initial sugar concentration of 15 % (w/v) at pH 7.0 was found to be optimal for the first two strains and 20 % (w/v) initial sugar at pH 7.0 was found to be optimal for the last two strains. The fermentation pattern of these strains on synthetic medium, cane juice and molasses were compared. Strain NRRL B 4286 showed maximum ethanol production on synthetic medium while on cane juice ATCC 10988 and ATCC 12526 performed well. However, all the strains fermented molasses poorly.     

Sambhar Salt Lake

The saline and alkaline brines from the Sambhar Salt Lake (SSL), both from the main lake and from the solar evaporation pans at Sambhar Salt Limited, Sambhar, Rajasthan, India, were studied with respect to their chemical composition and presence of red, extremely haloalkaliphilic archaebacteria. The brines had pH values of 9.5±0.2 and a total salt content ranging from 7% (w/v) to more than 30% (w/v). Sodium chloride, sodium carbonate, sodium bicarbonate and sodium sulphate were the principal salts present in these brines which lacked divalent cations (calcium and magnesium). Six strains of red, extremely haloalkaliphilic bacteria, designated SSL 1 to SSL 6, were isolated. All the isolates showed obligate requirements for sodium chloride (>15%, w/v) and high pH (>9.0). Magnesium ions were required in traces for maintaining morphological structure and pigmentation. All these strains possessed the diether core lipids, phosphatidylglycerol (PG), phosphatidylglycerophosphate (PGP), and bacterioruberins characteristic of halophilic archaebacteria. The strains were assigned to the newly proposed genus Natronobacterium.Part of the paper was presented by the authors at XIV International Congress of Microbiology 7–13 September 1986, Manchester, UK     

Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae – to produce sorbitol from Jerusalem artichokes

The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15. The cells of parental strains were pretreated with 0.1% EDTA (w/v) and 2-mercaptoethanol (0.1%, v/v) and then exposed to 2.0% (w/v) Zymolase at 30 °C for 30–40 min. The optimized fusion condition demonstrated that with the presence of 30% (w/v) polyethylene glycol 6000 (PEG-6000) and 10 mM CaCl₂ for 30 min, the fusion frequency reached 2.64 fusants/10⁶ parental cells. The fusants were screened by different characters between two parental strains and further identified by DNA contents, inulinase activity and sorbitol productivity. One of the genetically stable fusants, Strain F27, reached a maximal sorbitol production of 4.87 g/100 ml under optimal fermentation condition.     

Optimization of Process Parameters for the Production of Inulinase from a Newly Isolated Aspergillus niger AUP19

Summary Fifty strains were isolated from different soil samples on synthetic medium containing inulin as a sole carbon source for the production of extracellular inulinase. Of them, five isolates showed high inulinase activity and one of them was selected for identification and medium optimization studies. The isolate was identified as Aspergillus niger. Various physical and chemical parameters were optimized for inulinase production. Maximum productivity of inulinase (176 U ml⁻¹) was achieved by employing medium containing 5% (w/v) inulin, galactose as additional carbon source, corn steep liquor and (NH₄)H₂PO₄ as nitrogen sources, incubation period of 72 h, incubation temperature of 28 °C, pH 6.5, inoculum load at 10% (v/v) level and medium volume to flask volume ratio of 1:20 (v/v) with indented flasks.     

Thermus 2S from Thai hot springs: isolation and immobilization

Three strains of thermophilic bacteria producing extracellular protease have been isolated from hot springs in Chiang Mai, Thailand. The bacterium producing the highest amount of protease has been selected and identified as belonging to the genusThermus, and is tentatively calledThermus 2S. The isolate is a Gram-negative, rod shaped bacterium. It exhibited maximum growth around 60°C at pH 7. Entrapment of the microbial cells in calcium alginate maintained the cell viability. Protease production from immobilized cells using 2 g wet cells per 10 ml 3% (w/v) sodium alginate was higher than that from a free-cell system using 2% inoculum.     

A novel highly boron tolerant bacterium, <Emphasis Type="Italic">Bacillus boroniphilus</Emphasis> sp. nov., isolated from soil,that requires boron for its growth

Three strains of gram-positive, motile, rod-shaped and boron (B)-tolerant bacterium were isolated from naturally B containing soil of Hisarcik area in the Kutahya Province, Turkey. The strains, designated as T-14A, T-15Z^T and T-17s, produced spherical or ellipsoidal endospores in a terminal bulging sporangium. The strains required B for the growth and can tolerate more than 450 mM B. These also tolerated up to 7.0% (w/v) NaCl in the presence of 50 mM B in agar medium but grew optimally without NaCl. The temperature range for growth was 16–37°C (optimal of 30°C), whereas the pH range was 6.5–9.0 (optimal of 7.5–8.5). The DNA G + C content was 41.1–42.2 mol% and the predominant cellular fatty acid was iso-C_15:0. The major respiratory quinone system was detected as MK-7 and the diamino acid of the peptidoglycan was meso-diaminopimelic acid. Based on phenotypic and chemotaxonomic characteristics, phylogenetic analysis of 16S rRNA gene sequences data and DNA–DNA re-association values, we concluded that the three strains belong to a novel species of the genus Bacillus, the type strain of which is T-15Z^T and for which we proposed the name, B. boroniphilus sp. nov. (DSM 17376^T = IAM 15287^T = ATCC BAA-1204^T).     

Pilot-scale production of carboxymethylcellulase from rice hull by Bacillus amyloliquefaciens DL-3

Optimal conditions for pilot-scale production of the carboxymethylcellulase (CMCase) by Bacillus amyloliquefaciens DL-3 were investigated. The best carbon and nitrogen sources for the production of CMCase by B. amyloliquefaciens DL-3 were found to be rice hull and peptone and their optimal concentrations were 5.0 and 0.20% (w/v), respectively. Optimal temperature and initial pH for the production of CMCase were 37°C and 6.8. Optimal agitation speed and aeration rate for the production of CMCase were 300 rpm and 1.0 vvm in a 7 L bioreactor, which were different from those for the cell growth of B. amyloliquefaciens DL-3. The highest productions of CMCase by B. amyloliquefaciens DL-3 from 5.0% (w/v) rice hull as a carbon source under optimal conditions in a 7 or 100 L bioreactor were 220 and 367 U/mL, respectively.     

Production of Short Side Chain-Poly[Hydroxyalkanoate] by a Newly Isolated Ralstonia Pickettii Strain

In order to obtain better bacterial species or strains for production of short side chain-poly[hydroxyalkanoate](ssc-PHA) from cheap carbon sources, a bioprospecting programme was performed in a subtropical rainforest soil. From 398 bacterial isolates, one produced high amounts of ssc-PHA when grown on sugarcane molasses or sucrose as detected by spectrophotometric scanning and gas chromatography coupled to mass spectrometry. Also, the GC—MS analysis indicated that the polymer was composed basically of poly[3-hydroxybutyrate](PHB). Phylogenetic studies using 16S rDNA analysis showed that the isolated bacterium belonged to the Ralstonia pickettii species and had a high identity/similarity with 16S rDNA obtained from total DNA of uncultured strains of soils and with unidentified bacteria at species level. The new strain was named R. pickettii 61A6. Spectrofluorometric analysis showed that the best rates of ssc-PHA accumulation within the cells occurred in 10%(w/v) sucrose and in 5%(w/v) sugarcane molasses at the stationary phase, with a yield of 231 and 357 mg/l of ssc-PHA per g dry cell weight, respectively.     

Susceptibility of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> to antibiotics

A total of 74 Streptococcus thermophilus isolates collected between 1948 and 2005 from different environments were investigated to assess erythromycin, clindamycin, streptomycin, gentamicin, tetracycline and ampicillin susceptibility by means of microdilution, Etest and disk diffusion methods. For this purpose a new S. thermophilus Susceptibility test Medium (SSM) was developed. This medium allowed a better identification of strains with atypical tetracycline resistance. The recipe is a mixed formulation of Iso-Sensitest medium (90% v/v) and M17 medium (10% v/v) supplemented with lactose (0.5% w/v). The overall agreement of the techniques was good with exception of tetracycline, for which Etest provided lower MICs than the microdilution method. Most strains were susceptible to all the antibiotics tested while a few erythromycin, tetracycline and streptomycin resistant strains were detected.     

Isolation,Identification and Screening of Manganese Solubilizing Fungi From Low-Grade Manganese Ore Deposits

The present investigation reports the isolation, molecular identification and screening of manganese (Mn) solubilizing fungal strains from low-grade Mn mine tailings. Six morphologically distinct Mn solubilizing fungal strains were isolated on MnO₂-supplemented agar plates with Mn concentration of 0.1% (w/v). The biochemical characterization of the isolated fungal strains was carried out. The molecular identification by internal transcribed spacer (ITS) sequencing identified the strains as Aspergillus terreus, Aspergillus oryzae, Penicillium sp., Penicillium sp., Penicillium daleae and Penicillium sp. with GenBank accession numbers KP309809, KP309810, KP309811, KP309812, KP309813 and KP309814, respectively. The ability of the isolated fungal strains to tolerate and solubilize Mn was investigated by subculturing them on Mn-supplemented plates with concentration ranging from 0.1 to 0.5% (w/v). Mn solubilizing ability of the fungal isolates is possibly due to the mycelia production of biogenerated organic acids such as oxalic acid, citric acid, maleic acid and gluconic acid as revealed by ion chromatography. Our investigation signifies the role of fungi in biotransformation of insoluble Mn oxide.     

Isolation of thermotolerant,fermentative yeasts growing at 52°C and producing ethanol at 45°C and 50°C

Five thermotolerant, alcohol-producing yeast cultures were isolated from samples obtained from India. Two were identified as ofKluyveromyces marxianus. All five grew on plate-cultures up to 52°C, with maximum growth rates in liquid culture at 40°C. All produced relatively high alcohol concentrations: 5.7 to 7.0% (w/v) at 45°C and 5.0 to 5.5% (w/v) at 50°C when growing on 14.0% (w/v) glucose. All five isolates fermented diluted molasses containing 16.0% (w/v) total sugars, producing 5.6 to 6.0% (w/v) alcohol concentrations. Supplementing the molasses with P, K, Mg and Mn resulted in a 13 to 20% increase in alcohol production at 40°C. The maximum amounts of alcohol produced on supplemented molasses were 7.5 to 8.0 and 6.5 to 7.0% (w/v) at 37°C and 40°C, respectively.     

Production of cholesterol oxidase by a newly isolated Rhodococcus sp.

Fifteen strains of microorganisms with ability to degrade cholesterol were isolated. Among them a Gram-positive, non-motile, non-sporing bacterium with meso-DAP in the cell wall and with a rod-coccus cycle showed the highest ability for cholesterol degradation. It was identified as Rhodococcus sp. strain 2C and was deposited by code 1633 in Persian type culture collection (PTCC). This strain was able to produce high levels of both extracellular and cell-bound cholesterol oxidases in media containing cholesterol as a sole carbon source. The effects of medium composition and physical parameters on cholesterol oxidase production were studied. The optimized medium was found to contain cholesterol 0.15% (w/v), yeast extract 0.3% (w/v), diammonium hydrogen phosphate 0.1% (w/v), Tween 80 (0.05%). The optimum pH and temperature for cholesterol oxidase production in optimized medium were found to be 8–30 °C respectively. Triton X-100 showed the greatest effect in releasing the cell-bound enzyme. The first and most probably the main metabolite of cholesterol degradation was purified and identified as 4-cholestene-3-one.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

Molecular characterization of a lipase-producing <Emphasis Type="Italic">Bacillus pumilus</Emphasis> strain (NMSN-1d) utilizing colloidal water-dispersible polyurethane

Bacillus pumilus strain NMSN-1d isolated from polyurethane-contaminated water was found to grow in high salt concentration (NaCl 10%, w/v) and degrade Impranil-DLN, water-dispersible polyurethane. The genetic relatedness of the isolate has been established by standard molecular biological techniques and the enzyme(s) involved in polyurethane degradation were also studied. A total of nine bacterial strains were isolated from polyurethane-polluted sites and characterized by conventional, microbiological and biochemical methods. These isolates were subjected to 16S ribosomal RNA gene amplification by PCR using specific primers. The genetic relatedness of the isolates was also ascertained by ribotyping and BLAST analysis of the 16S ribosomal RNA gene sequences. The bacterial isolates were grown in yeast extract-salts minimal broth medium supplemented with water-dispersible polyurethane (Impranil DLN) as a sole source of carbon. The promising isolate utilizing polyurethane and producing lipase was identified as Bacillus pumilus NMSN-1d. The polyurethane degradation has been studied in polyurethane-Rhodamine-B and Luria-Bertani-polyurethane plate assays. The activity of hydrolytic enzymes such as lipase and esterase was confirmed on 2xYT-olive oil and tributyrin-Tween 20 plate assay. The newly isolated Bacillus pumilus appears promising in the management of polyurethane waste and in production of industrially important enzymes.