Novel Antifouling and Antimicrobial Compound from a Marine-Derived Fungus Ampelomyces sp.

In this study, using a bioassay-guided isolation and purification procedure, we obtained 3-chloro-2,5-dihydroxybenzyl alcohol from a marine-derived Ampelomyces species that effectively inhibited larval settlement of the tubeworm Hydroides elegans and of cyprids of the barnacle Balanus amphitrite. The inhibitive effect on larval settlement was nontoxic and the EC₅₀ of 3-chloro-2,5-dihydroxybenzyl alcohol ranged from 3.19 μg ml⁻¹ to 3.81 μg ml⁻¹ while the LC₅₀ was 266.68 μg ml⁻¹ for B. amphitrite cyprids; EC₅₀ ranged from 0.67 μg ml⁻¹ to 0.78 μg ml⁻¹, and LC₅₀ was 2.64 μg ml⁻¹ for competent larvae of H. elegans, indicating that inhibitive effect of this compound was nontoxic. At a concentration of 50 μg per disc, this compound showed strong inhibitive effects on the growth of 13 out of 15 marine bacterial species tested in disc diffusion bioassay. Overall, the high inhibitory activities against bacteria and larval settlement as well as the non- or low-toxic nature of this compound to the barnacle and polychaete larvae suggest this compound could be a potent antifoulant and/or antibiotic.     

Aerobic Gram-Positive and Gram-Negative Bacteria Exhibit Differential Sensitivity to and Transformation of 2,4,6-Trinitrotoluene (TNT)

A systematic evaluation of the ability of different bacterial genera to transform 2,4,6-trinitrotoluene (TNT), and grow in its presence, was conducted. Aerobic Gram-negative organisms degraded TNT and evidenced net consumption of reduced metabolites when cultured in molasses medium. Some Gram-negative isolates transformed all the initial TNT to undetectable metabolites, with no adsorption of TNT or metabolites to cells. Growth and TNT transformation capacity of Gram-positive bacteria both exhibited 50% reductions in the presence of approximately 10 μg TNT ml⁻¹. Most non-sporeforming Gram-positive organisms incubated in molasses media amended with 80 μg TNT ml⁻¹ became unculturable, whereas all strains tested remained culturable when incubated in mineral media amended with 98 μg TNT ml⁻¹, indicating that TNT sensitivity is linked to metabolic activity. These results indicate that the microbial ecology of soil may be severely impacted by TNT contamination. Received: 2 December 1996 / Accepted: 3 February 1997     

Effects of Rhamnolipids from Pseudomonas aeruginosa DS10-129 on Luminescent Bacteria: Toxicity and Modulation of Cadmium Bioavailability

In this study, the mixture of mono- and di-rhamnolipids produced by Pseudomonas aeruginosa DS10-129 was characterized for its toxicity and modulatory effects on Cd availability to different bacteria. Gram-negative naturally bioluminescent Vibrio fischeri and recombinant bioluminescent Pseudomonas fluorescens, P. aeruginosa, Escherichia coli, and Gram-positive Bacillus subtilis were used as model organisms. Rhamnolipids reduced the bioluminescence of these bacteria in less than a second of exposure even in relatively low concentrations (30-min EC₅₀ 45–167 mg l⁻¹). Toxicity of Cd to Gram-negative bacteria (30-min EC₅₀ values 0.16 mg l⁻¹ for E. coli, 0.96 mg l⁻¹ for P. fluorescens, and 4.4 mg l⁻¹ for V. fischeri) was remarkably (up to 10-fold) reduced in the presence of 50 mg l⁻¹ rhamnolipids. Interestingly, the toxicity of Cd to Gram-positive B. subtilis (30-min EC₅₀ value 0.49 mg l⁻¹) was not affected by rhamnolipids. Rhamnolipids had an effect on desorption of Cd from soil: 40 mg l⁻¹ rhamnolipids increased the water-extracted fraction of Cd twice compared with untreated control. However, this additionally desorbed fraction of Cd remained bound with rhamnolipids and was not available to bacteria. Hence, in carefully chosen concentrations (still effectively complexing heavy metals but not yet toxic to soil bacteria), rhamnolipids could be applied in remediation of polluted areas.     

Relation between phylogenetic position, lipid metabolism and butyrate production by different Butyrivibrio-like bacteria from the rumen

The Butyrivibrio group comprises Butyrivibrio fibrisolvens and related Gram-positive bacteria isolated mainly from the rumen of cattle and sheep. The aim of this study was to investigate phenotypic characteristics that discriminate between different phylotypes. The phylogenetic position, derived from 16S rDNA sequence data, of 45 isolates from different species and different countries was compared with their fermentation products, mechanism of butyrate formation, lipid metabolism and sensitivity to growth inhibition by linoleic acid (LA). Three clear sub-groups were evident, both phylogenetically and metabolically. Group VA1 typified most Butyrivibrio and Pseudobutyrivibrio isolates, while Groups VA2 and SA comprised Butyrivibrio hungatei and Clostridium proteoclasticum, respectively. All produced butyrate but strains of group VA1 had a butyrate kinase activity <40 U (mg protein)⁻¹, while strains in groups VA2 and SA all exhibited activities >600 U (mg protein)⁻¹. The butyrate kinase gene was present in all VA2 and SA bacteria tested but not in strains of group VA1, all of which were positive for the butyryl-CoA CoA-transferase gene. None of the bacteria tested possessed both genes. Lipase activity, measured by tributyrin hydrolysis, was high in group VA2 and SA strains and low in Group VA1 strains. Only the SA group formed stearic acid from LA. Linoleate isomerase activity, on the other hand, did not correspond with phylogenetic position. Group VA1 bacteria all grew in the presence of 200 μg LA ml⁻¹, while members of Groups VA2 and SA were inhibited by lower concentrations, some as low as 5 μg ml⁻¹. This information provides strong links between phenotypic and phylogenetic properties of this group of clostridial cluster XIVa Gram-positive bacteria.     

Inhibition of galectin-3 mediated cellular interactions by pectic polysaccharides from dietary sources

Pectic polysaccharides from dietary sources such as Decalepis hamiltonii—swallow root (SRPP), Hemidesmus indicus (HPP), Nigella sativa—black cumin (BCPP), Andrographis serpyllifolia—(APP), Zingiber officinale—ginger (GRPP) and, citrus pectin (CPP) were examined for galectin inhibitory activity. Inhibition of (a) galectin-3 of MDA-MB-231 cells induced hemagglutination of red blood cells; (b) galectin-3 mediated interaction between normal/metastatic human buccal cells (NBC)/(MBC) and; (c) invasion of MDA-MB-231 and MBC in the invasive chamber was assessed. Results indicated that SRPP inhibited hemagglutination at Minimum Inhibitory Concentration (MIC) of 1.86 μg ml⁻¹ equivalent of carbohydrate as apposed to those of BCPP (130 μg ml⁻¹), APP (40 μg ml⁻¹), HPP (40 μg ml⁻¹) and CPP (25 μg ml⁻¹). GRPP even at concentration >1–6 mg ml⁻¹ did not inhibit agglutination. Also SRPP showed ∼15 and 2 fold potent anti hemagglutination activity relative to that of galectin-3 specific sugars—galactose (MIC-27.1 μg ml⁻¹) and lactose (MIC-4.16 μg ml⁻¹) respectively. Further, SRPP at 10 μg ml⁻¹ inhibited agglutination of NBC by galectin-3 of MDA-MB-231 cells. Modified swallow root pectic polysaccharide (MSRPP) of 50 kDa retained anti hemagglutination activity (MIC of 1.03 μg ml⁻¹) and inhibited MDA-MB-231 and MBC invasion by 73 and 50% with an IC₅₀ of 136 and 200 μg ml⁻¹ respectively. Both SRPP and MSRPP induced apoptosis up to 80% at 100 μg ml⁻¹ concentration by activating ∼2 and 8 folds of Caspase-3 activity. Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content—arabinogalactan inhibited hemagglutination significantly.     

Antibacterial activity of a bacteriocin-like substance produced by Bacillus sp. P34 that targets the bacterial cell envelope

The objective of this study was to investigate the mode of action of BLS P34, a bacteriocin-like substance (BLS) produced by a novel Bacillus sp. strain P34 isolated from the Amazon basin. The effect of the BLS was tested against Listeria monocytogenes, showing a bactericidal effect at 200 AU (activity units) ml⁻¹, while no inhibition of spore outgrowth of Bacillus cereus was observed with a dose of 1,600 AU ml⁻¹. Growth of Escherichia coli and Salmonella Enteritidis was inhibited, but only when the chelating agent EDTA was co-added with the BLS. The effect of BLS P34 on L. monocytogenes was also investigated by Fourier transform infrared spectroscopy. Treated cells showed an important frequency increase in 1,452 and 1,397 cm⁻¹ and decrease in 1,217 and 1,058 cm⁻¹, corresponding assignments of fatty acids and phospholipids. Transmission electron microscopy showed damaged cell envelope and loss of protoplasmic material. BLS P34 was bactericidal to Gram-positive, and also showed inhibitory effect against Gram-negative bacteria. There is evidence that its mode of action corresponds to that of a membrane-active substance. The knowledge about the mode of action of this BLS is essential to determine its effective application as an antimicrobial agent.     

Endophytic fungi with anti-microbial,anti-cancer and anti-malarial activities isolated from Thai medicinal plants

A total of 81 Thai medicinal plant species collected from forests in four geographical regions of Thailand were examined for the presence of endophytic fungi with biological activity. Of 582 pure isolates obtained, 360 morphologically distinct fungi were selected for cultivation on malt Czapek broth and yeast extract sucrose broth, from which extracts were tested for biological activity. Extracts of 92 isolates could inhibit Mycobacterium tuberculosis (MIC 0.0625–200 μg ml⁻¹) when tested by the microplate Alamar blue assay, while extracts of six inhibited Plasmodium falciparum (IC₅₀ of 1.2–9.1 μg ml⁻¹) as determined by the [³H]hypoxanthine incorporation method. Strong anti-viral activity against Herpes simplex virus type 1 was observed in 40 isolates (IC₅₀ of 0.28–50 μg ml⁻¹). The sulphorhodamine B assay for activity against cancer cell lines revealed that 60 were active against human oral epidermoid carcinoma cells (EC₅₀ 0.42–20 μg ml⁻¹) and 48 against breast cancer cells (EC₅₀ 0.18–20 μg ml⁻¹). Bioactivity profile was affected by the type of culture medium. Given the high incidence of bioactive extracts and the fact that most of the isolated fungi could not be identified due to lack of spore formation, the results suggested that Thai medicinal plants can provide a wide variety of endophytes that might be a potential source of novel bioactive compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phytohormone production by three strains of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and possible physiological and technological implications

The aim of this work was to evaluate phytohormone biosynthesis, siderophores production, and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. Siderophore production and phosphate solubilization were evaluated in selective culture conditions, which had negative results. Indole-3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography–mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS–flame ionization detection and high-performance liquid chromatography (HPLC-UV), respectively. IAA, zeatin, and GA₃ were found in all three strains; however, their levels were significantly higher (p < 0.01) in SEMIA5080 (3.8 μg ml⁻¹), USDA110 (2.5 μg ml⁻¹), and E109 (0.87 μg ml⁻¹), respectively. ABA biosynthesis was detected only in USDA110 (0.019 μg ml⁻¹). Ethylene was found in all three strains, with highest production rate (18.1 ng ml⁻¹ h⁻¹) in E109 cultured in yeast extract mannitol medium plus l-methionine. This is the first report of IAA, GA₃, zeatin, ethylene, and ABA production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation.     

Isolation and characterization of dihydromonacolin-MV from <Emphasis Type="Italic">Monascus purpureus</Emphasis> for antioxidant properties

The methanolic extract of Monascus purpureus cultivated by solid-state fermentation on rice showed strong 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and better yield as compared to other polarity based extracted fractions. It was selected for further purification of the antioxidant. The activity-guided repeated fractionation of methanolic extract on a silica gel column chromatography yielded a compound that exhibited strong antioxidant activity. Based on the spectroscopic analysis by UV, IR, ¹H NMR, ¹³C NMR, 2D-HSQCT NMR, and MS, the antioxidant isolated was elucidated as a derivative of dihydromonacolin-K, where the ester group is 2-methyl propionate, designated as dihydromonacolin-MV. The DPPH radical was significantly scavenged by the dihydromonacolin-MV (IC₅₀ 20±1 μg ml⁻¹). The dihydromonacolin-MV showed strong inhibition of lipid peroxidation in a liposome model with an IC₅₀ value of 5.71±0.38 μg ml⁻¹ and superoxide radical scavenging activity with an IC₅₀ value of 163.97±2.68 μg ml⁻¹.     

Characterization of a cold-tolerant plant growth-promoting bacterium Pantoea dispersa 1A isolated from a sub-alpine soil in the North Western Indian Himalayas

Pantoea dispersa strain 1A is a Gram-negative rod-shaped, yellow-pigmented bacterium isolated on nutrient agar plates incubated at 4°C. The identity of the bacterium was confirmed by sequencing of the 16 S rRNA gene. It was capable of growing at temperatures ranging from 4 to 42°C, but maximum growth was observed at 30°C. It is endowed with multiple plant growth promotion attributes such as phosphate solubilization, IAA production, siderophore production and HCN production, which are expressed differentially at sub-optimal temperatures (15 and 4°C). It was able to solubilize phosphate (17.6 μg of P₂O₅ ml⁻¹ day⁻¹), and produce IAA (3.7 μg ml⁻¹ day⁻¹), at 15°C. Qualitative detection of siderophore production and HCN were also observed at 15°C. At 4°C it was found to express all the plant growth promotion attributes. This bacterial isolate was able to positively influence and promote the growth and nutrient uptake parameters of wheat (cv. VL.802) under glasshouse conditions. Hence in the context, of cold wheat-growing environments, it is proposed that Pantoea dispersa 1A (MTCC 8706), could be deployed as an inoculant to attain the desired results of bacterization.     

Antimicrobial activity of crude extracts from mangrove fungal endophytes

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml⁻¹). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml⁻¹). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml⁻¹ and 8–200/8–200 μg ml⁻¹, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml⁻¹). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml⁻¹against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products.     

Endocrinological investigation into the reproductive cycles of two sympatric skate species, Malacoraja senta and Amblyraja radiata, in the western Gulf of Maine

The smooth skate, Malacoraja senta, and thorny skate, Amblyraja radiata, are two commercially exploited batoids found within the Gulf of Maine. During the past five years, we conducted a large study to accurately describe important biological life history parameters previously lacking for these species. As part of that project, the current study reports our findings on the hormonal profiles associated with the reproductive cycles of M. senta and A. radiata. Blood samples were obtained from mature M. senta and A. radiata of both sexes from all months of the year, and plasma testosterone (T), estradiol (E₂) and progesterone (P₄) concentrations were determined using radioimmunoassay (RIA). In female M. senta and A. radiata, monthly T concentrations ranged from 4,522 pg ml⁻¹ to 1,373 pg ml⁻¹ and 31,940 pg ml⁻¹ to 14,428 pg ml⁻¹, E₂ concentrations from 831 pg ml⁻¹ to 60 pg ml⁻¹ and 8,515 pg ml⁻¹ to 2,902 pg ml⁻¹, and P₄ concentrations from 3,027 pg ml⁻¹ to 20 pg ml⁻¹ and 3,264 pg ml⁻¹ to 331 pg ml⁻¹, respectively. No statistical differences were detected between any months for any hormone. Estradiol concentrations were not correlated with ovary weight, shell gland weight, or diameter of the largest follicles in either species. Monthly T concentrations in male M. senta and A. radiata ranged from 23,146 to 12,660 pg ml⁻¹ and from 57,500pg ml⁻¹ to 24,737 pg ml⁻¹, while E₂ concentrations ranged from 7.5 pg ml⁻¹ to undetectable and 103 to 30 pg ml⁻¹, respectively. No statistical differences were observed between months for either steroid. Testosterone concentrations were weakly correlated with testes weight and percent of stage VI spermatocysts in A. radiata, however, no correlation was detected between T and stage VI spermatocysts in M. senta. Collectively, these data support the previous conclusion that M. senta and A. radiata of both sexes are capable of reproducing year round in the western Gulf of Maine.     

Induction of axenic culture of Arthrospira (Spirulina) platensis based on antibiotic sensitivity of contaminating bacteria

Arthrospira platensis SAG 21.99 and the isolated bacteria (Halomonas spp., Staphylococcus sp., etc.) from the culture of A. platensis SAG 21.99 were treated with five antibiotics to determine the minimal lethal concentrations. The combination of a washing step and a consecutive treatment with antibiotics, imipenem (100 μg ml⁻¹), neomycin (100 μg ml⁻¹) and cycloheximide (20 μg ml⁻¹), treatment step was highly effective in eliminating bacteria. An axenic culture of A. platensis SAG 21.99 could be induced within 3 days using this method. This technique is a simple and rapid method for obtaining axenic cultures of filamentous cyanobacteria.     

Antimicrobial activity of an endophytic <Emphasis Type="Italic">Xylaria</Emphasis> sp.YX-28 and identification of its antimicrobial compound 7-amino-4-methylcoumarin

An endophytic Xylaria sp., having broad antimicrobial activity, was isolated and characterized from Ginkgo biloba L. From the culture extracts of this fungus, a bioactive compound P3 was isolated by bioactivity-guided fractionation and identified as 7-amino-4-methylcoumarin by nuclear magnetic resonance, infrared, and mass spectrometry spectral data. The compound showed strong antibacterial and antifungal activities in vitro against Staphylococcus aureus [minimal inhibitory concentrations (MIC) 16 μg·ml⁻¹], Escherichia coli (MIC, 10 μg·ml⁻¹), Salmonella typhia (MIC, 20 μg·ml⁻¹), Salmonella typhimurium (MIC, 15 μg·ml⁻¹), Salmonella enteritidis (MIC, 8.5 μg·ml⁻¹), Aeromonas hydrophila (MIC, 4 μg·ml⁻¹), Yersinia sp. (MIC, 12.5 μg·ml⁻¹), Vibrio anguillarum (MIC, 25 μg·ml⁻¹), Shigella sp. (MIC, 6.3 μg·ml⁻¹), Vibrio parahaemolyticus (MIC, 12.5 μg·ml⁻¹), Candida albicans (MIC, 15 μg·ml⁻¹), Penicillium expansum (MIC, 40 μg·ml⁻¹), and Aspergillus niger (MIC, 25 μg·ml⁻¹). This is the first report of 7-amino-4-methylcoumarin in fungus and of the antimicrobial activity of this metabolite. The obtained results provide promising baseline information for the potential use of this unusual endophytic fungus and its components in the control of food spoilage and food-borne diseases.     

Characterization of phosphobacteria isolated from eutrophic aquatic ecosystems

Phosphobacteria are able to enhance phosphorus availability in soil and improve crop yields. To develop such biofertilizers, 14 predominant phosphobacteria were isolated from eutrophic aquatic ecosystems. Molecular identification and phylogenetic analysis revealed three groups among the nine isolates of inorganic phosphate-solubilizing bacteria (IPSB): IPSBl and IPSB2 belonged to the actinobacteria and flavobacteria, respectively, and the other seven belonged to the γ-proteobacteria. Among five isolates of organic phosphorus-mineralizing bacteria (OPMB), two groups were present: OPMB1 and OPMB3 belonged to the β-proteobacteria, while the other three belonged to the γ-proteobacteria. The IPSB isolates released 62.8–66.7 mg P 1⁻¹ from tricalcium phosphate under shaking conditions, and 26.8 to 43.7 mg P 1⁻¹ under static conditions; the OPMB strains released 23.5–30.2 mg P 1⁻¹ from lecithin under shaking conditions, and 16.7–27.6 mg P 1⁻¹ under static conditions. To the best of our knowledge, this is the first report indicating that IPSBl (designated Aureobacterium resistents) as a tricalcium phosphate-solubilizing bacterium and OPMB1 and OPMB3 (designated Acidovorax temperans and Achromobacter xylosoxidans, respectively) are lecithin-mineralizing bacteria. This investigation demonstrated that a eutrophic aquatic ecosystem is a selective source of phosphobacteria and the screened phosphobacteria are a potential alternative to the development of biofertilizers.     

Novel Antifoulants: Inhibition of Larval Attachment by Proteases

We investigated the effect of commercially available enzymes (α-amylase, α-galactosidase, papain, trypsin, and lipase) as well as proteases from deep-sea bacteria on the larval attachment of the bryozoan Bugula neritina L. The 50% effective concentrations (EC₅₀) of the commercial proteases were 10 times lower than those of other enzymes. Crude proteases from six deep-sea Pseudoalteromonas species significantly decreased larval attachment at concentrations of 0.03 to 1 mIU ml⁻¹. The EC₅₀ of the pure protease from the bacterium Pseudoalteromonas issachenkonii UST041101-043 was close to 1 ng ml⁻¹ (0.1 mIU ml⁻¹). The protease and trypsin individually incorporated in a water-soluble paint matrix inhibited biofouling in a field experiment. There are certain correlations between production of proteases by bacterial films and inhibition of larval attachment. None of the bacteria with biofilms that induced attachment of B. neritina produced proteolytic enzymes, whereas most of the bacteria that formed inhibitive biofilms produced proteases. Our investigation demonstrated the potential use of proteolytic enzymes for antifouling defense.     

Isolation of antifouling compounds from the marine bacterium, <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> SCH0402

Two compounds, 2-hydroxymyristic acid (HMA) and cis-9-oleic acid (COA), were isolated from a chloroform extract of the marine bacterium, Shewanella oneidensis SCH0402. In a spectrophotometer-based chemotaxis assay, HMA completely eliminated the optical density (OD) of Alteromonas marina SCH0401 and Bacillus atrophaeus SCH0408, motile, fouling bacteria, at 100 and 1000 μg ml⁻¹, respectively. COA similarly decreased the OD of A. marina and B. atrophaeus by 100% at 1000 μg ml⁻¹. The commercially available, highly toxic anti-fouling compound, tributyltin oxide (TBTO) never reduced the OD of the target bacteria by 100% even at higher concentration. Instead, all the test bacterial cells were killed at higher than 1000 μg ml⁻¹ of concentration. Both HMA and COA inhibited germination of Ulva pertusa spores completely at 10 and 100 μg ml⁻¹, respectively, while TBTO inhibited germination at 0.01 μg ml⁻¹. However, in field assays, both HMA and COA showed anti-fouling activities as potent as TBTO against a wide range of fouling organisms, including micro- and macro-algae, barnacles, and mussels. The average fouling coverage on the surface of the control panel was 93 ± 6% after 1.5 years but no fouling was observed on the surface of the test panel onto which each compound was applied separately. Thus, bacterial repellent compounds can be used as substitutes for potent toxic anti-fouling compounds, resulting in higher standards of environmental safety without loss of anti-fouling performance.     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Contamination of pear concentrate by Alicyclobacillus from recirculating flume water during fruit concentrate production

Alicyclobacillus and viable aerobic counts were monitored at nine different production stages of pear concentrate, with the functioning of either a recirculating or a one-pass flume water system. Significantly (P < 0.05) higher levels of Alicyclobacillus were detected in the final pasteurized product (102–104°C for 90 s) when the recirculating flume water system was operational. An average of 1.19 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 1.35 Log₁₀ c.f.u. ml⁻¹ endospores were recovered, whereas 0 c.f.u. ml⁻¹ vegetative cells and endospores were detected when the one-pass flume water system was operational. Alicyclobacillus levels did not differ significantly (P > 0.05) in condensate water during the functioning of the two flume water systems, with 1.81 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 1.01 Log₁₀ c.f.u. ml⁻¹ endospores (recirculating system) and 0.78 Log₁₀ c.f.u. ml⁻¹ vegetative cells and 0.42 Log₁₀ c.f.u. ml⁻¹ endospores (one-pass system) recovered, respectively. As a result, water treatment protocols should be established if untreated recirculating flume or condensate water is to be used in order to prevent Alicyclobacillus contamination and accumulation in the processing environment.     

Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005