Fungal endophytes from Acer ginnala Maxim: isolation, identification and their yield of gallic acid

Aims:  The aim of the study was to isolate the endophytic fungi from Acer ginnala and screen isolates rich in gallic acid.
Methods and Results:  After epiphytic sterilization, 145 fungal endophytes were isolated from the stem, annual twig and seed of Acer ginnala . The endophytes were grouped into ten different taxa, Phomopsis sp., Neurospora sp., Phoma sp., Epicoccum sp., Penicillium sp., Alternaria sp., Fusarium sp., Trichoderma sp., Cladosporium sp. and a species of Pleosporales Incertae Sedis , by their morphological traits and ITS-rDNA sequence analysis. The content and yield of gallic acid of 141 isolates were determined by HPLC. On average, the species of Pleosporales Incertae Sedis had the highest content and yield of gallic acid (13·28 mg g⁻¹ DW; 119·62 mg l⁻¹), while Alternaria sp. had the lowest.
Conclusions:  Of 141 fungal endophytes from A. ginnala , Phomopsis sp. isolate SX10 showed both the highest content and the highest yield of gallic acid (29·25 mg g⁻¹ DW; 200·47 mg l⁻¹).
Significance and Impact of the Study:  Endophytic fungi isolated from A. ginnala may be used as potential producers of gallic acid and other compounds with biological activities, or functioned as elicitors to produce natural compounds.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Antibacterial activity of sphagnum acid and other phenolic compounds found in Sphagnum papillosum against food-borne bacteria

Aims:  To identify the phenolic compounds in the leaves of Sphagnum papillosum and examine their antibacterial activity at pH appropriate for the undissociated forms.
Methods and Results:  Bacterial counts of overnight cultures showed that whilst growth of Staphylococcus aureus 50084 was impaired in the presence of milled leaves, the phenol-free fraction of holocellulose of S. papillosum had no bacteriostatic effect. Liquid chromatography–mass spectrometry analysis of an acetone–methanol extract of the leaves detected eight phenolic compounds. Antibacterial activity of the four dominating phenols specific to Sphagnum leaves, when assessed in vitro as minimal inhibitory concentrations (MICs), were generally >2·5 mg ml⁻¹. MIC values of the Sphagnum- specific compound 'sphagnum acid' [ p -hydroxy-β-(carboxymethyl)-cinnamic acid] were >5 mg ml⁻¹. No synergistic or antagonistic effects of the four dominating phenols were detected in plate assays.
Conclusions:  Sphagnum -derived phenolics exhibit antibacterial activity in vitro only at concentrations far in excess of those found in the leaves.
Significance and Impact of the Study:  We have both identified the phenolic compounds in S. papillosum and assessed their antibacterial activity. Our data indicate that phenolic compounds in isolation are not potent antibacterial agents and we question their potency against food-borne pathogens.     

Automated sperm morphology analysis in fishes: the effect of mercury on goldfish sperm

This study is the first to examine the morphology of fish sperm using automated sperm morphology analysis (ASMA). The technique was applied to investigate the effect of an environmental pollutant, mercury, on the sperm morphology of goldfish Carassius auratus , and the effects on sperm morphology were compared with those on sperm motility. Goldfish sperm flagellar length was significantly shortened after instant exposure to 100 mg l⁻¹ (368 µM) mercuric chloride, while curvilinear velocity (VCL) and the percentage of motile sperm were significantly decreased at mercuric chloride concentrations of 1 and 10 mg l⁻¹ (3·68 and 36·8 µM), respectively. After 24 h exposure to 0·001 mg l⁻¹ (0·0037 µM) mercuric chloride, flagellar length was significantly reduced in 38% of the spermatozoa. Following exposure to 0·1 mg l⁻¹ (0·37 µM) mercuric chloride for 24 h, however, the majority of spermatozoa (98%), had significantly shortened flagella and increased sperm head length, width and area. Sperm motility was also significantly decreased at 0·1 mg l⁻¹ (0·37 µM) mercuric chloride, probably due to the significantly reduced flagellar length at this concentration. This study shows that the morphological examination of fish sperm by ASMA provides, not only, an excellent tool for monitoring reproductive disruption caused by environmental pollution, but also has applications to other areas of fish reproductive biology, such as cryopreservation and aquaculture.     

Regulation of tacrolimus production by altering primary source of carbons and amino acids

Aims:  In the present communication, attempts have been made to regulate the tacrolimus production by supplementing commercial source of carbons and amino acids timely.
Methods and Results:  Tacrolimus production was regulated by supplying vegetable oils and amino acids, individually and in combination. Tacrolimus quantification was done by HPLC. Streptomyces spp. MA6858 B3178 was found to produce 115·3 mg l⁻¹ of tacrolimus. The rotation speed of shake flask, pH of the broth and supply of air were maintained at 7·1, 230 rev min⁻¹ and 2·0 vvm air respectively.
Conclusions:  The effect of carbons on tacrolimus production was noticed to be of diphasic manner. During the first 24 h of culture, monosaccharide is used for the growth of microbe. However, after the lapse of 36 h, addition of soya oil and l -lysine in combination enhanced the tacrolimus production to 115·3 mg l⁻¹. Besides this, pH of broth was also noticed as a critical factor in monitoring tacrolimus biosynthesis.
Significance and Impact of the Study:  The newly isolated mutant Streptomyces spp. MA6858 B3178 having high potential for tacrolimus production as compared to existing data can be well used for the commercialization of tacrolimus.     

Effect of alternative treatments on seed-borne Didymella lycopersici in tomato

Aims:  To quantify the phytotoxicity and effect of alternative seed treatments based on acidified nitrite and elicitors of plant resistance (Tillekur and Chitosan) against seed-borne inocula of Didymella lycopersici .
Methods and Results:  Treatments tested were: nitrite [sodium nitrite in citric acid buffer (pH 2)] at 30, 100 and 300 mmol l⁻¹ and three exposure times (10, 20 and 30 min); Tillekur (in water) at 12·5, 25, 50, 100 and 200 mg ml⁻¹; Chitosan (in 0·05% acetic acid) at 2·5, 5, 10 and 50 mg ml⁻¹. Efficacy of treatments was determined in growth chamber experiments. Nitrite at 300 mmol l⁻¹ was completely effective, as was the fungicide, at controlling disease when applied for less than 20 min. Tillekur was as effective as the fungicide postemergence, but proved to be phytotoxic pre-emergence. Chitosan was significantly less effective than the other treatments.
Conclusions:  The high efficacy and low cost of acidified nitrite indicates that it is a suitable alternative to fungicides.
Significance and Impact of the Study:  There is currently a lack of effective seed treatments that can be used in organic and low-input crops. Treatments identified in this study can be considered as an effective alternative to chemical control against seed-borne fungal pathogens.     

Efficacy of sodium hypochlorite and peracetic acid in sanitizing green coconuts

Aims:  To evaluate the efficacy of sanitizing green coconuts ( Cocos nucifera L.) through the treatment applied by juice industries using sodium hypochlorite and peracetic acid.
Methods and Results:  The surface of the fruits was inoculated with a mixture of five Listeria monocytogenes strains. The treatments consisted in immersing the fruits for 2 min at room temperature in sodium hypochlorite solution containing 200 mg l⁻¹ residual chlorine at pH 6·5, and 80 mg l⁻¹ solution of peracetic acid or sterile water. Bacterial populations were quantified by culturing on trypticase soy agar supplemented with yeast extract and Oxford selective culture medium; however, recovery was higher on the nonselective medium. Immersion in water produced a reduction in the L. monocytogenes population of 1·7 log₁₀ CFU per fruit, while immersion in sodium hypochlorite and peracetic acid solutions resulted in population reductions of 2·7 and 4·7  log₁₀ CFU per fruit respectively.
Conclusions:  The treatments studied are efficient to green coconuts.
Significance and Impact of the Study:  Sanitation of green coconut is one of the most important control measures to prevent the contamination of coconut water. This article provides information that shows the adequacy of sanitizing treatments applied by the juice industries.     

The efficacy of the inorganic copper-based biocide CuWB50 is compromised by hard water

Aims:  We sought to explain the unexpected failure of the inorganic copper-based biocide CuWB50 to effectively decontaminate microfibre cleaning cloths that became contaminated with Acinetobacter lwoffii .
Methods and Results:  CuWB50 was diluted using distilled water or tap water obtained from two different ICUs. Microtitre plate assays were used to determine the minimum inhibitory concentration (MIC) for the implicated A. lwoffii . pH and oxidation-reduction potential (ORP) tests were performed and representative water samples were chemically analysed. When diluted in distilled water, the CuWB50 MIC for A. lwoffii was 9 mg l⁻¹ but in tap water from each ICU it was 37 and 75 mg l⁻¹ at hardness levels of 246 and 296 mg CaCO₃ l⁻¹ respectively. CuWB50-distilled water solutions consistently had a lower pH and higher ORP than CuWB50-tap water solutions.
Conclusions:  Hard water adversely affects the biocidal efficacy of CuWB50.
Significance and Impact of the Study:  Unintentional environmental contamination is a risk when using wet microfibre cloths. This occurred when cloths were stored in CuWB50 overnight combined with the unintentional but erroneous use of tap water. This study emphasizes the need for clearly documented cleaning protocols embedded within a culture of adequate training and constant supervision of cleaning staff.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

Interactions between humic matter and bacteria when disinfecting water with UV light

Aims:  To investigate the impact of aquatic humic matter on the inactivation of Escherichia coli and Bacillus subtilis by ultraviolet (UV) light.
Methods and Results:  A bench-scale study investigated the potential for Aldrich^® humic acid (AHA) and Suwannee River natural organic matter (SR-NOM) to coat the surface of E. coli and B. subtilis and offer protection from low-pressure UV light. UV doses of 5 and 14 mJ cm⁻² were applied using a collimated beam at four concentrations of humic matter (0, 10, 50 and 120 mg l⁻¹) in reagent grade water. Both AHA and SR-NOM were found to offer statistically significant protection of both E. coli and B. subtilis at concentrations of 50 and 120 mg l⁻¹ for a UV dose of 14 mJ cm⁻².
Conclusions:  Both E. coli and B. subtilis are susceptible to coating by humic matter which can reduce the sensitivity of the cells to UV light.
Significance and impact of the study:  Micro-organisms in the environment may acquire characteristics through interaction with humic matter that render them more resistant to UV disinfection than would be predicted based on laboratory inactivation studies using clean cells.     

Biosurfactant production by Pseudomonas sp. and its role in aqueous phase partitioning and biodegradation of chlorpyrifos

Aim:  To study the effect of biosurfactant on aqueous phase solubility and biodegradation of chlorpyrifos.
Methods and Results:  A Pseudomonas sp. (ChlD), isolated from agricultural soil by enrichment culture technique in the presence of chlorpyrifos, was capable of producing biosurfactant (rhamnolipids) and degrading chlorpyrifos (0·01 g l⁻¹). The partially purified rhamnolipid biosurfactant preparation, having a CMC of 0·2 g l⁻¹, was evaluated for its ability to enhance aqueous phase partitioning and degradation of chlorpyrifos (0·01 g l⁻¹) by ChlD strain. The best degradation efficiency was observed at 0·1 g l⁻¹ supplement of biosurfactant, as validated by GC and HPLC studies.
Conclusion:  The addition of biosurfactant at 0·1 g l⁻¹ resulted in more than 98% degradation of chlorpyrifos when compared to 84% in the absence of biosurfactant after 120-h incubation.
Significance and Impact of the Study:  This first report, to the best of our knowledge, on enhanced degradation of chlorpyrifos in the presence of biosurfactant(s), would help in developing bioremediation protocols to counter accumulation of organophosphates to toxic/carcinogenic levels in environment.     

Mixed aerobic and anaerobic microbial communities in benzene-contaminated groundwater

Aims:  To investigate the factors affecting benzene biodegradation and microbial community composition in a contaminated aquifer.
Methods and Results:  We identified the microbial community in groundwater samples from a benzene-contaminated aquifer situated below a petrochemical plant. Eleven out of twelve groundwater samples with in situ dissolved oxygen concentrations between 0 and 2·57 mg l⁻¹ showed benzene degradation in aerobic microcosm experiments, whereas no degradation in anaerobic microcosms was observed. The lack of aerobic degradation in the remaining microcosm could be attributed to a pH of 12·1. Three groundwaters, examined by 16S rRNA gene clone libraries, with low in situ oxygen concentrations and high benzene levels, each had a different dominant aerobic (or denitrifying) population, either Pseudomonas , Polaromonas or Acidovorax species. These groundwaters also had syntrophic organisms, and aceticlastic methanogens were detected in two samples. The alkaline groundwater was dominated by organisms closely related to Hydrogenophaga .
Conclusions:  Results show that pH 12·1 is inimical to benzene biodegradation, and that oxygen concentrations below 0·03 mg l⁻¹ can support aerobic benzene-degrading communities.
Significance and Impact of the Study:  These findings will help to guide the treatment of contaminated groundwaters, and raise questions about the extent to which aerobes and anaerobes may interact to effect benzene degradation.     

Effects of a synbiotic product on blood antioxidative activity in subjects colonized with Helicobacter pylori

Aim:  To evaluate the impact of the consumption of a synbiotic product on the antioxidative activity markers of blood in asymptomatic H. pylori -colonized persons.
Methods and Results:  Fifty-three healthy adult volunteers without gastric symptoms participated in a randomized, double-blind placebo-controlled study. The crossover consumption of the enterocoated capsules containing antioxidative Lactobacillus fermentum ME-3, Lact. paracasei 8700:2 and Bifidobacterium longum 46 with Raftilose P95 lasted for 3 weeks and did not change the H. pylori colonization. In H. pylori -positive subjects the sera values of total antioxidative status (TAS) were significantly lower compared to H. pylori -negative subjects (0·97 vs 1·05 mmol l⁻¹, P  = 0·008). After the consumption of the synbiotic, TAS values (0·97 vs 1·03 mmol l⁻¹, P  = 0·004) increased, while the ratio between oxidized and reduced glutathione (0·035 vs 0·030, P  = 0·016) decreased in H. pylori -positive subjects.
Conclusions:  The consumption of a synbiotic containing an antioxidative probiotic strain improved the reduced systemic antioxidative activity in H. pylori -colonized asymptomatic subjects.
Significance and Impact of the Study:  A synbiotic product containing an antioxidative probiotic strain may be useful in the reduction of systemic oxidative stress in H. pylori infection.     

The antibacterial mechanism of carvacrol and thymol against Escherichia coli

Aims:  To investigate the antibacterial mechanism of carvacrol and thymol against Escherichia coli.
Methods and Results:  The time-kill curve results showed that carvacrol and thymol at 200 mg l⁻¹ could inhibit the growth of E. coli . Flow cytometry and fluorescent dyes were used to explore the effect of two components on membrane permeability and membrane potential. In membrane permeability experiment, the mean fluorescence intensity of cells treated with 200 mg l⁻¹ carvacrol or thymol were lower than nonexposed cells. The ratio of red to green fluorescence intensity of DiOC₂(3) reflected the change of membrane potential. Carvacrol and thymol at 200 mg l⁻¹ caused the ratio of red/green decreasing from 0·42 of control to 0·08 and 0·07, respectively.
Conclusions:  Carvacrol and thymol had desired antimicrobial effect on E. coli . The antibacterial effects were attributed to their ability to permeabilize and depolarize the cytoplasmic membrane.
Significance and Impact of the Study:  This study showed the potential use of flow cytometry as a suitable method to investigate the mode of antibacterial action of essential oil components.     

Potential of Bacillus sp. to produce polyhydroxybutyrate from biowaste

Aim:  To test the Bacillus strains for their abilities to produce polyhydroxybutyrate (PHB) from different sugars and biowaste (Pea-shells).
Methods and Results:  Six Bacillus strains were checked for their ability to produce PHB from GM2 medium supplemented with different sugars at the rate of 1% (w/v) and from biowaste and GM2 (BW : M) combinations (3 : 7, 1 : 1, 7 : 3). Glucose supplemented GM2 medium resulted in maximum PHB production of 435 mg l⁻¹ constituting 31–62% w/w of the total cell dry mass. Substituting GM2 medium to the extent of 50% with biowaste (pea-shell slurry) resulted in 945–1205 mg l⁻¹ PHB (55–65% w/w). Optimization for additional nitrogen supplementation, inoculum size resulted in a final PHB production of 3010–3370 mg l⁻¹ equivalent to 300 g kg⁻¹ biowaste (dry wt).
Conclusion:  The Bacillus strains were able to produce PHB from biowaste (Pea-shells) as cheap source of substrate.
Significance and Impact of the Study:  This is the first report on usage of pea-shells as feed for PHB production, opening new possibilities for its use for production of PHB and Bacillus as potential candidate for the purpose.     

Susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0·1, 0·2 and 0·5 mg l⁻¹), pH (6, 7 and 8) and temperature (4, 21 and 32 °C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH 6 than at pH 8 for both strains. Under the standard conditions of temperature 21 °C, pH 7 and chlorine concentration 0·2 mg l⁻¹, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Fed-batch culture of Candida guilliermondii FTI 20037 for xylitol production from sugar cane bagasse hydrolysate

A fed-batch culture system was used to study xylitol production by Candida guilliermondii FTI 20037 in a synthetic and a sugar cane bagasse hydrolysate medium. The values achieved for xylitol yield and volumetric productivity were, respectively, 0 · 84 g g⁻¹ and 0 · 64 g l⁻¹ h⁻¹ using the synthetic medium and 0 · 78 g g⁻¹ and 0 · 62 g l⁻¹ h⁻¹ using the hydrolysate medium.     

Degradation of linear alkylbenzene sulfonate by a bacterial consortium isolated from the aquatic environment of Argentina

Aims:  To isolate and identify linear alkylbenzene sulfonate (LAS)-degrading bacteria from Río de la Plata and adjacent waters, and to assay their degradation capability as a consortium and as single organisms.
Methods and Results:  A consortium consisting of four bacterial strains: Aeromonas caviae (two strains), Pseudomonas alcaliphila and Vibrio sp. was identified by 16S rRNA analysis. Isolates grown as a consortium produced higher biomass from LAS and CO₂ release (mineralization) than individual cultures, and degraded 86% of LAS (20 mg l⁻¹), whereas pure strains degraded between 21% and 60%. Bacterial desulfonation from LAS was evidenced in the consortium and A. caviae strains. A complete disappearance of LAS (10 mg l⁻¹) was accomplished, and LAS levels of 50 and 100 mg l⁻¹ led to a pronounced decrease in the biodegradation extent and inhibition of culture growth.
Conclusions:  A bacterial consortium capable of complete LAS degradation was isolated from the Río de la Plata and adjacent waters. This consortium was more efficient for LAS degradation than individual cultures, and was sensitive to high LAS concentrations.
Significance and Impact of the Study:  The autochthonous consortium with high effectiveness on LAS biodegradation is a useful tool for LAS depletion from these polluted ecosystems.     

Long-term ammonia exposure of turbot: effects on plasma parameters

Turbot juveniles were exposed to four ammonia concentrations [0·17 (L), 0·34 (M), 0·73 (MH) and 0·88 (H) mg l⁻¹ NH₃-N] for different exposure durations (28 days minimum to 84 days). Their physiological status and growth performances were compared to a control group [0·004 (C) mg l⁻¹ NH₃-N]. No growth was observed in the H group, and by day 57, mass increase in the MH group was only 15% of that in group C. During the first month growth in the L group was similar to that in control group while it was lower (33%) in the M group; afterwards the L and M groups had a similar growth (half that of controls). Accumulation of total ammonia nitrogen (TA-N) in plasma was dependent on ambient ammonia concentrations. Plasma urea levels in ammonia-exposed fish were lower, similar or greater than in controls (depending on ammonia concentration or exposure duration). Osmolarity, Cl^– and Na⁺ plasma concentrations were stable in the L and M groups. The increases in Na⁺, Cl^–, K⁺ and total Ca concentrations observed by the end of the experiment in the H and MH groups suggest that fish failed to adapt. There was an initial rise in plasma cortisol in all ammonia-exposed groups followed by a return to basal level (1·7–4 ng ml⁻¹) in the L and M groups. In group MH, plasma cortisol peaked at 42 ng ml⁻¹ by day 14, and after a decline at c . 1 month (14 ng ml⁻¹), it rose again.     

Inhibition of the tempe mould, Rhizopus oligosporus, by ammonia

The hyphal extension rate of Rhizopus oligosporus NRRL 2710 was slowed in the presence of 0·42 and 0·84 mmol NH3 l⁻¹ and inhibited by 1·3 mmol l⁻¹. Sporulation was prevented at NH3 concentrations of 0·42 mmol l⁻¹ andabove. There was no evidence of toxicity due to NH⁺₄ at concentrations up to 300 mmol l⁻¹.Independent of the concentrations of NH3 or NH⁺₄, the lower the pH value, in therange 6·0–9·0, the higher was the rate of hyphal extension. It is suggested that accumulationof toxic levels of NH3 could be responsible for the cessation of mould growth in tempe.