Isolation of a kojic acid-producing fungus capable of using starch as a carbon source

A fungal strain (S33-2), able to grow on cooked starch and produce a substantially high level of kojic acid, was isolated from morning glory flower ( Bixa orellana ). The fungus was characterized and identified as Aspergillus flavus. The effect of different types of starch (sago, potato and corn starch) on growth of strain S33-2 and kojic acid production was examined using shake flasks. It was found that strain S33-2 grew well on all types of starch investigated. However, kojic acid production was highest when corn starch was used, with the maximum kojic acid obtained being comparable to fermentation using glucose. The highest kojic acid production (19·2 g l⁻¹) was obtained when 75 g l⁻¹ corn starch was used. This gave a yield, based on starch consumed, and an overall productivity of 0·256 g g⁻¹ and 0·04 g l⁻¹ h⁻¹, respectively.     

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.     

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.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Statistical optimization of medium components for extracellular protease production by an extreme haloarchaeon, Halobacterium sp. SP1(1)

Aims:  Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach.
Methods and Results:  The significant factors influencing the protease production as screened by Plackett–Burman method were identified as soybean flour and FeCl₃. Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l⁻¹): NaCl, 250; KCl, 2; MgSO₄, 10; tri-Na-citrate, 1·5; soybean flour, 10 and FeCl₃, 0·16. This statistical optimization approach led to production of 69·44 ± 0·811 U ml⁻¹ of protease.
Conclusions:  Soybean flour and FeCl₃ were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3·9-fold increase in extracellular protease production.
Significance and Impact of the Study:  The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.     

Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes

Aims:  Isolation of novel alginate degrading bacteria for the disposal of seaweed waste in composting process.
Methods and Results:  Decomposition of alginate polymers was checked by the 3,5-dinitrosalicylic acid (DNS) method for reducing sugar, and absorbance at 235 nm for unsaturated sugar. A bacterium A7 was isolated from wakame compost and confirmed to belong to the genus Gracilibacillus by partial 16S rDNA analysis. The optimum condition for the growth of A7 in a medium containing 5 g l⁻¹ of sodium alginate is as follows: pH, 8·5–9·5; NaCl, 0·5 mol l⁻¹; temperature, 30°C and polypeptone as nutrient content, 2–5 g l⁻¹. In a laboratory-scale composting experiment, the alginate content in wakame compost decreased to 14·3% after 72 h of composting from an initial value of 36%, indicating the effectiveness of alginate decomposition of A7 in wakame composting.
Conclusions:  The bacterium A7 was found to be alginate lyase-producing in genus Gracilibacillus and effective in degrading alginate to oligosaccharides in wakame during composting process.
Significance and Impact of the Study:  Development of new methods for the disposal of marine wastes and production of functional products.     

The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5

Aims:  To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5.
Methods and Results:  Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l⁻¹ of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l⁻¹, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation.
Conclusions:  Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem.
Significance and Impact of the Study:  The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.     

Structural stability of Sclerotium rolfsii ATCC 201126 β-glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach

Aims:  Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences.
Methods and Results:  Fermenter-scale production led to productivity ( P _r) and yield ( Y _P/C) values higher at 48 h ( P _r = 0·542 g l⁻¹ h⁻¹; Y _P/C = 0·74) than at 72 h ( P _r = 0·336 g l⁻¹ h⁻¹; Y _P/C = 0·50). Both EPSs were neutral glucose-homopolysaccharides with a β-(1,3)-glycosidic backbone and single β-(1,6)-glucopyranosyl sidechains regularly attached every three residues in the main chain, as revealed by chemical analyses. The infra-red diagnostic peak at 890 cm⁻¹ confirmed β-glycosidic linkages, while gentiobiose released by β-(1,3)-glucanases confirmed single β-1,6-glycosidic branching for both EPSs.
Conclusions:  The true modular repeating unit of S. rolfsii ATCC 201126 scleroglucan could be resolved. Structural stability was corroborated and no structural differences could be detected as to account for the variations in EPSs behaviour.
Significance and Impact of the Study:  Recovery of S. rolfsii ATCC 201126 scleroglucan at 48 h might be considered based on better fermentation kinetic parameters and no detrimental effects on EPS structural features.     

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.     

Optimization of medium constituents for improved chitinase production by Paenibacillus sp. D1 using statistical approach

Aims:  Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1.
Methods and Results:  Urea, K₂HPO₄, chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett–Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l⁻¹): urea, 0·33; K₂HPO₄, 1·17; MgSO₄, 0·3; yeast extract, 0·65 and chitin, 3·75. This statistical optimization approach led to the production of 93·2 ± 0·58 U ml⁻¹ of chitinase.
Conclusions:  The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K₂HPO₄, chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2·56-fold increase in chitinase production.
Significance and Impact of the Study:  The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.     

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.     

Formation and microbial community analysis of chloroanilines-degrading aerobic granules in the sequencing airlift bioreactor

Aims:  This paper investigates a selection-based acclimation strategy for improving the performance and stability of aerobic granules at a high chloroanilines loading.
Methods and Results:  The experiments were conducted in a sequencing airlift bioreactor (SABR) to develop aerobic granules fed with chloroanilines (ClA). The evolution of aerobic granulation was monitored using image analysis and scanning electron microscopy, and PCR–DGGE analysis of microbial community was performed. The sludge granulation was apparently developed by decreased settling time and gradual increased ClA loading to 0·8 kg m⁻³ day⁻¹. A steady-state performance of the granular SABR was reached at last, as evidenced by biomass concentration of 6·3 g l⁻¹ and constant ClA removal efficiency of 99·9%. The mature granules had a mean size of 1·55 mm, minimal settling velocity of 68·4 m h⁻¹, specific ClA degradation rate of 0·181 g gVSS⁻¹ day⁻¹. Phylogenetic analysis of aerobic ClA-degrading granules confirmed the dominance of β - , γ -Proteobacteria and Flavobacteria.
Conclusions:  The chosen operating strategy involving step increase in ClA loading and enhancement of major selection pressures was successful in cultivating the aerobic ClA-degrading granules.
Significance and Impact of the Study:  This research could be helpful for improving the stability of aerobic granules via optimizing operating conditions and developing economic feasible full-scale granular bioreactor.     

Optimization of D-ribose production with a transketolase-affected Bacillussubtilis mutant strain in glucose and gluconic acid-based media

When the transketolase-deficient and D -ribose-producing Bacillus subtilis strain ATCC 21951 was grown in a glucose (200 g l⁻¹)-based medium (Kintaka et al. 1986), only 11 g l⁻¹ D -ribose was synthesized, in addition to acetic acid (12 g l⁻¹) and acetoin plus 2,3-butanediol (24 g l⁻¹), within 1 week of fermentation. After optimizing the process conditions at 2 l fermentor scale (simplified medium composition, pH 5·0 or 6·0, highly oxidative (1000 rev min⁻¹, 3 vvm)), 40 g l⁻¹ D -ribose was obtained from 200 g l⁻¹ D -glucose, in addition to 14·5 g l⁻¹ acetoin, during 1 week of fermentation. By partially substituting D -glucose with D -gluconic acid (100 g l⁻¹ D -glucose plus 50 g l⁻¹ D -gluconic acid) under highly oxidative (1000 rev min⁻¹, 3 vvm) and pH-controlled (pH 6·5) conditions, D -ribose productivity increased (45 g l⁻¹) and acetoin formation (7·5 g l⁻¹) dropped, as did the fermentation time (3·5 d). The mixed carbon substrate procedure here developed provides an excellent alternative to the less efficient glucose-based processes described so far.     

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.     

3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775

Aims:  3-Methylindole (3-MI) is a degradation product of l -tryptophan and is both an animal waste malodorant and threat to ruminant health. Culture conditions influencing 3-MI production in Clostridium scatologenes ATCC 25775 were investigated.
Methods and Results:  Extracellular 3-MI levels in cells cultured in brain heart infusion (BHI) medium (pH 7·0) at 33°C and 37°C for 72 h were 907 ± 38 and 834 ± 121  μ mol l⁻¹, respectively. Cells cultured in tryptone-yeast (TY) extract medium at 37°C for 48 h produced 104 ± 86  μ mol l⁻¹ 3-MI; however, addition of 1 mmol l⁻¹  l -tryptophan failed to increase extracellular levels (113 ± 50  μ mol l⁻¹ 3-MI). Specific activity of indole acetic acid decarboxylase measured in BHI, TY and TY plus 1 mmol l⁻¹ tryptophan-grown cells displayed 35-, 33- and 76-fold higher levels than in semi-defined medium-grown cells.
Conclusions:  When cultured in rich medium, at 33°C or 37°C and pH 7·0, Cl. scatologenes ATCC 25775 optimally produced 3-MI. Addition of l- tryptophan to medium did not lead to significant increases in extracellular 3-MI levels. Whole cell assays indicate growth in rich medium significantly up-regulated 3-MI production.
Significance and Impact of the Study:  Information presented here may prove useful in understanding what factors influence 3-MI production in malodorous animal wastes.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524

Aims:  To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced.
Methods and Results:  Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga . xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3·83 and 3·75 g l⁻¹ respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2·5 g l⁻¹. Solid state ¹³C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles.
Conclusions:  The production rate of bacterial cellulose by Ga . xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features.
Significance and Impact of the Study:  Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga . xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.     

Influence of phosphorus on biofilm formation in model drinking water distribution systems

Aims:  This study investigated the effects of phosphorus on biofilm formation via annular reactor systems in terms of biofilm cell growth, exopolysaccharide (EPS) production, biofilm structure and cell metabolic potential.
Methods and Results:  Drinking water biofilms were developed in annular reactors with supplement of carbon and different levels of phosphorus. The biofilm formation was monitored over a period of 30 days. Biofilm related parameters were examined by various methods, which included heterotrophic plate count, total carbohydrate content, confocal laser scanning microscopy and GN2 microplate assay. Our results showed that phosphorus addition can promote the biofilm cell growth (cell count increased about 1 log with addition of 30 and 300 μg l⁻¹ of phosphorus). However, the addition of 30 and 300 μg l⁻¹ of phosphorus caused 81% and 77% decrease in EPS production, respectively. The results of biofilm structure analysis showed that the addition of 30 and 300 μg l⁻¹ of phosphorus can induce thicker and less homogeneous biofilms with more biomass. Furthermore, the addition of 30 and 300 μg l⁻¹ of phosphorus dramatically increased the biofilm cell metabolic potential. The addition of 3 μg l⁻¹ of phosphorus was found to have minor effects on the parameters examined.
Conclusions:  The results indicate phosphorus addition to drinking water distribution system (DWDS) has a complicated effect on the biofilm formation.
Significance and Impact of the Study:  As the addition of phosphorus at certain levels can affect the biofilm growth in DWDS, care should be taken when phosphate-based corrosion inhibitors are used in the DWDS.     

Lipid formation and γ-linolenic acid production by Mucorales fungi grown on sunflower oil

Lipid formation and γ-linolenic acid (GLA) production by 48 species of Mucorales fungi grown on sunflower oil (which consists of 70% linoleic acid ; LA) were studied. The strains accumulated 42·7–65·8% lipid in biomass (7·66–13·39 g l⁻¹). Eight cultures produced more than 200 mg GLA l⁻¹. Highest GLA yields exhibited Mucor mucedo CCF-1384 and Cunninghamella echinulata CCF-103 (379 and 373 mg l⁻¹, respectively). Mortierella alpina CCF-185 synthesized 465 mg l⁻¹ arachidonic acid. While the decrease of LA utilization index (ratio of LA content of cell lipid/LA content of oil source) was accompanied with growth of delipidized biomass and with reduction of lipid accumulation within the cells, high lipid yield was as a consequence of the direct oil source incorporation into intracellular lipid.