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.     

Modification of culture conditions for production of the anti-tubercular hirsutellones by the insect pathogenic fungus Hirsutella nivea BCC 2594

Aims:  This work aimed to improve the production of anti-tubercular hirsutellones by the insect pathogenic fungus Hirsutella nivea BCC 2594.
Methods and Results:  The fungus was cultivated under different carbon/nitrogen sources and aerations (shake vs static flasks) to improve the production of the anti-tubercular alkaloids, hirsutellones A–D. Under the basal conditions, static cultivation at 25°C in minimum salt medium, only hirsutellone B and C were detected with maximum concentrations of 139·00 and 18·27 mg l⁻¹. Substitution of fructose for glucose and peptone for yeast extract increased the titres of hirsutellones A, B and C about two- to threefold. However, hirsutellone D was not detected in this medium. Culture agitation induced the production of hirsutellone D. As a result, the significant amounts of hirsutellones A–D were obtained with the concentration of 29·93, 169·63, 22·65 and 15·71 mg l⁻¹ within 15 days.
Conclusions:  Improved titres of hirsutellones in H. nivea BCC 2549 were achieved with an agitated (200 rev min⁻¹) fructose–peptone medium at 25°C.
Significance and Impact of the Study:  Improved yields of hirsutellones B–D will enable medicinal chemistry modifications leading to a development of a potential candidate for tuberculosis therapy.     

Development of balanced medium composition for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3

Aim:  To develop optimum fermentation environment for enhanced rifamycin B production by isolated Amycolatopsis sp. RSP-3.
Methods and Results:  The impact of different fermentation parameters on rifamycin B production by isolated Amycolatopsis sp. RSP-3 was investigated using Taguchi methodology. Controlling fermentation factors were selected based on one variable at a time methodology. The isolated strain revealed more than 25% higher production compared to literature reports. Five different nutritional components (soyabean meal, glucose, potassium nitrate, calcium carbonate and barbital) and inoculum concentration showed impact on rifamycin B production at individual and interactive level. At optimized environment, 65% contribution was observed from selected fermentation parameters.
Conclusions:  Soyabean meal and calcium carbonate were the most significant factors among the selected factors followed by barbital and potassium nitrate. Glucose, however, showed the least significance on rifamycin B production with this strain. A maximum of 5·12 g l⁻¹ rifamycin B production was achieved with optimized medium containing (g l⁻¹) soyabean meal, 27; glucose, 100; potassium nitrate, 4; calcium carbonate, 3 and barbital, 1·2.
Significance and Impact of the Study:  The present study signifies identification of balanced medium component concentrations for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3. This strain requires organic and inorganic nitrogen sources for effective product yield. Yet at individual level, organic nitrogen source has c. nine-fold higher influence compared to inorganic one.     

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.     

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.     

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.     

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.     

Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design

Aims:  The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp . , using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained.
Methods and Results:  Lipase activity values of 9·5 U ml⁻¹ in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l⁻¹) of 20·0, 5·0, 5·0 and of 10·0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4·9 to 5·5 and temperature from 37°C to 42°C. The crude extract maintained its initial activity at freezing temperatures up to 100 days.
Conclusions:  A newly isolated strain of Penicillium sp . used in this work yielded good lipase activities compared to the literature.
Significance and Impact of the Study:  The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).     

Itaconic acid production by Aspergillus terreus on raw starchy materials

Starchy materials such as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch, sweet potato and industrial potato flours, either acid or enzymatically hydrolysed, were used as substrates for itaconic acid production by Aspergillus terreus NRRL 1960. Both production and yield were highest on corn starch (18·4 g l⁻¹ and 34·0%, respectively). The degree of hydrolysis had a great influence on acid production which was highest when corn starch was saccharified at 85 DE (dextrose equivalent). In a 3 litre benchtop fermenter, itaconic acid production and productivity were 19·8 g l⁻¹ and 0·13 g l⁻¹ h⁻¹, respectively.     

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.     

A potent anti-oxidant property: fluorescent recombinant α-phycocyanin of Spirulina

Aims:  To express and product a fluorescent antioxidant holo-α-phycocyanin (PC) of Spirulina platensis ( Sp ) with His-tag (rHHPC; recombinant holo-α-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale.
Methods and Results:  A vector harbouring two cassettes was constructed: cpcA along with cpcE - cpcF in one cassette; ho1 - pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 ( S6 ) could catalyse the 82 site Cys in apo-α-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0·55 g l⁻¹ broth in 5-litre bench scale. rHHPC was purified by Ni²⁺ affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had λ_max at 621 and 650 nm, respectively. The IC₅₀ values of rHHPC were 277·5 ± 25·8 μ g ml⁻¹ against hydroxyl radicals and 20·8 ± 2·2  μ g ml⁻¹ against peroxyl radicals.
Conclusions:  Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals.
Significance and impact of the study:  A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.     

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 a new heteropolysaccharide production by a native isolate of Leuconostoc sp. CFR-2181

Aim:  This work is aimed at optimizing the production of a new heteropolysaccharide (HePS) of Leuconostoc sp. CFR-2181 by standardizing the fermentation conditions in a low cost semi-synthetic medium.
Methods and Results:  Both nutritional and cultural parameters, such as carbon source and its concentration, initial pH of the exopolysaccharide (EPS) medium, fermentation temperature and fermentation period were optimized. Fermentation of the EPS medium (pH 6·7), containing sucrose at 5% (w/v) and 5% (v/v) inoculum, at 25 ° C resulted in maximum production of HePS (18·38 g l⁻¹) by the isolate in 4 h of fermentation.
Conclusions:  The isolate was found to produce good amount of HePS in just 4 h in a low cost semi-synthetic EPS medium.
Significance and Impact of the Study:  This is the first report on rapid production of HePS by any lactic culture, which can significantly reduce the cost of the EPS.     

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.     

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.     

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.     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Two-stage fermentation process for alginate production by Azotobacter vinelandii mutant altered in poly-β-hydroxybutyrate (PHB) synthesis

Aims:  A two-stage fermentation strategy, based on batch cultures conducted first under non-oxygen-limited conditions, and later under oxygen-limited conditions, was used to improve alginate production by Azotobacter vinelandii (AT6), a strain impaired in poly-β-hydroxybutyrate (PHB) production.
Methods and Results:  The use of sucrose as carbon source, as well as a high oxygen concentration (10%), allowed to obtain a maximum biomass concentration of 7·5 g l⁻¹ in the first stage of cultivation. In the second stage, the cultures were limited by oxygen (oxygen close to 0%) and fed with a sucrose solution at high concentration. Under those conditions, the growth rate decreased considerably and the cells used the carbon source mainly for alginate biosynthesis, obtaining a maximum concentration of 9·5 g l⁻¹, after 50 h of cultivation.
Conclusion:  Alginate concentration obtained from the AT6 strain was two times higher than that obtained using the wild-type strain (ATCC 9046) and was the highest reported in the literature. However, the mean molecular mass of the alginate produced in the second stage of the process by the mutant AT6 was lower (400 kDa) than the polymer molecular mass obtained from the cultures developed with the parental strain (950 kDa).
Significance and Impact of the Study:  The use of a mutant of A. vinelandii impaired in the PHB production in combination with a two-stage fermentation process could be a feasible strategy for the production of alginate at industrial level.