Submerged Fermentation Conditions Research Articles

Isolation, partial purification, biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis, Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples.

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37 °C for 48 h with a constant agitation of 120 rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1 M ice cold CaCl2 and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60 s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.

Cellulase-free-thermo-alkali-solvent-stable xylanase from Bacillus altitudinis DHN8: Over-production through statistical approach, purification and bio-deinking/ bio-bleaching potential

The present study shows over-production of cellulase-free xylanase using Bacillus altitudinis DHN8 followed by its purification and applications. Response surface methodology, especially Box-Behnken Design was used to improve the enzyme yield by optimizing submerged fermentation conditions. Statistically optimized fermentation conditions to achieve higher xylanase production (492 IU/mL) were sorghum straw 1.0%, inoculum size 3.0%, incubation time 48 h, temperature 32 °C, agitation speed 200 rpm and gelatin 0.37%. Under these conditions, the resulted enzyme activity was two-fold high compared to the activity (245 IU/mL) resulted after one-factor-at-a-time optimization. The purified xylanase was of high molecular weight (~98 kDa) as revealed by SDS-PAGE. It showed optimum activity between 45 °C and 55 °C and at pH 8.0. Various metals, modulators, and organic solvents further improved the activity of purified enzyme. In this study, the crude xylanase was used for (1) bio-deinking of hand-written office waste papers having blue ink and (2) bio-bleaching of carton pulp. In the first case, 56.3% deinking of waste papers was achieved whereas in the second case, Kappa number of carton pulp was reduced to 10.85. Both results are suggesting the potential of B. altitudinis xylanase for the bio-deinking and bio-bleaching.

Optimization of cultural conditions for submerged state fermentation of di-gested biogas slurry for production of lignocellulolytic enzymes using Phanaerochaete chrysosporium MTCC 787

Growing environmental concerns and increasing demands from end-use sectors have increased the glob-al market for microbial products. Optimizations of production parameters hold great importance for the industry. The present study was aimed at optimization of submerged state fermentation conditions for production of lignocelluloly-tic enzymes from digested biogas slurry by Phanaerochaete chrysosporium MTCC 787. Enzyme activities for differ-ent enzymes i.e. endoglucanase, exoglucanase, ?-glucosidase; xylanase and mannanase; laccase, lignin peroxidase and manganese peroxidise, using P. chrysosporium MTCC 787 were maximum at 50% concentration of digested slur-ry and showed maximum value of xylanase i.e. 187.41U/ml. Effect of temperature (25°C, 30°C and 35°C) on lignocellu-losic bioconversion showed that at 30°C, maximum value of manganese peroxidise (167.5 U/ml) was obtained. High-est enzyme activites were obtained at selected inoculum size i.e. 10?spores/ml, e.g. 85.29 U/ml xylanase was ob-tained. Incubation period of 8 days and pH of 7.0 came out to be best conditions for P. chrysosporium MTCC787 to produce maximum enzyme activity e.g. xylanase 95.47U/ml at pH 7.0 and xylanase 144.96U/ml at 8 day incu-bation.This work presents a novel concept in optimization of fermentation process to produce lignocellulolytic en-zymes as this work is focussed on utilization of digested biogas slurry as a substrate for enzyme production and enhancement of the production with microbial source, which is environment friendly.

PRODUCTION OF ANTIBACTERIAL AGENT FROM FUNGI ISOLATED FROM PHARMACEUTICAL SOIL SAMPLE BY FERMENTATION UNDER OPTIMIZED CONDITIONS

Objective: This study aimed to isolate fungi having antibacterial activity from pharmaceutical site soil sample and production of antibacterial agents by solid and submerged state fermentation under optimized conditions. Antibacterial activity of laboratory isolated and produced antibacterial agent was compared with other commercialized antibiotics to check the efficiency of laboratory produced antibacterial agent. Methods: For isolation and characterization of fungal isolates American Public Health Association standard was followed. Antibacterial activity was determined using disc diffusion and agar disc diffusion method. Results: On the basis of morphological and microscopic characteristics six fungal isolates belongs to four different genus species, i.e., Aspergillus sp. (F1, F2, F3), Penicillium sp. (F4), Rhizopus sp. (F5), and Fusarium sp. (F6), and they were tested against six bacterial isolates, i.e., Streptococcus sp. (B1), Bacillus sp. (B2), Staphylococcus sp. (B3), Bacillus sp. (B4), Bacillus sp. (B5), and Enterococcus sp. (B6). Except B4 all bacterial isolates growth were inhibited by fungal isolates. Under optimized conditions maximum zone of inhibition, i.e., 78 mm against B1 and B5 shown by F1 and F6 at 2% and 1% glucose concentration, respectively, at 10 pH. When comparison was made between commercialized antibiotics and lab produced antibacterial agents, it was observed lab produced antibacterial agent was more efficient in terms of zone of inhibition. Conclusion: Lab isolated and produced antibacterial agents were more efficient than commercialized antibiotics. This study demonstrated that lab isolated antibacterial agents isolated from six fungal isolates seems to be a stable and potent antibacterial and can be used as alternative to expensive commercialized antibiotics.

Amylase Production from Thermophilic Bacillus sp. BCC 021-50 Isolated from a Marine Environment

The high cost of fermentation media is one of the technical barriers in amylase production from microbial sources. Amylase is used in several industrial processes or industries, for example, in the food industry, the saccharification of starchy materials, and in the detergent and textile industry. In this study, marine microorganisms were isolated to identify unique amylase-producing microbes in starch agar medium. More than 50 bacterial strains with positive amylase activity, isolated from marine water and soil, were screened for amylase production in starch agar medium. Bacillus sp. BCC 021-50 was found to be the best amylase-producing strain in starch agar medium and under submerged fermentation conditions. Next, fermentation conditions were optimized for bacterial growth and enzyme production. The highest amylase concentration of 5211 U/mL was obtained after 36 h of incubation at 50 °C, pH 8.0, using 20 g/L molasses as an energy source and 10 g/L peptone as a nitrogen source. From an application perspective, crude amylase was characterized in terms of temperature and pH. Maximum amylase activity was noted at 70 °C and pH 7.50. However, our results show clear advantages for enzyme stability in alkaline pH, high-temperature, and stability in the presence of surfactant, oxidizing, and bleaching agents. This research contributes towards the development of an economical amylase production process using agro-industrial residues.

Bostrycin production by agro-industrial residues and its potential for food processing.

Bostrycin, a red antibacterial agent produced by Nigrospora sp. no. 407, is considered for meat processing. To optimize production, the culture conditions of submerged fermentation (SmF) and solid-state fermentation (SSF) were investigated. The optimal SmF conditions were a medium containing 1.0% cane molasses and incubation at 30°C and 150rpm for 6days. In SSF, other than bostrycin, less pigment was produced and the optimal ratio of bagasse to water was 1:2 for 10days. The production and recovery rate of bostrycin by SmF were 120mg/L and 40%, respectively. Bostrycin exhibited thermostable, pH-dependent color change and dose-dependent antibacterial activity against Clostridium botulinum. Bostrycin-modified meat turned strong red for at least 24h and could not be removed by washing; bostrycin maintained its antibacterial activity with a bacteriostasis rate of 91% on Staphylcoccus aureus. This is an easy and inexpensive means of acquiring bostrycin from molasses and sugarcane.

Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune

Mono and dual cultures of four white-rot basidiomycete species were evaluated for cellulase and xylanase activity under submerged fermentation conditions. Co-cultivation of Pycnoporus coccineus or Trametes hirsuta with Schizophyllum commune displayed antagonistic interactions resulting in the decrease of endoglucanase and total cellulase activities. In contrast, increases in cellulase and xylanase activity were revealed through the compatible interactions of Irpex lacteus with S. commune. Co-cultivation conditions were optimized for maximum enzyme production by I. lacteus and S. commune, the best producers of cellulase/xylanase and β-glucosidase, respectively. An optimized medium for the target enzyme production by the mixed culture was established in a laboratory fermenter yielding 7U/mL total cellulase, 142U/mL endoglucanase, 104U/mL xylanase, and 5.2U/mL β-glucosidase. The dual culture approach resulted in an enzymatic mixture with 11% improved lignocellulose saccharification potential compared to enzymes from a monoculture of I. lacteus.

Proteomic characterization and schizophyllan production by Schizophyllum commune ISTL04 cultured on Leucaena leucocephala wood under submerged fermentation

In this study Schizophyllum commune ISTL04 was grown on Leucaena leucocephala wood (LLW) for secretome analysis and schizophyllan production. There is no report on extracellular protein profile and schizophyllan production on woody biomass by this fungus under submerged fermentation conditions. Leucaena leucocephala, a promising bioenergy crop having high holocellulose content was used as substrate without pretreatment. The maximum sugar, extracellular protein and exopolysaccharide (EPS) production during fermentation was found to be 8.53±0.07mgmL−1, 391±7.51mgL−1 and 4.2±0.1gL−1 or 0.21gg−1LLW on day 18 respectively. The secretome profile was dominated by glycoside hydrolases followed by carbohydrate esterase and other oxidative enzymes. EPS was further characterized by FTIR and GC–MS for functional group, monomer composition and linkage analysis and was identified as schizophyllan. The result indicated that LLW can be utilized as a low cost substrate for enzyme cocktail and schizophyllan production.

Phytase production by Aspergillus niger NCIM 563 for a novel application to degrade organophosphorus pesticides

The production of phytase using Aspergillus niger NCIM 563 under submerged fermentation conditions was studied using protein rich chickpea flour as substrate. Employing a hybrid statistical media optimization strategy of Plackett–Burman and Box-Behnken experimental designs in shake-flasks gave an increased phytase activity from an initial 66 IU/mL in 216 h to 160 IU/mL in a reduced time of 132 h. Productivity, thus increased by 3.97 times from 7.3 to 29 IU/mL/day. Using the optimized media, the production was successfully scaled-up further and improved up to 164 IU/mL in 96 h by studies carried out employing 2 and 10-L fermenters. The enzyme supernatant was recovered using centrifugal separation of biomass and the stability of the produced phytase was tested for animal feed applications under gastric conditions. In vitro degradation studies of water soluble monocrotophos, methyl parathion and water insoluble chlorpyrifos, pesticides used extensively in agriculture was carried out. It was observed by HPLC analysis that phytase could degrade 72% of chlorpyrifos at pH 7.0, 35 °C. Comparable results were obtained with monocrotophos and methyl parathion. With chlorpyrifos at higher temperature 50 °C as much as 91% degradation could be obtained. The degradation of chlorpyrifos was further validated by spraying phytase on harvested green chilli (Capsicum annuum L) under normal conditions of pH 7.0, 35 °C and the degradation products obtained analyzed by LCMS. Thus, the present study brings out a potentially novel application of phytase for biodegradation of organophosphorus pesticides.

Improvement of bacterial α-amylase production and application using two steps statistical factorial design

Abstract Five agro-industrial wastes (AIW) were selected to verify their potential as low cost substrate to produce α-amylase enzyme by bacterial strains using solid state fermentation (SSF) and submerged state fermentation (SMF). Among the AIW used strawberry leaves and watermelon rind produced the maximum α-amylase production under SMF by B.subtilis and B. licheniformis, respectively. Co-culture of B.subtilis and B. licheniformis (1:1) on watermelon rind (Citrullus lanatus) improved α-amylase production by 176.3% and 329.2%, respectively compared to mono culture of B.subtilis and B. licheniformis. Decreasing agitation speed to 100 rpm increased the production by 58.1%. In addition, the pretreatment of WMR with different methods before fermentation inhibited the enzyme production. Using two steps of statistical factorial design (Plackett-Burman and Central Composite) to optimize the production medium enhanced α-amylase production by 10.3-fold. The produced α-amylase could effectively hydrolyze AIW, however the highest saccharification yield with the highest reducing sugar (75% and 15 mg/ml) were obtained using cantaloupe peel (Cucumis melo). It is superior to use cantaloupe peel as a novel AIW for saccharification produced glucose (78.3%) and uronic acid (21.3%). Moreover, using Central Composite design to optimize saccharification condition of cantaloupe peel enhanced the yield of reducing sugar by 3.73-fold.

Cellulase with high β-glucosidase activity by Penicillium oxalicum under solid state fermentation and its use in hydrolysis of cassava residue.

In this study, we investigated cellulase production by Penicillium oxalicum EU2106 under solid-state fermentation (SSF) and its hydrolysis efficiency toward NaOH-H2O2-pretreated cassava residue (NHCR) produced after bioethanol fermentation. Optimization of SSF cultivation conditions for P. oxalicum EU2106 using a Box-behnken design-based response-surface methodology resulted in maximal cellulase activity of 34.0 ± 2.8 filter-paper units/g dry substrate, exhibiting a ~ twofold increase relative to activities obtained under non-optimized conditions. Furthermore, SSF-derived cellulase converted 94.3 ± 1.5% of NHCR cellulose into glucose within 96h. Interestingly, P. oxalicum EU2106 produced higher β-glucosidase activity under SSF conditions than that under submerged-state fermentation conditions, resulting in the elimination of cellobiose inhibition during the early stages of NHCR cellulose hydrolysis. Overall, this work provided an alternative for a potential cellulase source and a preferred option for cassava residue biotechnological application.

Pectinolytic Activity of Mutagenic Strain of Leuconostoc Mesenteroides Isolated From Orange and Banana Fruit Waste

The aim of this study was to isolate pectinolytic bacteria from fermented banana and orange peels. The bacterial isolates were identified using standard biochemical method. The bacteria isolates were screened on pectin agar plates. All the isolates showed pectinolytic activity in terms of making zone surrounding their colony on pectin agar medium. Pectinase activity was determined by dinitrosalicylic (DNS) acid method while protein concentration in the fermentation broth was quantified by Lowry method. The screened isolate designated OP6 tentatively identified as Leuconostoc mesenteroides with highest pectinase activity was subjected to mutagen (Ethidium bromide). The mutants of Leuconostoc mesenteroides generated were screened for pectinase production in comparison with the parent (wild) type in submerged state fermentation. All the mutant strains generated from Leuconostoc mesenteroides had their pectinolytic activities repressed in comparison with the wild strain. Out of mutants screened, mutant designated AB4 have the highest pectinolytic activity 1.54 U/mg. The pectinase activity produced by AB4 was approximately 32% lower than the wild strain. The pretreatment of Leuconostoc mesenteroides with Ethidium bromide caused enzyme repression. The appreciable yield in pectinase activity displayed by the mutant strains when compared with the wild type suggests its industrial relevance. Therefore, use of other chemical mutagens can be tested for further strains improvement.

Optimization of Physico-Chemical Parameters for Hyper Keratinase Production from a Newly Isolated Aspergillus sp. DHE7 using Chicken Feather as Substrate- Management of Biowaste

Objectives: Keratinases have recently gained biotechnological impetus due to their ability in degrading insoluble keratinous solid wastes into simplified forms, which used in the preparation of nitrogenous fertilizers, biodegradable films, glues and foils. Keratinases are widespread in nature and mainly produced by diverse microorganisms including bacteria, fungi and actinomycetes. Therefore, the aim of the present work was to isolate and identify a new potent fungal strain possessing extracellular keratinase production capacity using keratinous solid wastes as the sole carbon and nitrogen sources. Material and methods: Fifteen keratinolytic fungi, isolated from a poultry farm soil using white chicken feathers as substrate, were screened qualitatively and quantitatively for their ability to produce extracellular keratinase. Medium optimization for enzyme production was also studied under submerged fermentation conditions. Results: The results of the present work revealed that a new fungus identified as Aspergillus sp. DHE7 was reported as the most potent fungal strain for extracellular keratinase production under submerged shaking conditions (120 rpm). The highest yield of enzyme of 199+4.2 U/mL was observed after four days of incubation at pH 6.0 and temperature 30 °C using 2 % of chicken feathers as substrate. Medium supplementation with 0.5 % sucrose as an additional source of carbon increased keratinase yield to 226+5.4 U/mL. While using additional nitrogen sources could not promote a favorable effect on keratinase productivity as compared with the basal medium. Different keratinous wastes were used as substrate in the basal medium and better keratinase production was obtained with goat hair medium (452+12.3 U/mL) followed by turkey feather (435+9.2 U/mL) and sheep wool (322+13.4 U/mL) media Conclusions: The present data suggest Aspergillus sp. DHE7 as a potential keratinolytic strain for the bioconversion of keratinous wastes, through a non-polluting process.

Biochemical characterization and synergism of cellulolytic enzyme system from Chaetomium globosum on rice straw saccharification.

BackgroundEfficient hydrolysis of lignocellulosic materials to sugars for conversion to biofuels and chemicals is a key step in biorefinery. Designing an active saccharifying enzyme system with synergy among their components is considered a promising approach.ResultsIn this study, a lignocellulose-degrading enzyme system of Chaetomium globosum BCC5776 (CG-Cel) was characterized for its activity and proteomic profiles, and synergism with accessory enzymes. The highest cellulase productivity of 0.40 FPU/mL was found for CG-Cel under the optimized submerged fermentation conditions on 1% (w/v) EPFB (empty palm fruit bunch), 2% microcrystalline cellulose (Avicel®) and 1% soybean meal (SBM) at 30 °C, pH 5.8 for 6 d. CG-Cel worked optimally at 50–60 °C in an acidic pH range. Proteomics analysis by LC/MS/MS revealed a complex enzyme system composed of core cellulases and accessory hydrolytic/non-hydrolytic enzymes attacking plant biopolymers. A synergistic enzyme system comprising the CG-Cel, a β-glucosidase (Novozyme® 188) and a hemicellulase Accellerase® XY was optimized on saccharification of alkaline-pretreated rice straw by a mixture design approach. Applying a full cubic model, the optimal ratio of ternary enzyme mixture containing CG-Cel: Novozyme® 188: Accellerase® XY of 44.4:20.6:35.0 showed synergistic enhancement on reducing sugar yield with a glucose releasing efficiency of 256.4 mg/FPU, equivalent to a 2.9 times compared with that from CG-Cel alone.ConclusionsThe work showed an approach for developing an active synergistic enzyme system based on the newly characterized C. globosum for lignocellulose saccharification and modification in bio-industries.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0312-7) contains supplementary material, which is available to authorized users.

Effect of media components and morphology of Bacillus natto on menaquinone-7 synthesis in submerged fermentation

Vitamin K2 (menaquinone or MK) plays an important role in blood clotting, cardiovascular disease, and anti-osteoporosis. A novel bacterial strain was isolated and identified as Bacillus natto based on 16SrDNA sequencing and LC-MS analysis. The objective of this study was to improve the extraction efficiency and productivity of MK-7 from B. natto. Acid-heating method efficiently disrupted B. natto cells for MK-7 extraction. Bacillus natto had a wide range of pH (5.0 ~ 9.0) for optimal growth. Its MK-7 yield was increased when rotation speed was increased to 200 rpm. The highest MK-7 yield was obtained when glycerol and soy peptone were used in the growth media. Batch fermentation was subsequently tested in 5 L bioreactor, which gave a high productivity of MK-7 (at 0.60 mg/L/h). A positive correlation between MK-7 yield and sporulation ratio was also found. This study provides valuable information on the extraction and production of menaquinone-7 from B. natto under submerged fermentation condition.

ISOLATION, SCREENING AND CHARACTERIZATION OF ANTIBIOTIC PRODUCING ACTINOMYCETES FROM KAPULUPPADA PLASTIC WASTE DUMPING YARD, VISAKHAPATNAM

Objective: To isolate, screen and characterize antibiotic producing actinomycetes from Kapuluppada plastic waste dumping yard, Visakhapatnam.Methods: A total of 12 soil samples were collected, serially diluted and spread on starch casein agar supplemented with Rifampicin and Cycloheximide for inhibition of bacteria and fungi, respectively. Cross-streak method was used to check the antagonistic activity of isolated actinomycetes against bacteria and fungi. Crude extracts from submerged state fermentation were used for the production of antimicrobial compounds. Agar well diffusion method was used for antimicrobial activity of crude extracts against test organisms. The isolates were characterized by morphological, physiological and biochemical methods.Results: A total of 110 actinomycete isolates were isolated from plastic waste dumping yard. All isolates had shown antimicrobial activity against one or more tested bacteria/fungi. The crude extract of the isolates PD66 (12.2 mm), PD85 (11.5 mm) were most active against methicillin-resistant Staphylococcus aureus, PD4 (14.1 mm), PD66 (15.6 mm) were active against Pseudomonas aeruginosa, whereas the extracts of PD10 (19.2 mm), PD47 (19.8 mm), PD106 (19.1 mm) were active against Candida albicans, PD10 (14.6 mm), PD82 (15.7 mm) active against Saccharomyces cereviciae. The isolates had shown varying morphological, physiological and biochemical characteristics.Conclusion: The actinomycetes isolated from Kapuluppada plastic waste dumping yard were found to be most promising microorganisms for the production of antibacterial and antifungal antibiotics.

Isolation, purification, and characterization of xylanase produced by three species of bacillus under submerged fermentation conditions

This study focuses on the screening and identification of bacteria, which can produce alkaline xylanase at alkaline pH and high temperature. Bacterial isolates from enriched decaying soil, capable of hydrolyzing xylan were screened. Selected and purified 13 bacterial colonies (Bacilli and Kurthia) grown on xylan- nutrient agar slants, were activated and transferred into the fermentation medium. Three highest xylanase producing isolates (Bacillus badius, Kurthia gibsonii, Bacillus circulans) were selected for further studies and the xylanase produced by them were screened for their kinetic properties. The optimum temperature for the activity of the xylanase from Isolates A was 50oC; and for Isolate B was 40oC, while that of Isolate C was 30oC. The optimum pH value for the xylanase from isolate A and B was 9.0. In addition, the xylanase was also capable of producing high-quality xylo-oligosaccharides, which indicated its application potential not only in pulp bio-bleaching processes but also in the nutraceutical industry.

Development of a Cost Effective Medium for Enhanced Production of Bacillus thuringiensis ?-endotoxin

The present study was carried out to develop a sustainable production medium using locally available cheap raw materials for biopesticide production by Bacillus thuringiensis subsp. kurstaki (Btk) HD-73. In submerged fermentation (SmF) condition, the conventional Luria-Bertani (LB) medium which was enriched with nitrogen source (10% defatted soybean meal) supported 28.57% sporulation and 125% endotoxin increase over LB (alone). The effect of cystine on sporulation and endotoxin synthesis was highly pronounced in LB-soybean medium (LBS) with a range of 19.54% and 131.35% higher endotoxin yield respectively in SmF condition. While basal salts supplemented in soybean-cystine (SMc) medium, it resulted in 7.65% endotoxin yield compared to LB-soybean-cystine (LBSc) medium. Addition of molasses balanced the C: N ratio in the SMc medium thus helping 84.85% higher endotoxin synthesis after 24 hours fermentation. Substitution of basal salts with cost effective sea water yielded about 19.27% less endotoxin. The optimum medium thus obtained consisting of soybean extract-molasses-cystine with sea water was used in 3.0 L bioreactor cultivation for endotoxin synthesis by Btk HD-73 under 30% saturation of dO2 through cascade of agitation and aeration. The production rate obtained was 1.67 fold higher in bioreactor than in shake flask culture.Bangladesh J Microbiol, Volume 32, Number 1-2,June-Dec 2015, pp 1-6

Microbial production of four biodegradable siderophores under submerged fermentation

Four siderophore analogues were isolated and purified from Escherichia coli, Bacillus spp. ST13, and Streptomyces pilosus microorganisms under some specific submerged fermentation conditions. In order to evaluate the highest production of this siderophore analogues through the growth, a rapid spectrophotometric screening semi-quantitative method was used, in which interestingly the analogues were isolated in its own form not its iron chelate. After chromatographic separation, the chemical structures of the isolated and purified siderophores were illustrated using detailed spectroscopic techniques. The biodegradation studies were done on that four novel isolated and purified siderophores following OECD protocols. In addition, the bioactivities of these siderophores and their iron complexes were examined and evaluated.

Camptothecine production by mixed fermentation of two endophytic fungi from Nothapodytes nimmoniana

Two endophytic fungi isolated from the endangered plant Nothapodytes nimmoniana (Grah.) Mabb. were found to effectively synthesize CPT independent of their host plant under submerged fermentation conditions. Molecular characterization of fungi revealed their identity as Colletotrichum fructicola SUK1 (F1) and Corynespora cassiicola SUK2 (F2). Mixed fermentation experiments were carried out to study the effect of microbial signalling between the two fungal species on camptothecine production. Effect of culture parameters on CPT production was studied for both mono-cultures (F1 and F2) separately as well as for the mixed fermentation (F1 + F2). Further the most influencing ones were optimized statistically using response surface methodology. Statistical model based optimized parameters were whey (70 %), agitation rate (110 rpm), temperature (30 °C), and incubation period (7 d) for the mixed fermentation. Monocultures of the two fungal species F1 and F2 yielded CPT up to 33 ± 1.1 mg l−1and 69 ± 1.1 mg l−1, respectively; while their mixed fermentation under the optimized conditions yielded up to 146 ± 0.2 mg l−1. HPLC and LC-MS/MS techniques were used to analyze the products obtained.