Enhanced degradation of α-chitin materials prepared from shrimp processing byproduct and production of N-acetyl-D-glucosamine by thermoactive chitinases from soil mesophilic fungi.

Soil isolates of mesophilic Penicillium monoverticillium CFR 2, Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 were cultivated in solid state fermentation (SSF) using wheat bran solid medium supplemented with α-chitin in order to produce chitinolytic enzyme. Under SSF cultivation, maximum enzymes (U/g IDS) production was 41.0 (endo-chitinase) and 195.4 (ß-N-acetylhexosaminidase) by P. monoverticillium, 26.8 (endo-chitinase) and 222.1 (ß-N-acetylhexosaminidase) by A. flavus and 13.3 (endo-chitinase) and 168.3 (ß-N-acetylhexosaminidase) by F. oxysporum after 166 h of incubation. The crude endo-chitinase and ß-N-acetylhexosaminidase derived from A. flavus and F. oxysporum revealed optimum temperature at 62 ± 1°C, but the enzymes from P. monoverticillium showed optimum temperature at 52 ± 1°C for maximum activity. Several fold increase in endo-chitinase and ß-N-acetylhexosaminidase activities in the crude enzymes preparation was achieved after concentrating with polyethylene glycol. The concentrated crude chitinases from P. monoverticillium, A. flavus and F. oxysporum, respectively yielded 95.6, 96.6 and 96.1 mmol/l of N-acetyl-D: -glucosamine (GlcNAc) in 48 h of reaction from colloidal chitin. While, the crude enzyme preparations of P. monoverticillium, A. flavus and F. oxysporum produced 10.11, 6.85 and 10.7 mmol/l of GlcNAc respectively, in 48 h of reaction from crystalline α-chitin. HPLC analysis of colloidal chitin hydrolysates prepared with crude chitinases derived from P. monoverticillium, A. flavus and F. oxysporum revealed that the major reaction product was monomeric GlcNAc (~80%) and a small amount of (GlcNAc)(4) (~20%), indicating the potential of these enzymes for efficient production of GlcNAc from α-chitin.

Optimization of polyhydroxybutyrate production by Bacillus sp. CFR 256 with corn steep liquor as a nitrogen source.

Polyhydroxyalkanotes (PHAs), the eco-friendly biopolymers produced by many bacteria, are gaining importance in curtailing the environmental pollution by replacing the non-biodegradable plastics derived from petroleum. The present study was carried out to economize the polyhydroxybutyrate (PHB) production by optimizing the fermentation medium using corn steep liquor (CSL), a by-product of starch processing industry, as a cheap nitrogen source, by Bacillus sp. CFR 256. Response surface methodology (RSM) was used to optimize the fermentation medium using the variables such as corn steep liquor (5-25 g l(-1)), Na(2)HPO(4) 2H(2)O (2.2-6.2 g l(-1)), KH(2)PO(4) (0.5-2.5 g l(-1)), sucrose (5-55 g l(-1)) and inoculum concentration (1-25 ml l(-1)). Central composite rotatable design (CCRD) experiments were carried out to study the complex interactions of the variables.The optimum conditions for maximum PHB production were (g l(-1)): CSL-25, Na(2)HPO(4) 2H(2)O-2.2, KH(2)PO(4) - 0.5, sucrose - 55 and inoculum - 10 (ml l(-1)). After 72 h of fermentation, the amount of PHA produced was 8.20 g l(-1) (51.20% of dry cell biomass). It is the first report on optimization of fermentation medium using CSL as a nitrogen source, for PHB production by Bacillus sp.

Biodegradation of shrimp biowaste by marine Exiguobacterium sp. CFR26M and concomitant production of extracellular protease and antioxidant materials: production and process optimization by response surface methodology.

Twelve marine bacterial cultures were screened for extracellular protease activity, and the bacterium CFR26M which exhibited the highest activity on caseinate agar plate was identified as an Exiguobacterium sp. Significant amount of extracellular protease (5.9 ± 0.3 U/ml) and antioxidant materials, measured as 2,2'-diphenyl picrylhydrazyl (DPPH) radical scavenging activity (44.4 ± 0.5 %), was produced by CFR26M in submerged fermentation using a shrimp biowaste medium. Response surface methodology (RSM) was employed to optimize the process variables for maximum production of protease and antioxidant materials by CFR26M. Among the seven variables screened by two-level 2**(7-2) fractional factorial design, the concentration of shrimp biowaste, sugar, and phosphate was found to be significant (p ≤ 0.05). The optimum levels of these variables were determined by employing the central composite design (CCD) of RSM. The coefficient of determination (R (2)) values of 0.9039 and 0.8924 for protease and antioxidant, respectively, indicates the accuracy of the CCD models. The optimum levels of shrimp biowaste, sugar, and phosphate were 21.2, 10.5, and 2.3 % (w/v) for production of protease and 28.8, 12, and 0.32 % (w/v) for production of antioxidant material, respectively. The concentration of shrimp biowaste, sugar, and phosphate had linear and quadratic effect on both protease and antioxidant productions. RSM optimization yielded 6.3-fold increases in protease activity and 1.6-fold in antioxidant material production. The crude protease of CFR26M had a maximum activity at 32 ± 2 °C with pH 7.6. This is the first report on the use of marine Exiguobacterium sp. for concomitant production of protease and antioxidant materials from shrimp biowaste.

Thermoactive ß-N-acetylhexosaminidase production by a soil isolate of Penicillium monoverticillium CFR 2 under solid state fermentation: parameter optimization and application for N-acetyl chitooligosaccharides preparation from chitin.

Two fungal strains were evaluated for ß-N-acetylhexosaminidase production by solid state fermentation using different agro-industrial residues such as commercial wheat bran (CWB) and shrimp shell chitin waste (SSCW), of which Penicillium monoverticillium CFR 2 a local soil isolate showed significantly (P ≤ 0.001) higher ß-N-acetylhexosaminidase activity on CWB medium as compared with the activity of Fusarium oxysporum CFR 8. Fermentation parameters such as incubation temperature, incubation time, initial moisture content and inoculum concentration were optimized by statistically designed experiments, using 3**(4-1) fractional factorial design of Response Surface Methodology. The high R(2) (0.9512) observed during validation experiment showed the usefulness of the model. Highest level of enzyme activity (311.84 U/g IDS) was predicted at 75% (w/w) initial moisture content, 26 °C incubation temperature, 168 h incubation time and initial inoculum, at the highest concentration tested (2.95 ml spore suspension/5 g substrate). Statistical optimization yielded a 4.5 fold increase in ß-N-acetylhexosaminidase activity. The crude ß-N-acetylhexosaminidase showed optimum temperature of 57 ± 1 °C and pH of 3.6 and retained 50% activity after 1 h of incubation at 57 ± 1 °C. SDS-PAGE zymogram revealed crude enzyme was a monomer with an apparent molecular weight ~110 kDa. The crude enzyme formed 6.81 ± 0.03 mM/l of N-acetyl chitooligosaccharides from colloidal chitin in 24 h of incubation. HPLC analysis revealed hydrolysate contained 37.57% N-acetyl chitotriose and 62.43% N-acetyl chitohexose, indicating its potential for specific N-acetyl chitooligosaccharides production.

Bacterial synthesis of poly(hydroxybutyrate- co-hydroxyvalerate) using carbohydrate-rich mahua (Madhuca sp.) flowers.

AIMS: The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp-256. METHODS AND RESULTS: In the present work, three bacterial strains were tested for PHA production on mahua flower extract (to impart 20 g l(-1) sugar) amongst which, Bacillus sp-256 produced higher concentration of PHA in its biomass (51%) compared with Rhizobium meliloti (31%) or Sphingomonas sp (22%). Biosynthesis of poly(hydroxybutyrate-co-hydroxyvalerate) - P(HB-co-HV)--of 90 : 10 mol% by Bacillus sp-256 was observed by gas chromatographic analysis of the polymer. Major component of the flower is sugars (57% on dry weight basis) and additionally it also contains proteins, vitamins, organic acids and essential oils. The bacterium utilized malic acid present in the substrate as a co-carbon source for the copolymer production. The flowers could be used in the form of aqueous extract or as whole flowers. PHA content of biomass (%) and yield (g l(-1)) in a 3.0-l stirred tank fermentor after 30 h of fermentation under constant pH (7) and dissolved oxygen content (40%) were 54% and 2.7 g l(-1), respectively. Corresponding yields for control fermentation with sucrose as carbon source were 52% and 2.5 g l(-1). The polymer was characterized by proton NMR. CONCLUSIONS: Utilization of mahua flowers, a natural substrate for bacterial fermentation aimed at PHA production, had additional advantage, as the sugars and organic acids present in the flowers were metabolized by Bacillus sp-256 to synthesize P(HB-co-HV) copolymer. SIGNIFICANCE AND IMPACT OF THE STUDY: Literature reports on utilization of suitable cheaper natural substrate for PHA copolymer production is scanty. Mahua flowers used in the present experiment is a cheaper carbon substrate compared with several commercial substrates and it is rich in main carbon as well as co-carbon sources that can be utilized by bacteria for PHA copolymer production.