Tuning mesoporous molecular sieve SBA-15 for the immobilization of α-amylase

The present work describes the immobilization of α-amylase over well ordered mesoporous molecular sieve SBA-15 with different pore diameters synthesized by post synthesis treatment (PST) hydrothermally after reaction at 40°C. The materials were characterized by N 2 adsorption–desorption studies, small angle X-ray diffraction, scanning electron microscopy and high resolution transmission electron microscopy. Since α-amylase obtained from Bacillus subtilis has dimensions of 35 × 40 × 70 Å it is expected that the protein have access to the pore of SBA-15 (PST-120°C) with diameter 74 Å. The pore dimension is appropriate to prevent considerable leaching. The rate of adsorption of the enzyme on silica of various pore sizes revealed the influence of morphology, pore diameter, pore volume and pH.

Application of Fermentation Techniques in the Utilization of Prawn Shell Waste

The present study aimed at the utlisation of microbial organisms for the production of good quality chitin and chitosan. The three strains used for the study were Lactobacillus plantarum, Lactobacililus brevis and Bacillus subtilis. These strains were selected on the basis of their acid producing ability to reduce the pH of the fermenting substrates to prevent spoilage and thus caused demineralisation of the shell. Besides, the proteolytic enzymes in these strains acted on proteinaceous covering of shrimp and thus caused deprotenisation of shrimp shell waste. Thus the two processes involved in chitin production can be affected to certain extent using bacterial fermentation of shrimp shell.Optimization parameters like fermentation period, quantity of inoculum, type of sugar, concentration of sugar etc. for fermentation with three different strains were studied. For these, parameters like pH, Total titrable acidity (TTA), changes in sugar concentration, changes in microbial count, sensory changes etc. were studied.Fermentation study with Lactobacillus plantarum was continued with 20% w/v jaggery broth for 15 days. The inoculum prepared yislded a cell concentration of approximately 108 CFU/ml. In the present study, lactic acid and dilute hydrochloric acid were used for initial pH adjustment because; without adjusting the initial pH, it took more than 5 hours for the lactic acid bacteria to convert glucose to lactic acid and during this delay spoilage occurred due to putrefying enzymes active at neutral or higher pH. During the fermentation study, pH first decreased in correspondence with increase in TTA values. This showed a clear indication of acid production by the strain. This trend continued till their proteolytic activity showed an increasing trend. When the available sugar source started depleting, proteolytic activity also decreased and pH increased. This was clearly reflected in the sensory evaluation results. Lactic acid treated samples showed greater extent of demineralization and deprotenisation at the end of fermentation study than hydrochloric acid treated samples. It can be due to the effect of strong hydrochloric acid on the initial microbial count, which directly affects the fermentation process. At the end of fermentation, about 76.5% of ash was removed in lactic acid treated samples and 71.8% in hydrochloric acid treated samples; 72.8% of proteins in lactic acid treated samples and 70.6% in hydrochloric acid treated samples.The residual protein and ash in the fermented residue were reduced to permissible limit by treatment with 0.8N HCI and 1M NaOH. Characteristics of chitin like chitin content, ash content, protein content, % of N- acetylation etc. were studied. Quality characteristics like viscosity, degree of deacetylation and molecular weight of chitosan prepared were also compared. The chitosan samples prepared from lactic acid treated showed high viscosity than HCI treated samples. But degree of deacetylation is more in HCI treated samples than lactic acid treated ones. Characteristics of protein liquor obtained like its biogenic composition, amino acid composition, total volatile base nitrogen, alpha amino nitrogen etc. also were studied to find out its suitability as animal feed supplement.Optimization of fermentation parameters for Lactobacillus brevis fermentation study was also conducted and parameters were standardized. Then detailed fermentation study was done in 20%wlv jaggery broth for 17 days. Also the effect of two different acid treatments (mild HCI and lactic acid) used for initial pH adjustment on chitin production were also studied. In this study also trend of changes in pH. changes in sugar concentration ,microbial count changes were similar to Lactobacillus plantarum studies. At the end of fermentation, residual protein in the samples were only 32.48% in HCI treated samples and 31.85% in lactic acid treated samples. The residual ash content was about 33.68% in HCI treated ones and 32.52% in lactic acid treated ones. The fermented residue was converted to chitin with good characteristics by treatment with 1.2MNaOH and 1NHCI.Characteristics of chitin samples prepared were studied and extent of Nacetylation was about 84% in HCI treated chitin and 85%in lactic acid treated ones assessed from FTIR spectrum. Chitosan was prepared from these samples by usual chemical method and its extent of solubility, degree of deacetylation, viscosity and molecular weight etc were studied. The values of viscosity and molecular weight of the samples prepared were comparatively less than the chitosan prepared by Lactobacillus plantarum fermentation. Characteristics of protein liquor obtained were analyzed to determine its quality and is suitability as animal feed supplement.Another strain used for the study was Bacillus subtilis and fermentation was carried out in 20%w/v jaggery broth for 15 days. It was found that Bacillus subtilis was more efficient than other Lactobacillus species for deprotenisation and demineralization. This was mainly due to the difference in the proteolytic nature of the strains. About 84% of protein and 72% of ash were removed at the end of fermentation. Considering the statistical significance (P

Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps

The constitutive production of AMPs in shrimps ensures that animals are able to protect themselves from low-level assaults by pathogens present in the environment. As these molecules play important roles in the shrimp immune defense system, the expression level of these AMPs are possible indicators of the immune state of shrimps. The present study also indicates the antiviral property of AMPs, especially ALF, stressing the importance of their up-regulation through the application of immunostimulants/probiotics as a prophylactic strategy in aquaculture. The present study shows that shrimp defense system is equipped enough to evade WSSV infection to a certain extent, when the animals were maintained on marine yeast and probiotic diet, whereas the control diet fed group succumbed to WSSV infection. This study reveals that marine yeast and probiotic supplemented diet can delay the process of WSSV infection and confer greater protection to the animals. Particularly, the protection conferred by marine yeast, C. haemulonii S27 and Bacillus MCCB101 were highly promising imparting greater hope to the aquaculture community to overcome the prevailing disease problems in aquaculture. It may be inferred from the present study that up-regulation of AMP genes could be effected by the application of immunostimulants and probiotics. Also, AMP expression profile could be used as an effective tool for screening immunostimulants and probiotics for application in shrimp culture. Ultimately, it is likely that no single compound or strategy will provide a solution to the problem of disease within aquaculture and that, in reality, a suite of techniques will be required including the manipulation of the rearing environment, addition of probionts as a matter of routine during culture, and the use of immunostimulants and other supplements during vulnerable growth phases. Finally, the development of good management practices, the control of environmental variables, genetic improvement in the penaeid species, understanding of host-virus interaction, modulation of the shrimp immune system, supported by functional genomics and proteomics of this crustacean, as a whole suggests that the control of WSSV is not far.

Studies on Production of Bacterial Xylanases

Xylanases with hydrolytic activity on xylan, one of the hemicellulosic materials present in plant cell walls, have been identified long back and the applicability of this enzyme is constantly growing. All these applications especially the pulp and paper industries require novel enzymes. There has been lot of documentation on microbial xylanases, however, none meeting all the required characteristics. The characters being sought are: higher production, higher pH and temperature optima, good stabilities under these conditions and finally the low associated cellulase and protease production. The present study analyses various facets of xylanase biotechnology giving emphasis on bacterial xylanases. Fungal xylanases are having problems like low pH values for both enzyme activity and growth. Moreover, the associated production of cellulases at significant levels make fungal xylanases less suitable for application in paper and pulp industries.Bacillus SSP-34 selected from 200 isolates was clearly having xylan catabolizing nature distinct from earlier reports. The stabilities at higher temperatures and pH values along with the optimum conditions for pH and temperature is rendering Bacillus SSP-34 xylanase more suitable than many of the previous reports for application in pulp and paper industries.Bacillus SSP-34 is an alkalophilic thertmotolerant bacteria which under optimal cultural conditions as mentioned earlier, can produce 2.5 times more xylanase than the basal medium.The 0.5% xylan concentration in the medium was found to the best carbon source resulting in 366 IU/ml of xylanase activity. This induction was subjected to catabolite repression by glucose. Xylose was a good inducer for xylanase production. The combination of yeast extract and peptone selected from several nitrogen sources resulted in the highest enzyme production (379+-0.2 IU/ml) at the optimum final concentration of 0.5%. All the cultural and nutritional parameters were compiled and comparative study showed that the modified medium resulted in xylanase activity of 506 IU/ml, 5 folds higher than the basal medium.The novel combination of purification techniques like ultrafiltraton, ammonium sulphate fractionation, DEAE Sepharose anion exchange chromatography, CM Sephadex cation exchange chromatography and Gel permeation chromatography resulted in the purified xylanase having a specific activity of 1723 U/mg protein with 33.3% yield. The enzyme was having a molecular weight of 20-22 kDa. The Km of the purified xylanase was 6.5 mg of oat spelts xylan per ml and Vmax 1233 µ mol/min/mg protein.Bacillus SSP-34 xylanase resulted in the ISO brightness increase from 41.1% to 48.5%. The hydrolytic nature of the xylanase was in the endo-form.Thus the organism Bacillus SSP-34 was having interesting biotechnological and physiological aspects. The SSP-34 xylanase having desired characters seems to be suited for application in paper and pulp industries.

Proteases from an Environmental Isolate of Pseudomonas Aeruginosa MCCB 123 and Their Applications

In the present study,heterotrophic protease producing bacterial isolates were screened for protease activity and a potent protease producing bacterial isolate was selected,identified and coded as Pseudomonas aeruginosa MCCB 123.The organism was capable of producing three different types of enzymes each having potential industrial applications.The non-toxic nature of the bacterial strain and the relatively non-toxic nature of three enzymes suggested their poetential application in various industries.Application of LasA protease and beta-1,3 glucanase in DNA extraction is a promising area for commercial utilization. LasB protease can find its potential application in detergent and tanning industries.As on today Bacillus sp.has been the source of commercial proteases,and the ones produced form P.aeruginosa 123 can pave way for making the industrial and biomedical processes more cost effective and refined.

Studies on immobilization of Bacteria

Cell immnhilizatinn technology in a rapidly expanding arna in the endeavour of microbial fnrmentatiwn.During the lnmt 15 years anveral prnceafinn have been developed and more are in developmental atage of approaching commercial utilizatinn.In the present programme it was planned to develop an optimized process for the innobilization of alpha amylase producing Bacillus polymyxa (CBTB 25) an isolate obtained from Cochin University campus primarily for the production of alpha-amylase.Optimal concentration of support material that attributaa stability and maximal activity to the immobilized cell beads was determined using different concentrations of sodium aliginate as support and estimation of amylase production.An overeall assessment of the data obtained for the various studies conducted denotes that immobilized cells synthesize alpha-amylase at comparable rates with free cells and produce reducing sugara at a higher level than free cells.Results indicated that both phosphate and citrate buffers could be used for disrupting the immobilized beads since they enforced maximal release of cells through leaching from the beads within one hour.On comparative analysis it was observed that immobilized cells could synthesize alpha amylase at similar levels with free cells of B.polymyxa.On Co-immobilization of B.Polymyxa with S.cerevisiae,the co-immobilizate beads could effeciently convert starch directly to ethanol with a yield of 14.8% at 1 : 2 ratio.

L-Glutaminase production by Marine Bacteria

L-glutaminases (L—glutamine amidohydrolase EC.3.5.l.2) is proposed as a prospective candidate for enzyme therapy cnf cancer and also as zui important additive during enzymatic digestion of shoyu koji since it could enhance glutamate content of soysauce. Commercial production of glutaminase could make possible its wide application in these areas, which would demand availability of potential sources and suitable fermentation techniques. The ‘present investigation highlighted marine environment as a potential source of efficient glutaminase producing bacteria mainly species of pseudomonas, aeromonas ,vibrio,alcaligenes, acinetobacter bacillus and planococci.Among them pseudomonas fluorescens ACMR 267 and v.cholerae ACMR 347 were chosen as the ideal strains for glutaminase production.Extracellular glutaminase fraction from all strains were in higher titres than intracellular enzymes during growth in mineral media, nutrient broth and nutrient broth added with glutamine.Glutaminase from all strains were purified employing (NH4)2SO4 fractionation followed tnr dialysis and ion exchange chromatography. The purified glutaminase from all strains were observed to be active and stable over a wide range of gfii and temperature.Optimization studies cflf environmental variables that normally influence time yiehi of glutaminase indicated that the optimal requirements of these bacteria for maximal glutaminase production remained stable irrespective of the medium, they are provided with for enzyme production. However, solid state fermentation technique was observed to be the most suitable process for the production of Glutaminase.

Heterotrophic bacteria from the continental slope sediments of arabian sea

The marine environment is indubitably the largest contiguous habitat on Earth. Because of its vast volume and area, the influence of the world ocean on global climate is profound and plays an important role in human welfare and destiny. The marine environment encompasses several habitats, from the sea surface layer down through the bulk water column, which extends >10,000 meters depth, and further down to the habitats on and under the sea floor. Compared to surface habitats, which have relatively high kinetic energy, deep-ocean circulation is very sluggish. By comparison, life in the deep sea is characterized by a relatively constant physical and chemical environment. Deep water occupying the world ocean basin is a potential natural resource based on its properties such as low temperature, high pressure and relatively unexplored properties. So, a judicious assessment of the marine resources and its management are essential to ensure sustainable development of the country’s ocean resources. Marine sediments are complex environments that are affected by both physiological and biological factors, water movements and burrowing animals. They encompass a large extent of aggregates falling from the surface waters. In aquatic ecosystems, the flux of organic matter to the bottom sediments depend on primary productivity at the ocean surface and water depth. Over 50% of the earth’s surface is covered by deep-sea sediments that are primarily formed through the continual deposition of particles from the productive pelagic waters (Vetriani et al., 1999). These aggregates are regarded as ‘hot spots’ of microbial activity in the ocean (Simon et al., 2002). This represents a good nutritional substrate for heterotrophic bacteria and favours bacterial growth

Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags

The diversity and load of heterotrophic bacteria and fungi associated with the mangrove soil from Suva, Fiji Islands, was determined by using the plate count method. The ability of the bacterial isolates to produce various hydrolytic enzymes such as amylase, gelatinase and lipase were determined using the plate assay. The heterotrophic bacterial load was considerably higher than the fungal load. There was a predominance of the gram positive genus, Bacillus. Other genera encountered included Staphylococcus, Micrococcus, Listeria and Vibrio. Their effectiveness on the degradation of commercial polythene carry bags made of high density polyethylene (HDPE) and low density polyethylene (LDPE) was studied over a period of eight weeks in the laboratory. Biodegradation was measured in terms of mean weight loss, which was nearly 5 % after a period of eight weeks. There was a significant increase in the bacterial load of the soil attached to class 2 (HDPE) polythene. After eight weeks of submergence in mangrove soil, soil attached to class 1 and class 3 polythene mostly had Bacillus (Staphylococcus predominated in class 2 polythene). While most of the isolates were capable of producing hydrolytic enzymes such as amylase and gelatinase, lipolytic activity was low. Class 2 HDPE suffered the greatest biodegradation.

Growth enhancement of micro algae, Chaetoceros calcitrans and Nannochloropsis oculata, using selected bacterial strains

In natural systems phytoplankton interact with planktonic (free living) and attached epiphytic bacteria both synergistically and antagonistically. The specificity of the association with micro algae and bacteria differs in terms of adhesion mechanisms and metabolic cooperation. Present research was carried out to study the effect of bacterial isolates namely Bacillus sp. and Pseudomonas sp. from algal culture systems on the growth of micro algae such as Chaetoceros calcitrans and Nannochloropsis oculata. C. calcitrans (F= 15.34; P<0.05) and N. oculata (F=12.52; P<0.05) showed significantly higher growth, in treatments with Bacillus sp. and Pseudomonas sp when compared to control.

SPATIOTEMPORAL VARIATION AND ANTIBACTERIAL ACTIVITY OF ACTINOMYCETES ISOLATED FROM HIGH ALTITUDE GRASSLAND SOILS OF TROPICAL MONTANE FOREST – KERALA, INDIA

TThe invention of novel antibiotics and other bioactive microbial metabolites continues to be an important aim in new drug discovery programmes. Actinomycetes have the potential to synthesize lots of diverse biologically vigorous secondary metabolites and in the last decades actinomycetes became the most productive source for antibiotics. Therefore in the present study we analyze the antibacterial activity of the actinomycetes isolated from grassland soil samples of Tropical Montane forest. A total of 33 actinomycete strains isolated were characterized and screened for antibacterial activities using well diffusion method against six specific pathogenic organisms. Identification of the isolates revealed that the majority of them were belonging to Streptomycetes followed by Nocardia, Micromonospora, Pseudonocardia, Streptosporangium, Nocardiopsis and Saccharomonospora. Among the 33 isolates, Gr1 strain showed antagonistic activity against all checked pathogens. Nine strains showed antibacaterial activity against Listeria, Vibrio cholera, Bacillus cereus, Staphylococcus aureus and Salmonella typhi and only 2 strains (Gr1and Gr25) showed antagonism to E. coli. The overall percentage of activity of actinomycetes isolates against each pathogenic bacterium was also calculated. While 63.63% of the actinomycetes were antagoinistic against Listeria, Vibrio cholerae, and Bacillus cereus, 60.6% of them were antagonistic to Staphylococcus aureus. Very few isolates (6.06%) showed antibacterial activity against E. coli. In general most of the actinomycetes isolates were antagonistic to grampositive bacteria such as Listeria, Bacillus and Staphylococcus than Gram-negative bacteria Vibrio cholerae, E. coli and Salmonella

Molecular characterization of a crustinlike antimicrobial peptide in the giant tiger shrimp, Penaeus monodon, and its expression profile in response to various immunostimulants and challenge with WSSV

A crustinlike antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustinlike AMP from other shrimp crustins. The differential expression of the crustinlike AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two bglucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two grampositive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded preand postchallenge white spot syndrome virus (WSSV) by semiquantitative RTPCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustinlike AMP was found to be constitutively expressed in the animal and a significant downregulation could be noted postchallenge WSSV. Remarkable downregulation of the gene was observed in the immunostimulant fed animals prechallenge followed by a significant upregulation postchallenge WSSV. Tissuewise expression of crustinlike AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustinlike AMP and confer significant protection to P. monodon against WSSV infection

Penaeus monodon larvae can be protected from Vibrio harveyi infection by pre-emptive treatment of a rearing system with antagonistic or non-antagonistic bacterial probiotics

This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non-antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having su⁄cient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB111under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen.These inhibitory propertieswere further con¢rmed in the larval rearing systems of P. monodon. All these four probionts signi¢cantly improved larval survival in long-term treatments as well as when challengedwith a pathogenic strain ofV. harveyiMCCB111. We could demonstrate that Pseudomonas MCCB 102 andMCCB103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems throughactive antagonism of vibrios,whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme-supporting modes of action.

Efficacy of fermented prawn shell waste as a feed ingredient for Indian white prawn, Fenneropenaeus indicus

Prawn shell waste collected from shrimp-processing plants in Cochin, India, was subjected to fermentation using 20 chitinoclastic and proteolytic/non-proteolytic bacterial strains. The products generated were analysed for protein, lipid, total sugars, N-acetyl glucosamine, free amino acids and ash. Shrimp diets were prepared using these 20 fermented products and a control diet using raw prawn shell waste. Feeding experiment was conducted with postlarvae (PL21) of Indian white prawn, Fenneropenaeus indicus for a period of 21 days. Biogrowth parameters such as mean weight gain, feed conversion ratio, specific growth rate and protein efficiency ratio were estimated and the animals were challenged with white spot virus orally via diet. Enhanced growth could be observed in prawns fed F134 and F124, incorporated with the fermentation products generated using Bacillus spp., C134 and C124 respectively. The percentage survival of prawns after 7 days of challenge was found to be highest for groups fed diet F111 incorporated with fermentation product generated using Bacillus sp. These products of bacterial fermentation hold promise as growth enhancers and immunostimulants in aquaculture. KEY WORDS: biogrowth parameters, feed

Biogeochemistry of the shelf sediments of south eastern Arabian sea: Effect on benthic bacterial heterotrophs

The composition and variability of heterotrophic bacteria along the shelf sediments of south west coast of India and its relationship with the sediment biogeochemistry was investigated. The bacterial abundance ranged from 1.12 x 103 – 1.88 x 106 CFU g-1 dry wt. of sediment. The population showed significant positive correlation with silt (r = 0.529, p< 0.05), organic carbon (OC) (r = 0.679, p< 0.05), total nitrogen (TN) (r = 0.638, p< 0.05), total protein (TPRT) (r = 0.615, p< 0.05) and total carbohydrate (TCHO) (r = 0.675, p< 0.05) and significant negative correlation with sand (r = -0.488, p< 0.05). Community was mainly composed of Bacillus, Alteromonas, Vibrio, Coryneforms, Micrococcus, Planococcus, Staphylococcus, Moraxella, Alcaligenes, Enterobacteriaceae, Pseudomonas, Acinetobacter, Flavobacterium and Aeromonas. BIOENV analysis explained the best possible environmental parameters i.e., carbohydrate, total nitrogen, temperature, pH and sand at 50m depth and organic matter, BPC, protein, lipid and temperature at 200m depth controlling the distribution pattern of heterotrophic bacterial population in shelf sediments. The Principal Component Analysis (PCA) of the environmental variables showed that the first and second principal component accounted for 65% and 30.6% of the data variance respectively. Canonical Correspondence Analysis (CCA) revealed a strong correspondence between bacterial distribution and environmental variables in the study area. Moreover, non-metric MDS (Multidimensional Scaling) analysis demarcated the northern and southern latitudes of the study area based on the bioavailable organic matter