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.     

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.     

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.     

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).     

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

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

Enzymatic degradation of alginate by marine fungi

A total of 72 pre-selected strains of 19 species of marine fungi were tested for their ability to decompose sodium alginate, calcium alginate or freshly prepared calcium alginate gel. Active alginate decomposition was evident in 18 strains (25% of total tested). These belong to only three different species: Asteromyces cruciatus, Corollospora intermedia, and Dendryphiella salina. In broth culture, decomposition of sodium alginate by the two deuteromycetes was followed by gravimetric, electrometric, viscometric, photometric and chromatographic methods in order to characterize the alginase enzyme system and its degradation products. The alginase enzyme complex consisted of at least two different enzyme components: the already known alginate lyase (eliminase) and a new endo-alginate hydrolase. In summary, a model is presented on the alginase-mediated structural and molecular decomposition of sodium alginate by marine fungi.     

Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens

Aims:  To clone and characterize genes encoding novel cellulases from metagenomes of buffalo rumens.
Methods and Results:  A ruminal metagenomic library was constructed and functionally screened for cellulase activities and 61 independent clones expressing cellulase activities were isolated. Subcloning and sequencing of 13 positive clones expressing endoglucanase and MUCase activities identified 14 cellulase genes. Two clones carried two gene clusters that may be involved in the degradation of polysaccharide nutrients. Thirteen recombinant cellulases were partially characterized. They showed diverse optimal pH from 4 to 7. Seven cellulases were most active under acidic conditions with optimal pH of 5·5 or lower. Furthermore, one novel cellulase gene, C67-1, was overexpressed in Escherichia coli , and the purified recombinant enzyme showed optimal activity at pH 4·5 and stability in a broad pH range from pH 3·5 to 10·5. Its enzyme activity was stimulated by dl -dithiothreitol.
Conclusions:  The cellulases cloned in this work may play important roles in the degradation of celluloses in the variable and low pH environment in buffalo rumen.
Significance and Impact of the Study:  This study provided evidence for the diversity and function of cellulases in the rumen. The cloned cellulases may at one point of time offer potential industrial applications.     

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

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

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

Biodegradation of disperse textile dye Brown 3REL by newly isolated Bacillus sp. VUS

Aims:  To isolate the potential micro-organism for the degradation of textile disperse dye Brown 3 REL and to find out the reaction mechanism.
Methods and Results:  16S rDNA analysis revealed an isolate from textile effluent contaminated soil as Bacillus sp. VUS and was able to degrade (100%) dye Brown 3REL within 8 h at static anoxic condition. A significant increase in the activities of lignin peroxidase, laccase and NADH-DCIP reductase was observed up to complete decolourization of Brown 3REL. The optimum temperature required for degradation was 40°C and pH 6·5–12·0. Phyto-toxicity and chemical oxygen demand revealed nontoxic products of dye degradation. The biodegradation was monitored by UV–VIS, FTIR spectroscopy and HPLC. The final products 6,8-dichloro-quinazoline-4-ol and cyclopentanone were characterized by gas chromatography-mass spectrometry. This Bacillus sp. VUS also decolourized (80%) textile dye effluent within 12 h.
Conclusions:  This study suggests that Bacillus sp. VUS could be a useful tool for textile effluent treatment.
Significance and Impact of the Study:  The newly isolated Bacillus sp. VUS decolourized 16 textile dyes and textile dye effluent also. It achieved complete biodegradation of Brown 3REL. Phytotoxicity study demonstrated no toxicity of the biodegraded products for plants with respect to Triticum aestivum and Sorghum bicolor .     

Keratinolytic activity of Bacillus subtilis AMR using human hair

Aims:  To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides.
Methods and Results:  The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0·01% yeast extract increased the keratinolytic activity 4·2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0·01% yeast extract (HMY) at pH 8·0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50°C and pH 9·0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800–2600 Da.
Conclusions:  This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides.
Significance and Impact of the Study:  This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair . These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.     

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.     

Influence of amino acids, organic solvents and surfactants for phenylalanine ammonia lyase activity in recombinant Escherichia coli

Aim:  To improve phenylalanine ammonia lyase (E.C.4·3·1·5-PAL) activity in recombinant Escherichia coli . Some methods for enrichment of PAL activity in recombinant E. coli JM109 were described. In an effort to create a rich enzyme source these methods would lead to improvements in the production of l -phenylalanine.
Methods and Results:  The possibilities of enriching PAL activity in recombinant E . coli was investigated by using individual and combinations of amino acids, organic solvents and surfactants. PAL activity was induced by adding combination of l -phenylalanine and l -tyrosine, activities as high as 64·3 U g⁻¹of cells were obtained and enzyme activity was enriched by over 3·5-fold in comparison with the control. Permeabilization with cetyl trimethyl ammonium bromide or the acetone significantly enriched cellular PAL activity, which improved over 8·2- and 9·0-fold compared with the control, as high as 148·5 and 164·5 U g⁻¹of cells respectively.
Conclusion:  These efforts may provide some effective methods for enhancing l -phenylalanine ammonia lyase activity.
Significance and Impact of the Study:  These approaches for manipulating recombinant E . coli in an effort to create a rich enzyme source would serve as a biotechnologically important protocol for production of l -phenylalanine.     

Bacteriophage‐derived enzyme that depolymerizes the alginic acid capsule associated with cystic fibrosis isolates of Pseudomonas aeruginosa

Aims: To identify enzymes associated with bacteriophages infecting cystic fibrosis (CF) strains of Pseudomonas aeruginosa that are able to degrade extracellular alginic acids elaborated by the host bacterium. Methods and Results: Plaques produced by 21 Ps. aeruginosa‐specific phages were screened for the presence of haloes, an indicator of capsule hydrolytic activity. Four phages produced haloed plaques, and one (PT‐6) was investigated further. PT‐6 was shown by electron microscopy to belong to Podoviridae family C1, to reduce the viscosity of four alginate preparations using a rolling ball viscometer and to release uronic acid‐containing fragments from the polymers, as judged by spectrophotometry and thin layer chromatography. The alginase was partially purified by gel filtration chromatography and shown to be a 37 kDa polypeptide. Conclusions: Infection of CF strains of Ps. aeruginosa by phage PT‐6 involves hydrolysis of the exopolysaccharide secreted by the host. Significance and Impact of the Study: The alginase produced by PT‐6 has the potential to increase the well‐being of CF suffers by improving the surface properties of sputum, accelerating phagocytic uptake of bacteria and perturbing bacterial growth in biofilms.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Antimicrobial activity of two peptides casecidin 15 and 17, found naturally in bovine colostrum

Aims:  To isolate and characterize peptides from bovine colostrum with antimicrobial activity.
Methods and Results:  Three peptides were purified from fresh colostrum by a range of chromatographic methods using antimicrobial activity against Escherichia coli DH5α to screen for the most active fractions. Two peptides, with antimicrobial activity, casecidin 17 and casecidin 15, were identical to sequences in the C-terminal of bovine β-casein (YQEPVLGPVRGPFPIIV and YQEPVLGPVRGPFPI) and had corresponding molecular masses of 1881·00 and 1669·06 Da, respectively. The third peptide was the known peptide isracidin which has a mass of 2763·80 Da and sequence of RPKHPIKHQGLPQEVLNENLLRF. Casecidin 17 and casecidin 15 had identical minimal inhibition concentrations (MICs) against E. coli DPC6053 of 0·4 mg ml⁻¹. Structural modelling suggested amphiphilic structures having identical inhibitory and structural properties. The MIC value of isracidin against E. coli DPC6053 was 0·2 mg ml⁻¹.
Conclusions:  This study shows the presence of three antimicrobial peptides in colostrum which may contribute to a bioprotective role to limit pathogen contamination. Furthermore, the discovery of casecidin 17 and 15 may provide the basis for novel antimicrobial peptide design.
Significance and Impact of the Study:  This is the first study to characterize peptides with antimicrobial activity present in fresh bovine colostrum.     

Properties of an alginate-degrading Flavobacterium sp. strain LXA isolated from rotting algae from coastal China

A novel bacterium exhibiting alginolytic activity was isolated from rotten algae. The alginate-degrading activity was detected in the culture supernatant by measuring the decrease in alginate viscosity or the increase in reducing sugars. Basic characterization showed that it was gram negative, rod shaped, yellow pigmented, and positive for oxidase and catalase, with a DNA G+C content of 35.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain is related to members of the genus Flavobacterium. Sequence similarity values with their nearest phylogenetic neighbours ranged from 95.9% to 96.7%. Genotypic results, together with phenotypic characteristics, differentiated this species from related Flavobacterium organisms with validly published names, which suggests that the organism should be a new species of the genus Flavobacterium tentatively named as Flavobacterium sp. strain LXA.     

The antimicrobial effects of hops (Humulus lupulus L.) on ruminal hyper ammonia-producing bacteria

Aim:  To assess the antimicrobial effects of hops ( Humulus lupulus L.) on hyper ammonia producing-bacteria (HAB), which catabolize amino acids and peptides in the bovine rumen.
Methods and Results:  When media were amended with dried hops or hops extract (30·7% lupulone), ammonia production by mixed rumen bacteria was inhibited. The growth and ammonia production of pure cultures ( Peptostreptococcus anaerobius, Clostridium aminophilum, or Clostridium sticklandii ) was inhibited by 30 ppm lupulone at pH 6·7, and bactericidal activity was observed at pH 5·6. When hops extract was added to energized cell suspensions, the intracellular pH rapidly decreased and intracellular potassium was lost.
Conclusions:  The three HAB species were sensitive to the antimicrobial components in hops, and the inhibition of ammonia production by mixed rumen bacteria indicates that similar effects could be expected in the rumen.
Significance and Impact of the Study:  As much as half of the amino acids consumed by ruminants can be lost due to microbial degradation in the rumen. This study supports the idea that biologically active plant metabolites can be used to mitigate this wasteful process.