Film forming microbial biopolymers for commercial applications--a review.

Microorganisms synthesize intracellular, structural and extracellular polymers also referred to as biopolymers for their function and survival. These biopolymers play specific roles as energy reserve materials, protective agents, aid in cell functioning, the establishment of symbiosis, osmotic adaptation and support the microbial genera to function, adapt, multiply and survive efficiently under changing environmental conditions. Viscosifying, gelling and film forming properties of these have been exploited for specific significant applications in food and allied industries. Intensive research activities and recent achievements in relevant and important research fields of global interest regarding film forming microbial biopolymers is the subject of this review. Microbial polymers such as pullulan, kefiran, bacterial cellulose (BC), gellan and levan are placed under the category of exopolysaccharides (EPS) and have several other functional properties including film formation, which can be used for various applications in food and allied industries. In addition to EPS, innumerable bacterial genera are found to synthesis carbon energy reserves in their cells known as polyhydroxyalkanoates (PHAs), microbial polyesters, which can be extruded into films with excellent moisture and oxygen barrier properties. Blow moldable biopolymers like PHA along with polylactic acid (PLA) synthesized chemically in vitro using lactic acid (LA), which is produced by LA bacteria through fermentation, are projected as biodegradable polymers of the future for packaging applications. Designing and creating of new property based on requirements through controlled synthesis can lead to improvement in properties of existing polysaccharides and create novel biopolymers of great commercial interest and value for wider applications. Incorporation of antimicrobials such as bacteriocins or silver and copper nanoparticles can enhance the functionality of polymer films especially in food packaging applications either in the form of coatings or wrappings. Use of EPS in combinations to obtain desired properties can be evaluated to increase the application range. Controlled release of active compounds, bioactive protection and resistance to water can be investigated while developing new technologies to improve the film properties of active packaging and coatings. An holistic approach may be adopted in developing an economical and biodegradable packaging material with acceptable properties. An interdisciplinary approach with new innovations can lead to the development of new composites of these biopolymers to enhance the application range. This current review focuses on linking and consolidation of recent research activities on the production and applications of film forming microbial polymers like EPS, PHA and PLA for commercial applications.

Effect of carbon and nitrogen sources on simultaneous production of α-amylase and green food packaging polymer by Bacillus sp. CFR 67.

In this paper, effect of different carbon and nitrogen sources, including hydrolysates of rice bran and wheat bran, on simultaneous production of α-amylase (for hydrolysis of starch in food systems) and polyhydroxyalkanoates (PHA, a green biopolymer, which can be used as a packing material for foods) by Bacillus sp. CFR 67 was studied by submerged fermentation. Amongst various carbon sources tested, glucose and sucrose supported production of significantly (P < 0.05) higher amount of α-amylase (66 U/ml) and PHA (444 mg/l), respectively. Of the nitrogen sources tested, ammonium acetate and beef extract led to the production of maximum amount of amylase (36 U/ml) and PHA (592 mg/l), respectively. Supplementation of the production medium with wheat bran hydrolysate (50 ml/l) produced significantly higher amounts of amylase (73 U/ml) and PHA (524 mg/l). Thus this study indicated the potential of agro-residues for the production of value added biomolecules, which can reduce the cost of production of these molecules and enables to reduce the pollution mainly caused by the use of non biodegradable plastics.

Agro-industrial residues and starch for growth and co-production of Polyhydroxyalkanoate copolymer and alfa-amylase by Bacillus. SP CFR67îen

Polyhydroxyalkanoates (PHA) and α-amylase (α-1,4 glucan-4-glucanohydrolase, E.C. 3.2.1.1) were co-produced by Bacillus sp. CFR-67 using unhydrolysed corn starch as a substrate. Bacterial growth and polymer production were enhanced with the supplementation of hydrolysates of wheat bran (WBH) or rice bran (RBH) individually or in combination (5-20 g L-1, based on weight of soluble substrates-SS). In batch cultivation, a mixture of WBH and RBH (1:1, 10 g L-1 of SS) along with ammonium acetate (1.75 g L-1) and corn starch (30 g L-1) produced maximum quantity of biomass (10 g L-1) and PHA (5.9 g L-1). The polymer thus produced was a copolymer of polyhydroxybutyrate-co-hydroxyvalerate of 95:5 to 90:10 mol%. Presence of WBH and corn starch (10-50 g L-1) in the medium enhanced fermentative yield of α-amylase (2-40 U mL-1 min-1). The enzyme was active in a wide range of pH (4-9) and temperature (40-60ºC). This is the first report on simultaneous production of copolymer of bacterial PHA and α-amylase from unhydrolysed corn starch and agro-industrial residues as substrates.

Agro-industrial residues and starch for growth and co-production of polyhydroxyalkanoate copolymer and α-amylase by Bacillus sp. CFR-67.

Polyhydroxyalkanoates (PHA) and α-amylase (α-1,4 glucan-4-glucanohydrolase, E.C. 3.2.1.1) were co-produced by Bacillus sp. CFR-67 using unhydrolysed corn starch as a substrate. Bacterial growth and polymer production were enhanced with the supplementation of hydrolysates of wheat bran (WBH) or rice bran (RBH) individually or in combination (5-20 g L(-1), based on weight of soluble substrates-SS). In batch cultivation, a mixture of WBH and RBH (1:1, 10 g L(-1) of SS) along with ammonium acetate (1.75 g L(-1)) and corn starch (30 g L(-1)) produced maximum quantity of biomass (10 g L(-1)) and PHA (5.9 g L(-1)). The polymer thus produced was a copolymer of polyhydroxybutyrate-co-hydroxyvalerate of 95:5 to 90:10 mol%. Presence of WBH and corn starch (10-50 g L(-1)) in the medium enhanced fermentative yield of α-amylase (2-40 U mL(-1) min(-1)). The enzyme was active in a wide range of pH (4-9) and temperature (40-60°C). This is the first report on simultaneous production of copolymer of bacterial PHA and α-amylase from unhydrolysed corn starch and agro-industrial residues as substrates.

Extractability of polyhydroxyalkanoate synthesized by Bacillus flexus cultivated in organic and inorganic nutrient media.

Bacillus flexus was isolated from local soil sample and identified by molecular methods. In inorganic nutrient medium (IM) containing sucrose as carbon source, yield of biomass and polyhydroxyalkanoate (PHA) were 2 g/l and 1 g/l (50% of biomass), respectively. Substitution of inorganic nitrogen by peptone, yeast extract or beef extract resulted in biomass yields of 4.1, 3.9 and 1.6 g/l, respectively. Corresponding yields of PHA in biomass was 30%, 40% and 44%. Cells subjected to change in nutrient condition from organic to inorganic, lacked diaminopimelic acid in the cell wall and the concentration of amino acids also decreased. Under these conditions the extractability of the polymer from the cells by hot chloroform or mild alkali hydrolysis was 86-100% compared to those grown in yeast extract or peptone (32-56%). The results demonstrated that growth, PHA production and the composition of cell wall of B. flexus are influenced by the organic or inorganic nutrients present in the growth medium. Cells grown in inorganic medium lysed easily and this can be further exploited for easier recovery of the intracellular PHA.

Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy.

Rhizobium meliloti produced a copolymer of short chain length polyhydroxyalkanoate (scl-PHA) on sucrose and rice bran oil as carbon substrates. Recombinant Escherichia coli (JC7623ABC1J4), bearing PHA synthesis genes, was used to synthesize short chain length-co-medium chain length PHA (scl-co-mcl-PHA) on glucose and decanoic acid. Fourier transform infrared spectroscopy (FTIR) spectra of the PHAs indicated strong characteristic bands at 1282, 1723, and 2934 cm(-1) for scl-PHA and at 2933 and 2976 cm(-1) for scl-co-mcl-PHA polymer. Differentiation of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate-P(HB-co-HV) copolymer was obseverd using FTIR, with absorption bands at 1723 and 1281 for PHB, and at 1738, 1134, 1215 cm(-1) for HV-copolymer. The copolymers were analyzed by GC and (1)H NMR spectroscopy. Films of polymer blends of PHA produced by R. meliloti and recombinant E. coli were prepared using glycerol, polyethylene glycol, polyvinyl acetate, individually (1:1 ratio), to modify the mechanical properties of the films and these films were evaluated by FTIR and scanning electron microscopy.

Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus.

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated beta-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and (1)H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of beta-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.

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.

Effect of salinity on growth of green alga Botryococcus braunii and its constituents.

Growth of Botryococcus braunii (race 'A') and production of its constituents viz, hydrocarbon, carbohydrate, fatty acid, and carotenoids were influenced by different levels of salinity. Under salinity at 34 mM and 85 mM, 1.7-2.25-fold increase in the relative proportion of palmitic acid and two fold increase in oleic acid were observed. A twofold increase in carotenoid content was noticed at 85 mM salinity with lutein (75% of total carotenoid) as the major carotenoid followed by beta-carotene. The increase in biomass yields and changes in other constituents indicated the influence of salinity and the organism's adaptability to the tested levels of salinity (17 mM to 85 mM).

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.

Resistant starch derived from processed legumes: in vitro and in vivo fermentation characteristics.

The effect of processing of legumes on resistant starch formation, its rate of fermentation and the production of short chain fatty acids under in vitro and in vivo systems was assessed. The content of resistant starch in pressure-cooked Bengal gram, black gram and red gram was 3.59%, 1.58% and 3.34%, respectively. Fermentation in vitro of resistant starch derived from processed red gram showed higher amount of short chain fatty acids (2.38 mmol), especially butyric acid (2.22 mmol). Under in vivo conditions (in albino rats) all processed legumes showed a higher faecal bulking, and more short chain fatty acids, with a significant increase in the anaerobic bacterial counts. Compared with a processed legume diet, the caecum of animals fed a raw diet showed a preponderance of propionic acid.

Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. using the polymerase chain reaction (PCR).

AIMS: The aim of the work was to develop efficient method to identify polyhydroxyalkanoate (PHA)-producing species of Bacillus from numerous soil isolates of bacteria. Identification of the isolates and characterization of the PHA produced by strains positive on the polymerase chain reaction (PCR) was envisaged. METHODS AND RESULTS: Different bacteria isolated from soil were screened by PCR using two sets of primers designed for Bacillus megaterium. Amongst 23 isolates examined, the DNA of 12 isolates reacted positively with the primers giving amplicons identical in size to that obtained from B. megaterium. The isolates which were identified as strains of B. sphaericus, B. circulans, B. brevis and B. licheniformis, produced 11- 41% of PHA in biomass, in sucrose-containing medium, over a growth period of 24-72 h. The nature of the PHA thus produced was analyzed by Fourier transform infrared spectroscopy, gas chromatography and by nuclear magnetic resonance (NMR) and found to contain polyhydroxy butyrate and polyhydroxyvalerate. CONCLUSIONS: The results indicate that most of our isolates from different species contained the B. megaterium type of PHA synthase. Bacillus licheniformis appeared to belong to another group as it did not react with both sets of primers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the universality of the B. megaterium type of PHA synthase in soil isolates of Bacillus. Some variations were also found.

Stimulatory effect of honey on multiplication of lactic acid bacteria under in vitro and in vivo conditions.

The effect of honey and sucrose on lactic acid bacteria in vitro and in rat gut was studied to determine whether these organisms were affected differently by honey compared with sucrose. Under in vitro conditions, the number of Lactobacillus acidophilus and Lactobacillus plantarum counts increased 10-100 fold in the presence of honey compared with sucrose. Feeding of honey to rats also resulted in significant increase in counts of lactic acid bacteria. Although there was no significant difference in the counts of lactic acid bacteria in the small and large intestines of different groups, the honey-fed group showed a significant increase (P<0.05) in counts over the control and sucrose-fed animals. The results support the fact that consumption of honey has a beneficial effect on the physiological constitution of animals fed with it.

Studies on Scenedesmus acutus growth. I effect of autotrophic and mixotrophic conditions on the growth of Scenedesmus acutus.

Among sugars, glucose and mannose were found to be the most suitable substrates for mixotrophic growth, uptake of galactose and its influence on growth was negligible, and sucrose and fructose occupied intermediary positions. The optimum temperature for sugar uptake was 30 degrees C, both under light and in darkness. Enhancement in the photosynthetic oxygen-evolution rate, based on the utilization of substrates, was foremost in the presence of glucose, followed by mannose, sucrose, and fructose. Industrial by-products such as sugarcane molasses also were utilized to increase the algal growth under mixotrophic conditions. A maximum yield in biomass was obtained subsequent to the combined supply of sugarcane molasses with carbon dioxide to indoor as well as outdoor mixotrophic cultures. Doubling the carbon dioxide supply alone above a certain level, under autotrophic and mixotrophic outdoor conditions, did not produce a pronounced increase in the algal growth rate. The results on autotrophic and mixotrophic growth variations are discussed in the article.

Studies on Scenedesmus acutus growth II. Effect of autotrophic and mixotrophic growth on the amino acid and the carbohydrate composition of Scenedesmus acutus.

As a general rule an increase in carbohydrates occurs during the light phase of the cell cycle and that of protein during phase, although variations were found in these components under autotrophic and mixotrophic growth conditions. The results are based on the quantitative determination of carvohydrates as trimethylsilyl (TMS) derivatives and amino acids as N-trifluoroacetyl-n-butyl (TAB) esters in algal cells cultured in light and dark periods by gas-liquid chromatography (LC). Cells harvested during the dark period contained more amino acids as compared to similar cultures harvested during the light phase. In light, the production of amino acids of the aspartate family increased in cells cultivated with glucose and carbon dioxide. With glucose as sole carbon source, the carbohydrate content was higher in the dark than in the light period. Under continuous light conditions, in the presence of carbon dioxide, there was a decrease in the carbohydrate content also. Gas-liquid chromatography analysis of the extract of the purified cell walls showed that they are made up of 0.076% carbohydrates and 0.28% amino acids on the dry weight (DW) basis of whole cells. The results on the metabolism of cells, under autotrophic and mixotrophic conditions, are discussed in this article.

Studies on Scenedesmus acutus growth. The incorporation of 14C-glucose by Scenedesmus acutus under light and dark periods.

Qualitative and quantitative estimations of alcohol-extractable compounds from (14)C-glucose-incorporated Scendesmus acutus cells were performed at various intervals of time. After two hours of incubation with (14)C-glucose in light, amino acids were synthesized at 65% and sucrose at 26% of the total input label. In the dark incorporation, 30% of the total radioactivity was found in amino acids and 46% in sucrose. Leucine and valine were detected only during the oxidation of glucose in light. The concentration of serine increased more in the presence of light, as compared to dark. These results on the oxidation of glucose are discussed in this article.