Production of poly-3-hydroxyalkanoates from CO and H2 by a novel photosynthetic bacterium

A novel process is described to efficiently photoconvert low-grade organic materials such as waste biomass into natural biological plastics. When heterogeneous forms of dry biomass are thermally gasified, relatively homogeneous synthesis gas mixtures composed primarily of carbon monoxide and hydrogen are produced. Unique strains of photosynthetic bacteria were isolated that nearly quantitatively photoassimilate the carbon monoxide and hydrogen components of synthesis gas into new cell mass. Under unbalanced culture conditions when cellular growth is limited by shortages of nitrogen, calcium, magnesium, iron, or essential vitamins, up to 28% of the new cell mass is found as granules of poly-3-hydroxyalkanoate (PHA), a highmolecular-weight thermoplastic that can be solvent-extracted. The dominant monomeric unit of PHAs is 3-hydroxybutyrate (3HB), which is polymerized into the homopolymeric poly-3-hydroxybutyrate (PHB). PHB is marketed as a biodegradable plastic with physical properties similar to polystyrene. When a green alga was cocultured with the photosynthetic bacterium in light-dark (day-night) cycles, the bacteria synthesized a polymer of poly-3-hydroxybutyrate-3-hydroxyvalerate (PHB-V) with a composition of 70% 3HB and 30% 3-hydroxyvalerate (3HV) to an extent of 18% of the new cell mass. PHB-V is commercially marketed as Biopol and has physical properties similar to polypropylene or polyethylene. Our results demonstrate that a strain of photosynthetic bacteria capable of photoassimilating synthesis gas or producer gas is a potential candidate for large-scale production of biological polyesters.     

Production of citronellyl acetate in a fed-batch system using immobilized lipase

Several reports exist in the literature citing the decrease in conversion rates of organic-phase catalytic synthesis reactions when acetic acid is present as a reaction component. This inhibition is thought to result from damage to either the hydration layer-protein interaction or the overall enzyme structure. In this work, the inhibitory effect of acetic acid on lipase enzyme activity was ameliorated by conducting syntheses under acetic acid-limiting conditions in a fed-batch system, resulting in higher product yields. Periodic additions of acetic acid at levels of 40 mM or less gave maximum yields of 65% conversion for the reaction of citronellol and acetic acid to form citronellyl acetate. The enzyme used was a fungal lipase fromMucor miehei, and was immobilized on macroporous synthetic resin (a Novo lipozyme Novo Nordisk, Denmark). These results represent a fourfold improvement over batch runs reported in the literature for direct esterification of terpene alcohol with acetic acid using lipozyme as a catalytic agent.     

Production of exocellular polysaccharide by azotobacter chroococcwn

Environmental conditions affect the production of extracellular polysaccharide by Azotobacter chroococcum ATCC 4412. Production of exocellular polymer from a variety of carbon sources depended on the air flow rate. A high sucrose concentration in medium (8%) markedly favored expopolysaccharide production, which reached 14 g/L in about 72 h. In cell suspensions incubated in the presence of 8% sucrose in a nitrogen-free medium, biopolymer final concentration of 9 g/L corresponds to 68 g/g biomass. Maximum efficiency of sucrose conversion into exopolysaccharide peaked at 70% for initial disaccharide concentration of 6%. High performance liquid chromatography and gas liquid chromatography of acid hydrolysates of the exopolymer revealed the presence of mannuronosyl, guluronosyl, and acetyl residues, but not neutral sugars. The infrared spectrum corroborated the presence of carboxylate anions and O-acetyl groups in the exopolymer. Though the presence of more than one kind of polysaccharide cannot be ruled out, these data suggest that, under the experimental conditions used in this work, only a type of alginate-like exopolysaccharide is produced by A. chroococcum ATCC 4412.     

Xylanase recovery by ethanol and Na2SO4 precipitation

Xylans are the major components of the hemicellulosic fraction of lignocellulosic biomass and their hydrolysis can be obtained using xylanases fromPenicillium janthinellum. In this work, sugarcane bagasse hemicellulosic hydrolysate was used as the substrate for producing xylanase. The precipitation of these enzymes was studied using ethanol and Na₂SO₄ as precipitating agents. Ethanol precipitation experiments were performed batchwise in concentrations ranging from 10 to 80%, pH 4.0 to 7.0, at 4áC. The concentrations used in the precipitations with Na₂SO₄ were from 5 to 60% at pH 5.5 and 25áC. Solubility curves as a function of xylanase activity and total protein for both precipitating agents were made. According to the results, Na₂SO₄ is not appropriate for precipitating xylanases in this medium since at salt concentrations higher than 25%, the enzyme was denaturated and at this concentration less than 80% of the enzyme and total protein were precipitated. Because of differences in xylanase and total protein solubility, a fractionated precipitation using ethanol can be performed, since with 40% ethanol, 49% of the total protein was precipitated and more than 95% of the enzyme was kept in solution. On the other hand approx 100% of the xylanases were recovered by precipitation after adding 80% ethanol.     

Factors affecting coal solubilization by the BacteriumStreptomyces setonii 75Vi2 and by alkaline buffers

Streptomyces setonii 75Vi2 produces an extracellular coal-solubilizing component(s) in the absence of coal. The heat stability, relatively low molecular weight, and insensitivity to proteases of the substance(s) responsible for coal solubilization indicate that the process is nonenzymatic. This report describes factors affecting the production and activity of this substance(s) and the similarity in its action to alkaline buffer solutions in solubilizing coal.     

Production of methyl ester fuel from microalgae

Microalgae are unique photosynthetic organisms that are known to accumulate storage lipids in large quantitites and thrive in saline waters. Before these storage lipids can be used, they must be extracted from the microalgae and converted into usable fuel. Transesterification of lipids produces fatty acid methyl esters that can be used as a diesel fuel substitute. Three solvents, 1-butanol, ethanol, and hexane/2-propanol, were evaluated for extraction efficiency of microalgal lipids. Type of catalyst, concentration of catalyst, time of reaction, temperature of reaction, and quality of lipid were examined as variables for transesterification. The most efficient solvent of the three for extraction was 1-butanol (90% efficiency), followed by hexane/2-propanol and ethanol. Optimal yield of fatty acid methyl esters was obtained using 0.6N hydrochloric acid in methanol for 0.1 h at 70°C.     

Production of succinic acid by anaerobiospirillum succiniciproducens

The effect of an external supply of carbon dioxide and pH on the production of succinic acid byAnaerobiospirillum succiniciproducens was studied. In a rich medium containing yeast extract and peptone, when the external carbon dioxide supply was provided by a 1.5M Na₂CO₃ solution that also was used to maintain the pH at 6.0, no additional carbon dioxide supply was needed. In fact, sparging CO₂ gas into the fermenter at 0.025 L/L-min or higher rates resulted in significant decreases in both production rate and yield of succinate. Under the same conditions, the production of the main by-product acetate was not affected by sparging CO₂ gas into the fermenter. The optimum pH (pH 6.0) for the production of succinic acid was found to be in agreement with results previously reported in the literature. Succinic acid production also was studied in an industrial-type inexpensive medium in which light steep water was the only source of organic nutrients. At pH 6.0 and with a CO₂ gas sparge rate of 0.08 L/L-min, succinate concentration reached a maximum of 32 g/L in 27 h with a yield of 0.99 g succinate/g glucose consumed.     

Production of l-lactic acid byRhizopus oryzae in a bubble column fermenter

Two distinctive forms of growth (mycelial filamentous and mycelial pellets) ofRhizopus oryzae were obtained by manipulating the initial pH of the medium with the controlled addition of CaCO₃ in a bubble fermenter. In the presence of CaCO₃, diffused filamentous growth was obtained when the initial pH of the substrate was 5.5. In the absence of CaCO3, mycelial pellet growth was obtained when the initial pH was 2.0. The fermentation study indicated that the mycelial growth has a shorter lag period before the onset of acid formation. Both physical forms of growth ofRhizopus exhibited a high yield of L-lactic acid in the bubble fermenter when the initial glucose concentration exceeded 70 g/L. A final lactic acid concentration of 62 g/L was produced by the filamentous form ofRhizopus from 78 g/L glucose after 27 h. This showed a weight yield of 80% of glucose consumed, with an average specific productivity of 1.46 g/h/g. Similarly, the pellet form ofRhizopus produced a final lactic acid concentration of 66 g/L from 76 g/L glucose after 43 h, with a weight yield of 86% and an average specific productivity of 1.53 g/h/g.     

Development of a novel,two-step process for treating municipal biosolids for beneficial reuse

Modern municipal sewage waste treatment plants use conventional mechanical and biological processes to reclaim wastewaters. This process has an overall effect of converting a water pollution problem into a solid waste disposal problem (sludges or biosolids). An estimated 10 million tons of biosolids, which require final disposal, are produced annually in the United States. Although numerous disposal options for biosolids are available, including land application, landfilling, and incineration, disposal costs have risen, partly because of increased federal and local environmental restrictions(1). A novel, thermomechanical biosolids pre-treatment process, which allows for a variety of potential value-added uses, was developed. This two-step process first employs thermal explosive decompression to inactivate or kill the microbial cells and viruses. This primary step also results in the rupture of a small amount of the microbial biomass and increases the intrinsic fluidity of the biosolids. The second step uses shear to effect a near-complete rupturing of the microbial biomass, and shears the nondigested organics, which increases the overall surface area. Pretreated biosolids may be subjected to a secondary anaerobic digestion process to produce additional fuel gas, and to provide for a high-quality, easily dewatered compost product. This novel biosolids pretreatment process was recently allowed a United States patent.     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

Evaluation of O2 and CO2 transfer coefficients in a locally integrated tubular hollow fiber bioreactor

We have studied the transfer coefficients for O₂ transport to and CO₂ removal from a model cell-free system using microporous hydrophobic hollow fibers axially placed along a tubular bioreactor for ethanol production with immobilized yeast in the shell. In this locally integrated bioreactor, O₂ and CO₂ transfer rates depend strongly on the shell side liquid flow rate; O₂ flow rate in the fiber bore influences O₂ transport only at very low flow rates. Diffusion of CO₂ does not affect O₂ transport. Local and overall O₂ and CO₂ transfer coefficients have been determined for a wide range of Reynolds Numbers. The efficiency of such transfers has been demonstrated for alcohol fermentation.     

Production and purification of CGTase of alkalophylicBacillus isolated from Brazilian soil

Alkalophylic bacilli that produce cyclodextringlycosyltransferase (CGTase) were isolated from Brazilian soil, with a scheme of two plating steps. In the first step, the bacterial isolate forms a halo in the cultivation medium that contains γ-cyclodextrin (CD) complexing dyes. The CGTase of an isolate was purified 157-fold by biospecific affinity chromatography, with β-CD showing a mol wt of 77,580 Daltons. It produces a γ- to β-CD ratio of 0.156 and a small amount of α-CD, using maltodextrin 10% as substrate, at 50°C, pH 8.0 and 22 h reaction time, reaching 21.4% conversion of the substrate to cyclodextrins. In the second screening step, the isolates chosen give larger halos with β-CD complexing dyes, and smaller halos with β-CD complexing dyes, leading to a 30% improvement in γ-CD selectivity, although at lower total yield for cyclodextrins (11.5%).     

A novel process for anaerobic composting of municipal solid waste

A novel process has been developed and evaluated in a pilotscale program for conversion of the biodegradable fraction of municipal solid waste (MSW) to methane via anaerobic composting. The sequential batch anaerobic composting (SEBAC) process employs leachate management to provide organisms, moisture, and nutrients required for rapid conversion of MSW and removal of inhibitory fermentation products during start-up. The biodegradable organic materials are converted to methane and carbon dioxide in 21–42 d, rather than the years required in landfills.     

Urease purification from the seeds ofCajanus Cajan and its application in a biosensor construction

Urease has been purified from the seeds of Cajanus Cajan. The purification process involves three solvent extraction steps followed by DEAE-cellulose column chromatography. The specific activity of the purified enzyme is found to be 1920 U/mg with the recovery of 8%. The application of the purified enzyme in a biosensor construction is discussed.     

Synthesis of Cyclodextrin Glucosyl Transferase byBacillus cereus for the production of cyclodextrins

A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h⁻¹ resulting in the productivity of 23.0 kU/L/h.     

Mode of action and synergism of cellulases fromPenicillium funiculosum

A 1,4-β-d-glucan cellobiohydrolase (EC 3.2.1.91) and l,4-β-d-glucan glucanohydrolase (EC 3.2.1.4) were purified from the culture filtrates ofPenicillium funiculosum by using preparative isoelectric focusing. Both the enzymes were homogeneous on polyacrylamide gel with and without sodium dodecyl sulphate. The mol wt of the cellobiohydrolase and endoglucanase were 14,400 and 25,000 respectively. The purified enzymes were free of β-glucosidase activity. Acting in isolation, the cellobiohydrolase had little capacity for solubilizing Avicel or Walseth cellulose, but showed increased rates of hydrolysis when combined with endoglucanase. Cellobiose inhibition (50%) was observed in the initial rate of the hydrolysis of Walseth cellulose. It was also observed that cellobiohydrolase initiates the attack on crystalline cellulose. † NCL communication no. 3898.     

Production of ethanol from starch by co-immobilizedZymomonas mobilis-glucoamylase in a fluidized-bed reactor

The production of ethanol from starch was studied in a fluidized-bed reactor (FBR) using co-immobilizedZymomonas mobilis and glucoamylase. The FBR was a glass column of 2.54 cm in diameter and 120 cm in length. TheZ. mobilis and glucoamylase were co-immobilized within small uniform beads (1.2-2.5 mm diameter) of κ-carrageenan. The substrate for ethanol production was a soluble starch. Light steep water was used as the complex nutrient source. The experiments were performed at 35κC and pH range of 4.0-5.5. The substrate concentrations ranged from 40 to 185 g/L, and the feed rates from 10 to 37 mL/min. Under relaxed sterility conditions, the FBR was successfully operated for a period of 22 d, during which no contamination or structural failure of the biocatalyst beads was observed. Volumetric productivity as high as 38 g ethanol/(Lh), which was 74% of the maximum expected value, was obtained. Typical ethanol volumetric productivity was in the range of 15-20 g/(Lh). The average yield was 0.49 g ethanol/g substrate consumed, which was 90% of the theoretical yield. Very low levels of glucose were observed in the reactor, indicating that starch hydrolysis was the rate-limiting step.     

Degradation of organic sulfur compounds by a coal-solubilizing Fungus

Paecilomyces sp. TLi, a coal-solubilizing fungus, was shown to degrade organic sulfur-containing coal substructure compounds. Di-benzothiophene was degraded via a sulfur-oxidizing pathway to 2,2′-dihydroxybiphenyl. No further metabolism of that compound was observed. Ethyl phenyl sulfide and diphenyl sulfide were degraded to the corresponding sulfones. A variety of products were formed from dibenzyl sulfide, presumably via free radical intermediates. Diphenyl disulfide and dibenzyl disulfide were cleaved to the corresponding thiols and other single-ring products. It was concluded that degradation of organic sulfur compounds byPaecilomyces involves an oxidative attack localized at the sulfur atom.