James P. Nakas

Production and characterization of poly‐3‐hydroxybutyrate from biodiesel‐glycerol by Burkholderia cepacia ATCC 17759

Glycerol, a byproduct of the biodiesel industry, can be used by bacteria as an inexpensive carbon source for the production of value‐added biodegradable polyhydroxyalkanoates (PHAs). Burkholderia cepacia ATCC 17759 synthesized poly‐3‐hydroxybutyrate (PHB) from glycerol concentrations ranging from 3% to 9% (v/v). Increasing the glycerol concentration results in a gradual reduction of biomass, PHA yield, and molecular mass (Mn and Mw) of PHB. The molecular mass of PHB produced utilizing xylose as a carbon source is also decreased by the addition of glycerol as a secondary carbon source dependent on the time and concentration of the addition. 1H‐NMR revealed that molecular masses decreased due to the esterification of glycerol with PHB resulting in chain termination (end‐capping). However, melting temperature and glass transition temperature of the end‐capped polymers showed no significant difference when compared to the xylose‐based PHB. The fermentation was successfully scaled up to 200 L for PHB production and the yield of dry biomass and PHB were 23.6 g/L and 7.4 g/L, respectively.

Xylanase production by Trichoderma longibrachiatum

Abstract The effects of carbon source, substrate concentration, culture pH, and spore inoculum concentration on production of extracellular xylanase and cellulase were examined. Very low enzyme activities were obtained with growth on glucose, xylose, and cellobiose, while significantly higher levels were produced from lactose and arabinose. Higher levels of both enzymes were generated from alpha cellulose, wood pulp, and fibrous paper waste than from purified xylan. However, the ratio of xylanase to cellulase activity was higher with pure xylan. High levels of both xylanase and cellulase activity were obtained when the culture was grown on lactose plus xylan. A factorial experiment showed that spore and substrate concentration had significant effects on xylanase yield, and that an interaction existed between these two variables. Highest levels of xylanase were generated with cultures grown on 1% wood pulp at pH 7.0 using an inoculum of 10 5 spores ml -1 . Maximal xylanase activity was observed at pH 4.8-5.5 at 55°C using a 30-min assay. The type of xylan used as substrate and the method of reducing sugar detection significantly affected measured xylanase activity.

Bacterial PCB biodegradation

The environment has become polluted with a variety of xenobiotics, including PCBs, as a result of the industrial development of useful halogenated compounds. While the PCBs may not exhibit the acute toxicity originally ascribed to them, they and their attendant byproducts remain as significant factors for adverse effects in the ecological food-chain. The use of microorganisms for bioremediation of PCBs is reviewed. This paper further details three new isolates obtained by conventional enrichment technics which show significant degradation capabilities for Aroclor 1242. These were identified by morphology, staining, and fatty acid analysis as Comamonas testosteroni, Rhodococcus rhodochrous, and a strain of Pseudomonas putida. These isolates demonstrated somewhat selective degradations of the congeners within Aroclor 1242; comprising total losses of 13.8, 19.1, and 24.6%, respectively. Each organism can attack dichloro-through tetrachlorobiphenyls. Analysis of chromatographic patterns from anaerobically digested Aroclor 1242 samples treated by these bacteria demonstrated decreases in di- through penta-substituted biphenyls. Each of these isolates, with discrete specificities, showed preferences for ‘open’ 2,3-sites, indicative of the action of 2,3-dioxygenase enzymes. The identification of many intermediates in the foregoing transformations was established by GC-MS analyses. Several variations in metabolic pathways, centering on the meta cleavage product 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) were suggested from these data. It is concluded that the described strains may be of future bioremediation use in processes which have an initial anaerobic dechlorination stage.

Production and Characterization of Poly-β-hydroxyalkanoate Copolymers from Burkholderia cepacia Utilizing Xylose and Levulinic Acid

Poly(β‐hydroxybutyrate ‐ co‐β‐hydroxyvalerate) (P(3HB ‐ co‐3HV)) copolymers were prepared via shake‐flask fermentations of Burkholderia cepacia(formerly Pseudomonas cepacia) containing 2.2% (w/v) xylose and concentrations of levulinic acid ranging from 0.07% to 0.67% (w/v). Periodic harvest of shake‐flask cultures from 48 to 92 h post‐inoculation yielded 4.4–5.3 g/L of dry cell biomass, containing 42–56% (w/w) P(3HB ‐ co‐3HV), with optimal product yield occurring between 66 and 74 h. Growth and PHA accumulation enhancement were observed with concentrations of levulinic acid from 0.07 to 0.52% (w/v), producing dry cell biomass and P(3HB ‐ co‐3HV) yields of 9.5 and 4.2 g/L, respectively, at the 0.52% (w/v) concentration of levulinic acid. Representative samples were subjected to compositional analysis by 300 MHz 1H and 150 MHz 13C NMR, indicating that these random copolymers contained between 0.8 and 61 mol % 3‐hydroxyvalerate (3HV). Solvent‐cast film samples were characterized by differential scanning calorimetry, which demonstrated melting temperatures ( Tm) to decrease in a pseudoeutectic fashion from 174.3 °C (0.8 mol % 3HV) to a minimum of 154.2 °C (25 mol % 3HV) and the glass transition temperatures ( Tg) to decrease linearly from 2.1 to –11.9 °C as a function of increasing mol % 3HV. Thermogravimetric analysis of the copolymer series showed the temperature for onset of thermal decomposition ( Tdecomp) to vary as a function of mol % 3HV from 273.4 to 225.5 °C. Intrinsic viscosities (η) varied from 3.2 to 5.4 dL/g, as determined by dilute solution viscometry. Viscosity average molecular weights ( Mv) of the copolymers were determined to range from 469 to 919 kDa, indicating that these P(3HB ‐ co‐3HV) copolymers are of sufficient molecular mass for commercial application.

Microfloral and faunal interactions in natural and agro-ecosystems

1. Introduction.- 2. Nutrient cycling and decompoisition in natural terrestrial ecosystems.- 3. Decomposition and nutrient cycling in agro-ecosystems.- 4. Biodegradation of organic residues in soil.- 5. Root and soil microbial interactions which influence the availability of photoassimilate carbon in the rhizosphere.- 6. The role of microorganisms in the soil nitrogen cycle.- 7. Role of microflora in terrestrial sulfur cycling.- 8. The role of microfloral and faunal interactions in affecting soil processes.- 9. Effects of management on soil decomposers and decomposition processes in grasslands and croplands.- 10. Recent advances in quantitative soil biology.- 11. The role of modeling in research on microfloral and faunal interactions in natural and agroecosystems.

Origin and expression of phosphatase activity in a semi-arid grassland soil

Abstract The phosphatase activity of a grassland soil and some of the microorganisms producing phosphatase were characterized. Soil supplemented with casamino acids (1% w/w) had increased alkaline phosphatase activity (pH 8.5) after 2 days of incubation, which decreased to the original level after 24 days. No increase in phosphatase activity was observed at pH values of 5.5 or 6.5. Antibiotics were added to several soil samples in order to suppress either bacterial or fungal activity. Alkaline phosphatase activity did not increase in soils supplemented with casamino acids when the fungi were suppressed. These data may help to explain why correlations between phosphatase activity and crop yield are inconsistent.

Mediator-assisted selective oxidation of lignin model compounds by laccase from Botrytis cinerea

Laccase from Botrytis cinereacatalyzes benzylic oxidations and cleavage of lignin-related diphenylmethanes. Selective reactions with non-phenolic monomeric or b-1-dimeric model compounds using O 2 and redox mediators can also be carried out. At substrate to mediator ratios of 5:1, 1-hydoxybenzotriazole (HOBT) is 8-20 fold more effective than 2,2-azinobis-3-ethylthiazoline-6-sulfonate (ABTS) for such biotransformations. With unblocked phenols, ring couplings are the dominant endproducts either with or without mediators.

Purification and characterization of an intracellular carboxylesterase from Arthrobacter viscosus NRRL B-1973

Abstract An intracellular carboxylesterase (EC 3.1.1.1) was isolated from Arthrobacter viscosus NRRL B-1973 and purified to homogeneity. Purification of extracted enzyme was achieved by ammonium sulfate precipitation followed by separation with Phenyl-Sepharose, DEAE-Sepharose, and Sepharose CL-6B. This carboxylesterase exhibits a specific activity of 55.4 μmol min −1 mg −1 based on the hydrolysis of p -nitrophenyl acetate at pH 7.4 and 30°C. The apparent molecular mass was 16.7 ± 0.4 kDa as determined by SDS-PAGE. The isoelectric point was estimated to be 5.6 and optimum activity for the enzyme was found at pH 7.4 and 40°C. The enzyme removed acetyl residues from xanthan, alginate, glucose pentaacetate, cellobiose octaacetate, and the native exopolysaccharide produced by A. viscosus and also deacetylated p -nitrophenyl propionate, naphthyl acetate, isopropenyl acetate, and triacetin. This enzyme demonstrates a restricted chain-length specificity for deacetylation of a wide variety of low and high molecular weight esters. Preliminary investigations indicate that it can also catalyze transesterifications from selected acyl donors to polymeric acceptors, notably cellulose.

Preliminary characterization of laminarinase from Trichoderma longibrachiatum

Abstract Trichoderma longibrachiatum, when cultivated on untreated ground mushroom (Agaricus bisporus) fruiting bodies as the only carbon substrate, exhibited extracellular laminarinase (endo-1,3(4)-s-glucanase EC 3.2.1.6) activity. The general physicochemical and catalytic properties of that enzyme preparation were examined. The apparent Kmand Vmax values were determined to be 137 mg ml−1) and 0.62 nmol s−1, respectively. The optimum pH and temperature for enzyme activity were 5.0 and 50°C, and among several buffers tested at pH 5.0, sodium acetate was found to be optimum. The energy of activation, Ea, and temperature coefficient, Q10 (between 30 and 40°C), were calculated to be 3.7 kcal mol−1 and 1.02, respectively. The enzyme was stable between pH 3.0 and 9.0 when stored at 4°C for 48 h. In the absence of substrate, enzyme activity was unaffected at 30°C for 1 h but lost complete activity within 10 min at 60°C. Among a number of metal ions and enzyme inhibitors tested, only Mn2+, Hg2+, N-bromosuccinimide and potassium permanganate showed prominent inhibition.

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid by Clostridium thermoaceticum

SummaryClostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO2, typically produced 14gl−1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl−1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.