Evaluation of thioesterases from Acinetobacter baylyi for production of free fatty acids.

Acinetobacter baylyi is one of few Gram-negative bacteria capable of accumulating storage lipids in the form of triacylglycerides and wax esters, which makes it an attractive candidate for production of lipophilic products, including biofuel precursors. Thioesterases play a significant dual role in the triacylglyceride and wax ester biosynthesis by either providing or removing acyl-CoA from this pathway. Therefore, 4 different thioesterase genes were cloned from Acinetobacter baylyi ADP1 and expressed in Escherichia coli to investigate their contribution to free fatty acids (FFAs) accumulation. Overexpression of the genes tesA' (a leaderless form of the gene tesA) and tesC resulted in increased accumulation of FFAs when compared with the host E. coli strain. Overexpression of tesA' showed a 1.87-fold increase in production of long-chain fatty acids (C16 to C18) over the host strain. Unlike TesC and the other investigated thioesterases, the TesA' thioesterase also produced shorter chain FFAs (e.g., myristic acid) and unsaturated FFAs (e.g., cis-vaccenic acid (18:1Δ11)). A comparison of the remaining 3 A. baylyi ADP1 thioesterases (encoded by the tesB, tesC, and tesD genes) revealed that only the strain containing the tesC gene produced statistically higher levels of FFAs over the control, suggesting that it possesses the acyl-ACP thioesterase activity. Both E. coli strains containing the tesB and tesD genes produced levels of FFAs similar to those of the plasmid-free control E. coli strain, which indicates that TesB and TesD lack the acyl-ACP thioesterase activity.

Biodegradation of a mixture of mononitrophenols in a packed-bed aerobic reactor.

Aerobic biodegradation of individual mononitrophenols (4-, 3- and 2-NPs) and their mixture in simulated wastewater was investigated in a packed-bed bench scale bioreactor continuously operated in a flow mode, with a mixed microbial culture adsorbed on expanded slate. Under a low, suboptimal hydraulic retention time (HRT) of 30 min the reactor removed more than 3 g.L(-1).day(-1) of the NP mixture while maintaining a > 85-90% removal efficiency (RE). Under higher HRT values, starting at 45 min, more than 2 g.L(-1).day(-1) of the NP mixture were removed with an RE > 98%. Significant substrate interactions were observed; the addition of other NPs caused the saturation of 2-NP catabolic capacity whereas the addition of 2-NP caused the de-saturation of the 4- and 3-NP catabolic capacity. 3- and 4-NPs appeared to be removed independently, i.e., by different enzyme systems. After ten months of operation, the biofilm composition was significantly altered to become predominantly bacterial. Only one originally inoculated strain remained indicating microbial contamination followed by a genetic material exchange.

CH···π interactions do not contribute to hydrogen transfer catalysis by glycerol dehydratase.

The role of the nonbonded CH···π interaction in the hydrogen abstraction from glycerol by the coenzyme B(12)-independent glycerol dehydratase (GDH) was examined using the QM/MM (ONIOM), MP2, and CCSD(T) methods. The studied CH···π interaction included the hydrogen atom of the -C(2)H(OH)- group of the glycerol substrate and the tyrosine-339 residue of the dehydratase. A contribution of this interaction to the stabilization of the transition state for the transfer of a hydrogen atom from the adjacent terminal C(1)H(2)(OH) group to cysteine 433 was determined by ab initio HF, MP2, and CCSD(T) calculations with the aug-cc-pvDZ basis set for the corresponding methane/benzene, methanol/phenol, and glycerol radical/phenol subsystems. The calculated CH···π distance, defined as the distance between the H atom and the center of the phenol ring, shortened from 2.62 to 2.52 Å upon going from the ground- to the transition-state of the GDH-catalyzed reaction. However, this shortening was not accompanied by the expected lowering of the CH···π interaction free energy. Instead, this interaction remained weak (about -1 kcal/mol) along the entire reaction coordinate. Additionally, the mutual orientation of the CH group and the phenol ring did not change significantly during the reaction. These results suggest that the phenol group of the tyrosine-339 does not contribute to lowering the activation barrier in the enzyme, but do not exclude the possibility that tyrosine 339 facilitates proper orientation of glycerol for the electrostatic catalysis, or inhibits side-reactions of the reactive glycerol radical intermediate.

Microbial lipids from renewable resources: production and characterization.

A number of microorganisms belonging to the genera of algae, yeast, bacteria, and fungi have ability to accumulate neutral lipids under specific cultivation conditions. The microbial lipids contain high fractions of polyunsaturated fatty acids and have the potential to serve as a source of significant quantities of transportation fuels. This paper reviews the current state of the art of this field. It summarizes the various microorganism used, feed stocks available, environmental factors that influence growth of cells and accumulation of lipids, major fatty acid composition of lipids, and the technology.

Glycerol Dehydratases: Biochemical Structures, Catalytic Mechanisms, and Industrial Applications in 1,3-Propanediol Production by Naturally Occurring and Genetically Engineered Bacterial Strains.

To date, two types of glycerol dehydratases have been reported: coenzyme B12-dependent and coenzyme B12-independent glycerol dehydratases. The three-dimensional structure of the former is a dimer of αßγ heterotrimer, while that of the latter is a homodimer. Their mechanisms of reaction are typically enzymatic radical catalysis. Functional radical in both the glycerol dehydratases is the adenosyl radical. However, the adenosyl radical in the former originates from coenzyme B12 by homolytic cleavage, and that in the latter from S-adenosyl-methionine. Until some years ago, Clostridium butyricum VPI 1718 was the only microorganism known to possess B12-independent glycerol dehydratase, but since then, several other bacteria with this unique capability have been identified. This article focuses on the glycerol dehydratases and on 1,3-propanediol production from glycerol by naturally occurring and genetically engineered bacterial strains containing glycerol dehydratase.

Aerobic nitrification-denitrification by heterotrophic Bacillus strains.

Twenty-four Bacillus strains predominantly outgrown in a night soil treatment system were isolated and characterized. Under various culture conditions, cell interactions took place among them and cell population changed. Maximum removal of NH4+-N and cell production by the isolates occurred under the conditions of 30% DO and C/N ratio of 8. Five dominant isolates were identified to be species of Bacillus cereus, Bacillus subtilis and Bacillus licheniformis with similarities of 78-94%. Additions of 0.8% peptone and 0.3% yeast extract to a basal medium influenced the growth of isolates and the removal of NH4+-N in flask culture. Metal ions such as Ca2+, Fe2+ and Mg2+ had a similar effect. The specific growth rates of the five isolates were found to be in a range of 0.43-0.55 h(-1). During the flask experiment of nitrogen removal under aerobic growth conditions, active nitrification by the isolates occurred largely in 1h with a decrease of COD and alkalinity reduced to only 74.6% of theoretical value. From the nitrogen balance, the percentage of nitrogen lost in the flask culture was estimated to be 33.0%, which was presumed to convert to N2 gas. This conversion of ammonia to N2 without formation of nitrous oxide under aerobic growth conditions was confirmed by GC analysis. From all the results, it has been found that the Bacillus strains were able to occur simultaneously aerobic nitrification/denitrification and the B3 process using the Bacillus strains seemed to possess some economic advantages.

Extended bioremediation of PAH/PCP contaminated soils from the POPILE wood treatment facility.

A study was conducted using two pilot-scale land-treatment units (LTUs) to evaluate the efficacy of different cultivation and maintenance schedules during bioremediation of contaminated soil from a wood treatment facility using landfarming technology. The soil contained high concentrations of polycyclic aromatic hydrocarbons (PAHs, approximately 13000 ppm) as well as of pentachlorophenol (PCP, approximately 1500 ppm). An initial 6-month intensive-treatment phase was followed by 24 months of less-intensive treatment. During the first phase, traditional landfarming practice of regular cultivation was compared with a gas-phase composition based cultivation strategy, and both the landfarming units were intensively monitored and maintained with respect to moisture control and delivery of nutrients. The two strategies resulted in similar contaminant concentration profiles with time during this phase, although different microbial populations developed in the two-landfarming units. The second (less-intensive) treatment phase involved no moisture control and nutrient delivery beyond the initial adjustments, and compared natural attenuation (no cultivation) with quarterly cultivation of soil. Both the strategies showed similar behavior again. GC/MS analysis of the soil samples showed PAH removal including four-ring homologues. Leachability tests at zero time and after 6 and 22 months of operation showed significant reductions in leaching of PCP and low molecular weight PAHs. Extended treatment resulted in some leaching of high molecular weight PAHs. Significant biological activity was demonstrated, even at the high contaminant concentrations. Phospholipid ester-linked fatty acid (PLFA) analysis showed an increase in biomass and a divergence in community composition in soils depending on the treatment conducted.

Alkali hydrolysis of trinitrotoluene.

Data for alkali hydrolysis of 2,4,6-trinitrotoluene (TNT) in aqueous solution at pH 12.0 under static (pH-controlled) as well as dynamic (pH-uncontrolled) conditions are reported. The experiments were conducted at two different molar ratios of TNT to hydroxyl ions at room temperature. The TNT disappeared rapidly from the solution as a first-order reaction. The complete disappearance of aromatic structure from the aqueous solution within 24 h was confirmed by the ultraviolet-visible (UV-VIS) spectra of the samples. Cuvet experiments in a UV-VIS spectrophotometer demonstrated the formation of Meisenheimer complex, which slowly disappeared via formation of aromatic compounds with fewer nitro groups. The known metabolites of TNT were found to accumulate only in very small quantities in the liquid phase.

The diversity and molecular modelling analysis of B12 and B12-independent glycerol dehydratases.

To broaden our knowledge on the diversity of glycerol dehydratases, comprehensive sequence and molecular modelling analyses of these enzymes were performed. Our sequence analysis showed that B12-dependent and B12-independent glycerol dehydratases are not related, suggesting that they evolved from different ancestors. Second, our study demonstrated that a gene fusion event occurred between α and ß subunits of B12-dependent glycerol dehydratases in several bacteria during enzyme evolution. In addition, our sequence and molecular modelling analyses revealed more B12-independent glycerol dehydratases including hypothetical proteins. Furthermore, we found that some microorganisms contain both B12-dependent and B12-independent glycerol dehydratases in their genomes.

Dual feeding strategy for the production of alpha-amylase by Bacillus caldolyticus using complex media.

In this study, the objective was to investigate an exponential feeding strategy for fed-batch production of thermostable alpha-amylase (E.C. 3.2.1.1.) from the Bacillus caldolyticus (DSM405). The parameters for establishing compositions of feed media and feeding rate were obtained by statistical analysis of batch and continuous shake flask experiments. These parameters were casitone to starch ratio of 2.67g(casitone)g(starch)(-1), maintenance coefficient 0.174g(casitone)g(DW)(-1)h(-1), cell yield 0.62g(DW)g(casitone)(-1) and mu(opt)=0.2h(-1). The exponentially fed fermentation resulted in yield of 120Uml(-1) alpha-amylase that was thermostable up to 105 degrees C. Results of the exponentially fed fermentation have been discussed in the light of a feed-back controlled fed-batch fermentation reported earlier by the authors. A comparison of the temperature and pH effects on amylase produced by B. caldolyticus and on several other commercially available amylases has also been presented.

The Fate of Hydrogen Peroxide as an Oxygen Source for Bioremediation Activities within Saturated Aquifer Systems.

In situ bioremediation is an innovative technique for the remediation of contaminated aquifers that involves the use of microorganisms to remediate soils and groundwaters polluted by hazardous substances. During its application, this process may require the addition of nutrients and/or electron acceptors to stimulate appropriate biological activity. Hydrogen peroxide has been commonly used as an oxygen source because of the limited concentrations of oxygen that can be transferred into the groundwater using above-ground aeration followed by reinjection of the oxygenated groundwater into the aquifer or subsurface air sparging of the aquifer. Because of several potential interactions of H2O2 with various aquifer material constituents, its decomposition may be too rapid, making effective introduction of the H2O2 into targeted treatment zones extremely difficult and costly. Therefore, a bench-scale study was conducted to determine the fate of H2O2 within subsurface aquifer environments. The purpose of this investigation was to identify those aquifer constituents, both biotic and abiotic, that are most active in controlling the fate of H2O2. The decomposition rates of H2O2 were determined using both equilibrated water samples and soil slurries. Results showed H2O2 decomposition to be effected by several commonly found inorganic soil components; however, biologically mediated catalytic reactions were determined to be the most substantial.