Potassium deficiency results in accumulation of ultra-high molecular weight poly-beta-hydroxybutyrate in a methane-utilizing mixed culture.

AIMS: To investigate the effect of various single nutrient deficiencies on poly-beta-hydroxybutyrate (PHB) biosynthesis in a methane-utilizing mixed culture (dominant species Methylocystis sp. GB 25 DSM 7674). METHODS AND RESULTS: Poly-beta-hydroxybutyrate accumulation experiments were performed in 7 and 70 l bioreactors and initiated by potassium, sulfur or iron deficiency. After 24 h the PHB content reached levels of 33.6%, 32.6% and 10.4% respectively. Interestingly a polymer with an ultra-high average-weight molecular weight (M(w)) of 3.1 MDa was accumulated under potassium-limited conditions. When sulfur and iron were lacking M(w) were lower by 20.6 and 41.6%. Potassium-deficiency experiments were furthermore characterized by a maximum specific PHB formation rate 0.08 g g(-1)residual biomass (R) h(-1) and a yield coefficient of 0.45 g PHB g(-1) CH(4). CONCLUSIONS: Biosynthesis of an ultra-high M(w) PHB in a methane-utilizing mixed culture can be induced by potassium deficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: This study greatly extends the knowledge in the field of bacterial biopolymer formation with gaseous substrates. The special system used here combines the use of methane a low-cost substrate available from natural and renewable sources with the possibility of employing a mixed-culture in an open system for the synthesis of a high-value product.

White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses.

Functionalized compounds, which are difficult to produce by classical chemical synthesis, are of special interest as biotechnologically available targets. They represent useful building blocks for subsequent organic syntheses, wherein they can undergo stereoselective or regioselective reactions. "White Biotechnology" (as defined by the European Chemical Industry [ http://www.europabio.org/white_biotech.htm ], as part of a sustainable "Green Chemistry,") supports new applications of chemicals produced via biotechnology. Environmental aspects of this interdisciplinary combination include: Use of renewable feedstock Optimization of biotechnological processes by means of: New "high performance" microorganisms On-line measurement of substrates and products in bioreactors Alternative product isolation, resulting in higher yields, and lower energy demand In this overview we describe biotechnologically produced pyruvic, 2-oxopentaric and 2-oxohexaric acids as promising new building blocks for synthetic chemistry. In the first part, the microbial formation of 2-oxocarboxylic acids (2-OCAs) in general, and optimization of the fermentation steps required to form pyruvic acid, 2-oxoglutaric acid, and 2-oxo-D-gluconic acid are described, highlighting the fundamental advantages in comparison to chemical syntheses. In the second part, a set of chemical formula schemes demonstrate that 2-OCAs are applicable as building blocks in the chemical synthesis of, e.g., hydrophilic triazines, spiro-connected heterocycles, benzotriazines, and pyranoic amino acids. Finally, some perspectives are discussed.

Effects of plants and microorganisms in constructed wetlands for wastewater treatment.

Constructed wetlands are a natural alternative to technical methods of wastewater treatment. However, our understanding of the complex processes caused by the plants, microorganisms, soil matrix and substances in the wastewater, and how they all interact with each other, is still rather incomplete. In this article, a closer look will be taken at the mechanisms of both plants in constructed wetlands and the microorganisms in the root zone which come into play when they remove contaminants from wastewater. The supply of oxygen plays a crucial role in the activity and type of metabolism performed by microorganisms in the root zone. Plants' involvement in the input of oxygen into the root zone, in the uptake of nutrients and in the direct degradation of pollutants as well as the role of microorganisms are all examined in more detail. The ways in which these processes act to treat wastewater are dealt with in the following order: Technological aspects; The effect of root growth on the soil matrix; Gas transport in helophytes and the release of oxygen into the rhizosphere; The uptake of inorganic compounds by plants; The uptake of organic pollutants by plants and their metabolism; The release of carbon compounds by plants; Factors affecting the elimination of pathogenic germs.

Annual cycle of nitrogen removal by a pilot-scale subsurface horizontal flow in a constructed wetland under moderate climate.

The annual course of nitrogen removal in a stable operating subsurface horizontal flow constructed wetland (SSF) in a moderate climate was evaluated using a large pool of data from 4 years of operation. In spring and autumn removal efficiencies were found to depend on the nitrogen load in a linear mode. The efficiencies in winter and summer differed extremely (mean removal rates of 0.15/0.7 g m(-2) d(-1) (11%/53%) in January/August) and were independent of the nitrogen load (0.7-1.7 g m(-2) d(-1)) in principle. Oscillations of the removal rates in spring, forming several maxima, suggest seasonal specific effects caused by the dynamics of the plant-physiology finally determining the nitrification efficiency, i.e. via O(2)-supply. Nitrification is limited by temperature during all seasons and surprisingly in midsummer additionally restricted by other seasonal aspects forming a clear-cut relative nitrification minimum (mean rate of 0.43 g m(-2) d(-1) (32%)) in July. The importance and the effect of the plants' gas exchange and oxygen input into the rhizosphere are discussed. Denitrification was nearly complete in midsummer and was clearly restricted at seasonal temperatures below 15 degrees C.

Producing poly-3-hydroxybutyrate with a high molecular mass from methane.

Poly-3-hydroxybutyrate (PHB) and other polyesters can be produced by various species of bacteria. Of the possible carbon sources, methane could provide a suitable substrate for the production of PHB. Methane is cheap and plentiful - not only as natural gas, but also as biogas. The methanotrophic strain Methylocystis sp. GB 25 DSMZ 7674 is able to accumulate PHB in a brief non-sterile process. The studies were carried out using a 7-l and a 70-l pressure bioreactor. Cultivation was performed in two stages: a continuous growth phase (dilution rate 0.17 h(-1)) and a PHB accumulation phase under deficiency conditions of an essential nutrient (ammonium, phosphorus or magnesium) in batch culture. The PHB content of biomass was as high as 51%; efficiency was highest during the first 5 h of the product formation process. The maximum PHB yield relative to the methane consumed was estimated to be 0.55 g g(-1). The PHB produced is of very high quality, having a high molecular mass of up to 2.5x10(6) Da.

Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds.

A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.

Comparison of API 20NE and Biolog GN identification systems assessed by techniques of multivariate analyses.

The increasing use of commercial multitest systems for identification of environmental bacteria creates the problem of how to compare the identification results obtained from different systems. The limited use of species designations in such comparisons is caused by low usage of environmental bacteria in the development of commercial identification schemes. Two multivariate statistical methods, the Mantel's test and the co-inertia analysis, were applied to analyze data derived from the Biolog GN and the API 20NE systems of identification for 50 environmental bacterial strains. We found these two methods to be useful for revealing the relationship between the two sets of numerical taxonomic traits. Both of these methods showed that the distances according to the Biolog GN results between the studied strains were related to those derived from the API 20NE results, despite the differences in the test sets of the two systems. In addition, the co-inertia analysis allowed us to visualise the relationships between classifications of strains derived from the two identification systems and, simultaneously, to estimate the contribution of particular tests to the differentiation of bacterial strains.

Functional and structural successions in arbitrary samples of heterotrophic bacteria during aerobic treatments of lignite-carbonization wastewater in in situ enclosures.

In situ mesocosm experiments were performed in Lake Schwelvollert (located in the district of Weissenfels, Saxony-Anhalt, Germany), an anaerobic lignite-carbonization effluent lake containing phenolic compounds and their autoxidation products (anthropogenic humic matter). In the aeration enclosure, the anaerobic Schwelvollert wastewater was aerated and in the flocculation enclosure, it was flocculated to precipitate the oxygen-trapping anthropogenic humic matter to enhance the input of oxygen by diffusion. To gain an insight into the metabolic state of the aerobic heterotrophic microbiota during the treatments, arbitrary samples of bacterial isolates were taken from a general agar medium and tested for their abilities to cleave predominant phenolic contaminants by a procedure called the isolate sample assay. In this way, successions of degradation potentials were observed in both mesocosms, with degradation abilities for meta- and para-alkylated phenols appearing before degradation abilities for ortho-substituted phenols as a common phenomenon. To examine the structure of samples, the respective isolates were characterized using the Biolog GN MicroPlate system, the random amplified polymorphic DNA nucleic acid (RAPD) fingerprinting technique, and amplified ribosomal DNA restriction analysis (ARDRA). Although similar functional patterns occurred in both mesocosms, the compositions and diversities of the respective bacterial communities varied significantly, even at different depths from the same enclosure, with members of the Pseudomonas RNA group I being predominant.

Phosphine by bio-corrosion of phosphide-rich iron.

Phosphine is a toxic agent and part of the phosphorus cycle. A hitherto unknown formation mechanism for phosphine in the environment was investigated. When iron samples containing iron phosphide were incubated in corrosive aquatic media affected by microbial metabolites, phosphine was liberated and measured by gas chromatography. Iron liberates phosphine especially in anoxic aquatic media under the influence of sulfide and an acidic pH. A phosphine-forming mechanism is suggested: Phosphate, an impurity of iron containing minerals, is reduced abioticly to iron phosphide. When iron is exposed to the environment (e.g. as outdoor equipment, scrap, contamination in iron milled food or as iron meteorites) and corrodes, the iron phosphide present in the iron is suspended in the medium and can hydrolyze to phosphine. Phosphine can accumulate to measurable quantities in anoxic microbial media, accelerating corrosion and preserving the phosphine formed from oxidation.

Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species.

Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation.

Microbial phenol degradation of organic compounds in natural systems: Temperature-inhibition relationships.

The combined influence of high phenol concentrations and low temperatures on aerobic and anaerobic phenol degradation kinetics was investigated in microbial enrichment cultures to evaluate temperature-inhibition relationships with respect to the ambient conditions in polluted habitats. The inhibition of microbial phenol degradation by excess substrate was found to be temperature-dependent. Substrate inhibition was intensified when temperatures were lower. This results in an elevated temperature sensitivity of phenol degradation at inhibitory substrate concentrations. The synergistic amplification of substrate inhibition at low temperatures may help to explain the limited self-purification potential of contaminated habitats such as soils, sediments and groundwater aquifers where high pollutant concentrations and low temperatures prevail.

Degradation efficiency and molecular size alteration during the aerobic microbial treatment of lignite pyrolysis deposit water.

Investigation into aerobic biological degradation were carried out as part of an extensive programme designed to facilitate the cheap remediation of a pyrolysis waste-water deposit. Attention was focused on the processes of carbon conversion by different populations. The susceptibility of a body of lignite-processing deposit water to microbiological degradation was examined in batch investigations in a Sapromat system and in continuous bench-scale fermenter cultivations, with respect to nutrient supply, inoculation culture and molecular size distribution. It was found that degradation best occurs with an adapted mixed culture. The autochthonous culture removes 30% less dissolved organic carbon (DOC) and has a 40% higher specific oxygen demand. A shortage of phosphorus, investigated with a view to avoiding additional eutrophication problems in the open water in the case of in situ remediation, causes reduced DOC degradation and significantly higher specific oxygen demand. The biological process is overlapped by abiotic oxidation. During aerobic treatment, a concentration of colour-giving aromatic substances of between 0.5 kDa and 5 kDa was observed. This phenomenon is caused by the oxidation of low- and high-molecular-mass compounds. The removal of DOC is limited to 65% and mainly occurs in the range below 0.5 kDa (30%) and in the 0.5-1 kDa range (12%); the removal is negligible in the ranges 1-3 kDa (0.8%) and 3-5 kDa (2%) and a little higher in the ranges 5 kDa-0.3 micron (5%) and above 0.3 micron (6%). In the investigations it was discovered that DOC removal causes in the ranges below 0.5 kDa, 0.5-1 kDa and 5 kDa-0.3 micron mainly as a result of degradation, but the range above 0.3 micron is chiefly caused by bioadsorption. Aerobic microbiological treatment is able to remove most low-molecular-mass substances. In order to remove the macromolecular and colour-giving part of the deposit water, an additional treatment stage, e.g. flocculation, is required.

Free phosphine from the anaerobic biosphere.

The possible liberation of highly toxic and mutagenic phosphine from putrefying media raises the question of its significance as a problem of hygiene. Free phosphine was established by gas chromatography as a universal trace component in gas emitted from the anaerobic biosphere. Sources of phosphine include landfills, compost processing, sewage sludge, animal slurry and river sediments. We detected maximum concentrations in the order of 20 ppb(v/v).

Toxic oxide deposits from the combustion of landfill gas and biogas.

Oxide deposits found in combustion systems of landfill gas fired power stations contain relatively high concentrations of elements which form volatile species such as P, As, Sb and Sn. These deposits should be handled with care because of their potential toxicity. By contrast, deposits in biogas system engines were found to contain much lower levels of such elements. The enrichment of these elements can be attributed to a hypothetical multistage process. The elements form volatile species in the landfill body. They are selectively transported as part of the landfill gas into the gas-burning devices. Inside the burners, they are immobilized as nonvolatile oxides.

[Utilization of paraffins and other noncarbohydrate carbon sources for microbial citric acid synthesis]. / Nutzung von Paraffinen und anderen Nichtkohlenhydrat-Kohlenstoffquellen zur mikrobiellen Citronensäuresynthese.

This article reviews the developments achieved in citrate and isocitrate accumulation with non-carbohydrate substrates by microorganisms presented as well in academic publications as in patients. The efficiency of citrate and isocitrate overproducing microorganisms and of mutants obtained thereof with respect to different carbon sources (n-alkanes, triglycerides, organic acids, etc.) is discussed. The influence of environmental conditions (media, pH etc.) and biochemical mechanisms which lead to metabolic overflow are emphasized. The kinetics of fermentation processes are described, calculations concerning carbon balances are involved. The production of by-products and the conversion of isocitrate to citrate is considered. The production of citric acid by yeasts which utilize different carbon sources may be economically feasible and an accession to the practized molasse-Aspergillus-process.

[Effect of oxygen partial pressure on citric acid synthesis in Saccharomycopsis lipolytica using n-alkanes]. / Einflusseta des Sauerstoffpartialdrucks durch Saccharomycopsis liplytica aus n-Paraffinen.

The dependence of growth formation of citric acids (citrate: isocitrate = 1:1) on oxygen parital pressure of an alkane utilising yeast Saccharomycopsis lipolytica was investigated. During growth oxygen corresponds to a Michaelis-Menten-kinetics (Ks = 2.0 . 10(-5) M). The respiration quotient RQ for a dissolved oxygen concentration in the range of 10-100% (air saturation) is 0.46 +/- +/- 0.04. The phase of product formation is characterized by 3 sections. Immediately after N-exhaustion the cell activities are the highest. They decline during the first 30 hours of production. Besides the production of reserve material in this first section the highest production rate for citrate and isocitrate is observed. The rate of citric acid production depends on the oxygen partial pressure and is governed by Michaelis-Menten-kinetics. The specific production rate and the rate of oxygen consumption correspond to KS-values of 4.0 X 10(-5) and 3.3 X 10(-5) M, respectively. The RQ-value declines to a constant value of 0.23 +/- 0.02 and is not influenced by oxygen partial pressures in the range of 10--100% (related to air saturation). During the second section cell activities remain nearly constant for about 100 h. Due to this constancy the following equation could be derived: 14 O2 + C15H32 leads to 2 C6H8O7 + 3 CO2 + 8 H2O. In the third section the cell activities decline again.