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The three pillars of sustainability framework is an applied and solutions oriented approach to sustainable development, which at the broadest and most important scale supports the creation of new economic and political institutions that embed (from start to finish) the key inputs, stakeholders, and incentive structures necessary for sustainability planning and projects to be feasible and successful. The three pillars framework is based upon the key and connected roles of: (1) technology and innovation; (2) laws and governance; and (3) economics and financial incentives. Through the lens of a review of the evolution of sustainability models over the last several decades, it is proposed that the three pillars framework can more effectively help us translate complex sustainability issues into ideas and an applied focus that can be better understood and acted upon by community and economic stakeholders. This, combined with full transparency, creates the necessary, and often sufficient, foundation for successful, scalable, more rapidly deployable, and culturally acceptable sustainability solutions. As demonstrated in practice and in numerous case studies, sustainability solutions that engage all three pillars at once—good governance, technology implementation, and creating market incentives—are most effective and durable.

Fair fresh water distribution among humans and nature and among all sectors will be one of the main challenges of the 21^st century. There is a complex interplay between the different water users, and clear systematics are needed for efficient decision making. Water uses can be divided into four sectors, (i) water for people, services and industries, (ii) water for agriculture, (iii) water for nature, and (iv) water for energy production. A number of water related issues are relevant for each sector, though not all with the same importance. The issues relate either to water quality, water quantity, (urban) water infrastructure and integrated water management, and socio-economics and institutional aspects. Depending on the sector and the issues, there is an appropriate level for actions. Responsibilities for providing water for people, services, and industries must be taken at the local level (communities, cities, districts). Water for nature and the provision of ecosystem services ask for a more regional, national, or even multinational decision-making structure. The demographic development of the coming 25 years will be a challenge for agriculture to satisfy the food needs of all humans. The adequate and just access to agricultural products needs to be internationally guaranteed. Decisions for all sectors and on all levels imply formidable economic challenges, which will accompany human societies for the next decennia.

The glutathione peroxidase homologous gene (Gpxhgene) in Chlamydomonas reinhardtiiis up-regulated under oxidative stress conditions. The Gpxhgene showed a remarkably strong and fast induction by the singlet oxygen-generating photosensitizers neutral red, methylene blue and rose Bengal. The GpxhmRNA levels strongly increased, albeit much more slowly, upon exposure to the organic hydroperoxides tert-butyl hydroperoxide (t-BOOH) and cumene hydroperoxide. In contrast, the GpxhmRNA levels were only weakly induced by exposure to the superoxide-generating compound paraquat and by hydrogen peroxide. A comparison of the GpxhmRNA levels with those of the heat shock protein HSP70A and the iron superoxide dismutase gene showed qualitative and quantitative differences for the three genes under oxidative stress conditions tested. The Gpxhgene is specifically induced by singlet-oxygen photosensitizers and the relative induction by other compounds is much weaker for Gpxhthan for the other genes investigated. Using Gpxhpromoter fusions with the arylsulfatase reporter gene, we have shown that the Gpxhwas transcriptionally up-regulated by singlet-oxygen photosensitizers. It is also shown that the Gpxhpromoter contains a region between 104 and 179 bp upstream of the transcription start that is responsible for the mRNA up-regulation upon exposure to 1O2but not t-BOOH. Within this region a regulatory sequence homologous to the mammalian cAMP response element (CRE) and activator protein 1 (AP-1) binding site was identified within a 16 bp palindrome.

Free hydroxyl radicals (free ^·OH), singlet oxygen (¹O2), or ^·OH produced by DNA-copper-hydroperoxo complexes are possible DNA-damaging reactive oxygen species (ROS) in the reaction system containing copper, catechol, and DNA. para-Chlorobenzoic acid (pCBA) degradation studies revealed that CuCl2 mixed with catechol produced free ^·OH. In the presence of DNA, however, inhibition of the pCBA degradation suggested that another ROS is responsible for the DNA degradation. Of a series of ROS scavengers investigated, only KI, NaN3, and Na-formate—all of the salts tested—strongly inhibited the DNA degradation, suggesting that the ionic strength rather than the reactivity of the individual scavengers could be responsible for the observed inhibition. The ionic strength effect was confirmed by increasing the concentration of phosphate buffer, which is a poor ^·OH scavenger, and was interpreted as the result of destabilization of DNA-copper-hydroperoxo complexes. Piperidine-labile site patterns in DNA degraded by copper and catechol showed that the mixture of Cu(II) and catechol degrades DNA via the intermediate formation of a DNA-copper-hydroperoxo complex. Replacement of guanine by 7-deazaguanine did not retard the DNA degradation, suggesting that the DNA-copper-hydroperoxo complexes do not bind to the guanine N-7 as proposed in the literature. Environ. Mol. Mutagen. 36:5–12, 2000. © 2000 Wiley-Liss, Inc.

Free hydroxyl radicals (free ·OH), singlet oxygen (1O2), or ·OH produced by DNA‐copper‐hydroperoxo complexes are possible DNA‐damaging reactive oxygen species (ROS) in the reaction system containing copper, catechol, and DNA. para‐Chlorobenzoic acid (pCBA) degradation studies revealed that CuCl2mixed with catechol produced free ·OH. In the presence of DNA, however, inhibition of the pCBA degradation suggested that another ROS is responsible for the DNA degradation. Of a series of ROS scavengers investigated, only KI, NaN3, and Na‐formate—all of the salts tested—strongly inhibited the DNA degradation, suggesting that the ionic strength rather than the reactivity of the individual scavengers could be responsible for the observed inhibition. The ionic strength effect was confirmed by increasing the concentration of phosphate buffer, which is a poor ·OH scavenger, and was interpreted as the result of destabilization of DNA‐copper‐hydroperoxo complexes. Piperidine‐labile site patterns in DNA degraded by copper and catechol showed that the mixture of Cu(II) and catechol degrades DNA via the intermediate formation of a DNA‐copper‐hydroperoxo complex. Replacement of guanine by 7‐deazaguanine did not retard the DNA degradation, suggesting that the DNA‐copper‐hydroperoxo complexes do not bind to the guanine N‐7 as proposed in the literature. Environ. Mol. Mutagen. 36:5–12, 2000. © 2000 Wiley‐Liss, Inc.

Two new detection methods for the determination of poly-β-hydroxybutyrate (PHB) and -valerate (PHV) are described. Both methods are based on depolymerization of PHB/PHV to 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Depolymerization was achieved by either propanolic or hydrolytic digestion. Propanolic digestion transformed commercial PHB/PHV stoichiometrically into 3HB/3HV and yielded apparently complete overies of bacterial PHB/PHV from activated sludge. Hydrolytic digestion was suitable only for PHB determination. For quantification of 3HB and 3HV directly from digested sludge, a method based on ion-exchange chromatography and conductivity detection was developed (IC-method). Alternatively, the total of 3HB and 3HV was quantified using a commercial enzymatic test kit and colorimetric detection (enzyme method). Both detection methods are easier to perform than previous methods and are suitable for complex matrices such as activated sludge. The IC-method is recommended for high sample throughputs or if distinction between PHB and PHV is essential. Enzymatic detection is recommended if a few samples per day have to be measured immediately or if an ion chromatograph is unavailable.

Activated sludge communities which performed enhanced biological phosphate removal (EBPR) were phylogenetically analyzed by 16S rRNA-targeted molecular methods. Two anaerobic-aerobic sequencing batch reactors were operated with two different carbon sources (acetate vs. a complex mixture) for three years and showed anaerobic-aerobic cycles of polyhydroxybutyrate- (PHB) and phosphate-accumulation characteristic for EBPR-systems. In situhybridization showed that the reactor fed with the acetate medium was dominated by bacteria phylogenetically related to the Rhodocyclus-group within the β-Proteobacteria(81% of DAPI-stained cells). The reactor with the complex medium was also predominated by this phylogenetic group albeit at a lesser extent (23% of DAPI-stained cells). More detailed taxonomic information on the dominant bacteria in the acetate-reactor was obtained by constructing clone libraries of 16S rDNA fragments. Two different types of Rhodocyclus-like clones (R1 and R6) were retrieved. Type-specific in situhybridization and direct rRNA-sequencing revealed that R6 was the type of the dominant bacteria. Staining of intracellular polyphosphate- and PHB-granules confirmed that the R6-type bacterium accumulates PHB and polyphosphate just as predicted by the metabolic models for EBPR. High similarities to 16S rDNA fragments from other EBPR-sludges suggest that R6-type organisms were present and may play an important role in EBPR in general. Although the R6-type bacterium is closely related to the genus Rhodocyclus, it did not grow phototrophically. Therefore, we propose a provisional new genus and species CandidatusAccumulibacter phosphatis.

Chlorocatechols introduced into the environment directly or as a result of degradation processes are highly toxic, particularly when combined with heavy metals. With in vitro DNA degradation assays, the high reactivity of chlorocatechols combined with heavy metals could be shown, whereby copper was shown to be more active than iron. Structure–activity analysis showed that the degradation potential of the chlorocatechols decreased with an increasing number of chloratoms. The addition of reactive oxygen species scavengers allowed the identification of hydrogen peroxide as an important agent leading to DNA damage in this reaction. The potential of other reactive compounds, however, can neither be determined nor excluded with this approach. Exposure of Escherichia coliand Salmonella typhimuriumcultures to the same mixtures of chlorocatechols and copper surprisingly did not lead to an enhanced mutation rate. This phenomenon was explained by doing marker gene expression measurements and toxicity tests with E.colimutants deficient in oxidative stress defense or DNA repair. In catechol‐copper‐exposed cultures an increased peroxide level could indeed be demonstrated, but the highly efficient defense and repair systems of E.coliavoid the phenotypical establishment of mutations. Increased mutation rates under chronic exposure, however, cannot be excluded. Environ. Mol. Mutagen. 33:202–210, 1999 © 1999 Wiley‐Liss, Inc.

Chlorocatechols introduced into the environment directly or as a result of degradation processes are highly toxic, particularly when combined with heavy metals. With in vitro DNA degradation assays, the high reactivity of chlorocatechols combined with heavy metals could be shown, whereby copper was shown to be more active than iron. Structure–activity analysis showed that the degradation potential of the chlorocatechols decreased with an increasing number of chloratoms. The addition of reactive oxygen species scavengers allowed the identification of hydrogen peroxide as an important agent leading to DNA damage in this reaction. The potential of other reactive compounds, however, can neither be determined nor excluded with this approach. Exposure of Escherichia coli and Salmonella typhimurium cultures to the same mixtures of chlorocatechols and copper surprisingly did not lead to an enhanced mutation rate. This phenomenon was explained by doing marker gene expression measurements and toxicity tests with E. coli mutants deficient in oxidative stress defense or DNA repair. In catechol-copper-exposed cultures an increased peroxide level could indeed be demonstrated, but the highly efficient defense and repair systems of E. coli avoid the phenotypical establishment of mutations. Increased mutation rates under chronic exposure, however, cannot be excluded. Environ. Mol. Mutagen. 33:202–210, 1999 © 1999 Wiley-Liss, Inc.

Toluene degradation occurred in anaerobic flow‐through sediment columns filled with contaminated sediment and sludge to which either amorphous or highly crystalline manganese oxide was added. An enrichment culture from these sediment columns was able to grow on toluene under strictly anaerobic conditions in the presence of manganese oxide. The oxidation of toluene was coupled to the production of CO2and to the reduction of Mn(IV). Of the different manganese oxides tested, the rate was slowest with crystalline manganese oxide. After several transfers of the enrichment culture, its ability to degrade toluene became less and it was ultimately lost, unless sterilized Rhine river sediment was present in the medium. Direct contact between the bacteria and the manganese oxide was found to be advantageous for a rapid toluene degradation. The degradation rate could be further increased by adding organic ligands such as oxalic acid or nitrilotriacetic acid.

The influence of pH on the denitrification activity of a continuous culture of Paracoccus denitrificans was studied in relation to the presence of nitrite. After a transition from aerobic to anaerobic conditions at the suboptimal pH of 6.8, P. denitrificans was not able to build up a functional denitrification pathway. Nitrite accumulated in the medium as the predominant denitrification product. Although the nitrite reductase gene was induced properly, the enzyme could not be detected at sufficient amounts in the culture. These observations indicate that either translation was somehow inhibited, or once synthesized nitrite reductase was inactivated, possibly by the high concentrations of nitrous acid (HNO2. Interestingly, when a P. denitrificans culture which was grown to steady-state under anaerobic conditions was then exposed to suboptimal pHs, cells exhibited a reduced overall denitrification activity, but neither nitrite nor any other denitrification intermediate accumulated.

A sequence analysis of the 16S-rRNA of Methanococcoides euhalobius revealed that this organism was highly related to members of the genus Methanohalophilus. On the basis of sequence data, an oligonucleotide probe specific to Methanohalophilus species was designed. Hybridization studies with this probe confirmed close relationship of Methanococcoides euhalobius to Methanohalophilus species. Therefore, we propose that Methanococcoides euhalobius should be transferred to the genus Methanohalophilus as Methanohalophilus euhalobius.

Similarities between the nitrogen-fixing systems of the archaebacteriumMethanosarcina barkeri (strain Fusaro) and a number of eubacteria were investigated. Using antibodies againstRhizobium leguminosarum nitrogenase and a probe of clonednif-HDK genes of this species, homology withM. barkeri was demonstrated on the protein level and to a greater extent on the DNA level.

Respiration of Dehalobacter restrictusis based on reductive dechlorination of tetrachloroethene. The terminal component of the respiratory chain is the membrane‐bound tetrachloroethene reductase. The metal prosthetic groups of the purified enzyme have been studied by optical and EPR spectroscopy. The 60‐kDa monomer contains one cobalamin with Em(Co1+/2+)=−350 mV and Em(Co2+/3+)>150 mV and two electron‐transferring [4Fe–4S](2+;1+)clusters with rather low redox potentials of Em≈−480 mV. The cob(II)alamin is present in the base‐off configuration. A completely reduced enzyme sample reacted very rapidly with tetrachloroethene yielding base‐off cob(II)alamin rather than trichlorovinyl‐cob(III)alamin.

The xylose metabolism of Bacteroides xylanolyticus X5-1 was studied by determining specific enzyme activities in cell free extracts, by following 13C-label distribution patterns in growing cultures and by mass balance calculations. Enzyme activities of the pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway were sufficiently high to account for in vivo xylose fermentation to pyruvate via a combination of these two pathways. Pyruvate was mainly oxidized to acetyl-CoA, CO2 and a reduced cofactor (ferredoxin). Part of the pyruvate was converted to acetyl-CoA and formate by means of a pyruvate-formate lyase. Acetyl-CoA was either converted to acetate by a combined action of phosphotransacetylase and acetate kinase or reduced to ethanol by an acetaldehyde dehydrogenase and an ethanol dehydrogenase. The latter two enzymes displayed both a NADH- and a NADPH-linked activity. Cofactor regeneration proceeded via a reduction of intermediates of the metabolism (i.e. acetyl-CoA and acetaldehyde) and via proton reduction. According to the deduced pathway about 2.5 mol ATP are generated per mol of xylose degraded.

An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139±7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an a2ß2 oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri-and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg2+ was required for activity. The pH optimum was 8 at 1 mM PPi and 5 mM Mg2+. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent Km for PPi of 0.1 mM in the presence of 5 mM Mg2+. The Vmax was 590 µmol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a Kcat of 1400 per second.

A new species of sporulating saccharolytic anaerobe, designated as Clostridium quiniisp. nov., is described. A gram-positive strain BS1, was isolated from the granular metanogenic sludge (UASB) from a waste-water treatment plant at a sugar refinery. The strain exhibits a series of morphological stages, developing from a spore to a small rod to a motile rod (peritrichous flagella) in the exponential growth phase, and then swelling to form cigar-shaped cells, exhibiting tumbling movements, in the late exponential growth phase before finally becoming large nonmotile ovoid cells in the stationary phase. Swelling occurs as a result of glucose being taken up and stored as a glycogen-like substance. The main fermentation products when growing on glucose is H2, CO2, formate, acetate and ethanol as well as small amounts of butyrate during exponential growth. Lactate is formed during the stationary phase, when glucose is abundant. Optimal conditions for growth is 40–45°C and pH of around 7.4. The type strain BS1 contains 28.0% mol G+C.

Rhizobium leguminosarumbiovar trifoliiTA-1 produced high molecular weight extracellular (EPS) and capsular polysaccharides (CPS) as the main carbohydrate products in a medium (10 g of mannitol and 1 g of glutamic acid per liter) with low osmotic pressure of 0.20 MPa. By increasing the osmotic pressure of the medium with the addition of NaCl or other osmolytes up to 1.44 MPa, the synthesis of EPS and CPS was suppressed. Cyclic β-(1,2)-glucans were excreted instead. Concentrations of over 1500 mg of glucans/l medium were produced by a biomass of 520 mg protein at 200 mM NaCl (1.20 MPa). Intracellular cyclic β-(1,2)-glucan concentrations remained at 45 to 100 mg/g protein during the stationary phase, independent of the osmotic strength of the medium. Parallel to the increasing osmotic pressure of the medium, the disaccharide trehalose accumulated in the cells as osmo-protectant. Concentrations of up to 130 mg/g protein were reached. Strain TA-1 could tolerate 350 mM NaCl.

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h-1) nitrate and nitrite reductase activities of 200 nmol · mg-1 protein · min-1 and 250 nmol · mg-1 protein · min-1 were measured, respectively. At a high growth rate (0.55 h-1) both enzyme activities were considerably lower (25 and 12 nmol mg-1 · protein · min-1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.

A new genus of methanogenic bacteria is described, which was isolated from a mesophilic sewage digester. It is most probably the filamentous bacterium, earlier referred to asMethanobacterium soehngenii, “fat rod” or “acetate organism”. The single non-motile, non-sporeforming cells are rod-shaped (0.8×2 µm) and are normally combined end to end in long filaments, surrounded by a sheath-like structure. The filaments form characteristic bundles.Methanothrix soehngenii decarboxylates acetate, yielding methane and carbon dioxide. Other methanogenic substrates (H2-CO2, formate, methanol, methylamines) are not used for growth or methane formation. Formate is split into hydrogen and carbon dioxide. The temperature optimum for growth and methane formation is 37°C and the optimal pH range is 7.4–7.8. Sulfide and ammonia serve as sulfur and nitrogen source respectively. Oxygen completely inhibits growth and methane formation, but the bacteria do not loose their viability when exposed to high oxygen concentrations. 100 mg/l vancomycin showed no inhibition of growth and methanogenesis. No growth and methane formation was observed in the presence of: 2-bromoethanesulfonic acid, viologen dyes, chloroform, and KCN. The bacterium has a growth yield on acetate of 1.1–1.4 g biomass per mol acetate. The apparent “KS” of the acetate conversion system to methane and carbon dioxide is 0.7 mmol/l. The DNA base composition is 51.9 mol% guanine plus cytosine. The nameMethanothrix is proposed for this new genus of filamentous methane bacterium. The type species,Methanothrix soehngenii sp. nov., is named in honor of N. L. Söhngen.

Recently it has been shown that the initial stages of bacterial adhesion to a model-surface of sulphated polystyrene can best be described using hydrophobic and electrostatic parameters. In the present study it is tested whether these parameters can generally be applied to predict bacterial adhesion by using (i) glass, as a model for hydrophilic and natural surfaces of silicates and oxides, (ii) polystyrene coated with proteins, as a model for a surface coated with an organic layer, and (iii) river Rhine sediment, as an example of a natural surface. Adhesion to glass was dominated by electrostatic interaction, whereas adhesion to polystyrene coated with various types of proteins depended on the surface characteristics of the bacteria and the type of protein. By relating Van der Waals interactions to hydrophobicity of the interacting species, the adhesion of bacteria to the various surfaces including the river Rhine sediments could be interpreted in terms of the DLVO-theory. It is therefore concluded that the conceptual principles of the DLVO-theory (interplay of Van der Waals and electrostatic interactions) are suitable to describe, at least qualitatively, the initial processes of bacterial adhesion to a wide range of surfaces.

The adhesion of bacteria to solid surfaces was studied using a physicochemical approach. Adhesion to negatively charged polystyrene was found to be reversible and could be described quantitatively using the DLVO theory for colloidal stability, i.e., in terms of Van der Waals and electrostatic interactions. The influence of the latter was assessed by varying the electrolyte strength. Adhesion increased with increasing electrolyte strength. The adhesion Gibbs energy for a bacterium and a negatively charged polystyrene surface was estimated from adhesion isotherms and was found to be 2–3 kT per cell. This low value corresponds to an adhesion in the secondary minimum of interaction as described by the DLVO theory. The consequences of these findings for adhesion in the natural environment are discussed.

As part of a study of the biogas production from cattle waste, xylanolytic bacteria were isolated from enrichments of fermenting cattle manure. From 34 isolates, mostly Gram-negative rods, a typical strain was investigated in more detail. It was an anaerobic non-sporeforming, Gramnegative rod, which was motile with peritrichous flagella. This organism fermented xylan and many soluble sugars (glucose, cellobiose, mannose, xylose, arabinose). Other hemicelluloses such as gum xanthan, laminaran, locust bean gum, and gum arabic were not utilized. It also could not use cellulose. Fermentation products were carbon dioxide, hydrogen, acetate and ethanol. The bacterium produced carboxymethylcellulase and xylanase, especially when growing on xylan. Growth was optimal between 25°C and 40°C and between pH 6.5 and 7.5. The guanine plus cytosine content of the DNA was 34.8±0.8%. The isolate was identified as a member of the genus Bacteroides, and a new species is proposed: Bacteroides xylanolyticus (xylan dissolving). The type strain of B. xylanolyticus is strain X5-1 (DSM 3808).

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H2/CO2 converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.

A bacterial culture (LET-13) was enriched, which uses toluene as sole carbon and energy source, and manganese oxide as terminal electron acceptor. The culture is able to degrade a variety of substituted monoaromatic compounds like p-hydroxy-benzylalcohol, p-hydroxy-benzaldehyde, p-hydroxy-benzoate, phenol and the three isomers of cresol. Benzene, ethylbenzene, all xylenes and naphthalene were not degraded under the experimental conditions used. Based on the results of growth experiments and the detection of intermediates, it is concluded that toluene is degraded via a methyl hydroxylation. A possible side reaction can lead to the formation of cresol. The organisms in the culture look similar; motile rods, which are Gram-negative, oxidase-negative and catalase-negative. The culture was partly identified by phylogenetic analysis of cloned rDNA sequences. The phylogenetic analysis showed that at least two major groups of bacteria are present. One group of bacteria shows 70–80% similarity (based on 16S rRNA gene sequence data) with the Bacteroides-Cytophagagroup, and one group consists of members of the β-subclass of the Proteobacteria.

Toluene degradation occurred in anaerobic flow-through sediment columns filled with contaminated sediment and sludge to which either amorphous or highly crystalline manganese oxide was added. An enrichment culture from these sediment columns was able to grow on toluene under strictly anaerobic conditions in the presence of manganese oxide. The oxidation of toluene was coupled to the production of CO2and to the reduction of Mn(IV). Of the different manganese oxides tested, the rate was slowest with crystalline manganese oxide. After several transfers of the enrichment culture, its ability to degrade toluene became less and it was ultimately lost, unless sterilized Rhine river sediment was present in the medium. Direct contact between the bacteria and the manganese oxide was found to be advantageous for a rapid toluene degradation. The degradation rate could be further increased by adding organic ligands such as oxalic acid or nitrilotriacetic acid.

A bacterial culture (LET‐13) was enriched, which uses toluene as sole carbon and energy source, and manganese oxide as terminal electron acceptor. The culture is able to degrade a variety of substituted monoaromatic compounds like p‐hydroxy‐benzylalcohol, p‐hydroxy‐benzaldehyde, p‐hydroxy‐benzoate, phenol and the three isomers of cresol. Benzene, ethylbenzene, all xylenes and naphthalene were not degraded under the experimental conditions used. Based on the results of growth experiments and the detection of intermediates, it is concluded that toluene is degraded via a methyl hydroxylation. A possible side reaction can lead to the formation of cresol. The organisms in the culture look similar; motile rods, which are Gram‐negative, oxidase‐negative and catalase‐negative. The culture was partly identified by phylogenetic analysis of cloned rDNA sequences. The phylogenetic analysis showed that at least two major groups of bacteria are present. One group of bacteria shows 70–80% similarity (based on 16S rRNA gene sequence data) with the Bacteroides‐Cytophagagroup, and one group consists of members of the β‐subclass of the Proteobacteria.

Abstract: The regulation of and the optimum conditions for polyphosphate accumulation inAcinetobacter sp. were determined.Acinetobacter strain 210A accumulated polyphosphate in the presence of an intra- or extracellular energy source. The accumulation of polyphosphate during endogenous respiration was stimulated by streptomycin and inhibited by KCN. The highest amount of polyphosphate was found in cells in which energy supply was not limited, namely at low growth rates under sulphur limitation, and in the stationary phase of growth when either the nitrogen or the sulphur source was depleted. The phosphorus accumulation was not affected by the pH between 6.5 and 9. There was a pronounced effect of the temperature on phosphorus accumulation but is varied from strain to strain.Acinetobacter strain 210A accumulated more phosphate at low temperatures, strain B8 showed an optimum accumulation at 27.5 C, while strain P accumulated phosphorus independently of the temperature. The optimum temperature for growth ofAcinetobacter strains tested ranged from 25 to 33 C, and the optimum pH was between 6 and 9.

In an attempt to obtain bacteria growing on 1,2-dimethylbenzene as sole carbon and energy source two different strains were isolated. One was identified as an Arthrobacter strain, the other as a Corynebacterium strain. Corynebacterium strain C125 was further investigated. The organism was not capable to grow on 1,3- and 1,4-dimethylbenzene. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 3,4-dimethylcatechol-2,3-dioxygenase activity was found in cell extracts. When 3,4-dimethylcatechol was added to cell extract of 1,2-dimethylbenzene-grown cells, first a compound with the spectral properties of 2-hydroxy-5-methyl-6-oxo-2,4-heptadienoate was formed and subsequently acetate was produced. It is proposed that dioxygenases are involved in the initial steps of 1,2-dimethylbenzene degradation, and ring opening proceeds via meta-cleavage.

Abstract: The highly enriched anaerobic bacterium that couples the reductive dechlorination of tetrachloroethene to growth, previously referred to as PER-K23, was obtained in pure culture and characterized. The bacterium, which does not form spores, is a small, gram-negative rod with one lateral flagellum. It utilized only H2 as an electron donor and tetrachloroethene and trichloroethene as electron acceptors in an anaerobic respiration process; it could not grow fermentatively. Acetate served as a carbon source in a defined medium containing iron as the sole trace element, the two vitamins thiamine and cyanocobalamin, and the three amino acids arginine, histidine, and threonine. The cells contained menaquinones and b-type cytochromes. The G+C content of the DNA was 45.3 ± 0.3 mol%. The cell wall consisted of type-A3γ peptidoglycan with ll-diaminopimelic acid and one glycine as an interpeptide bridge. The cells are surrounded by an S-layer; an outer membrane was absent. Comparative sequence analysis of the 16S rRNA sequence showed that PER-K23 is related to gram-positive bacteria with a low G+C content of the DNA. Based on the cytological, physiological, and phylogenetic characterization, it is proposed to affiliate the isolate to a new genus, Dehalobacter, with PER-K23 as the type strain of the new species Dehalobacter restrictus.

The conductivity of bacterial cell suspensions has been studied over a wide range of ionic strengths and is interpreted in terms of their cell wall properties. The experimental data have been analyzed after improving the high κadouble-layer theory of Fixman, by accounting for ionic mobility in the hydrodynamically stagnant layer, i.e., in the bacterial wall. Static conductivity and dielectric dispersion measurements both show that the counterions in the porous gel-like cell wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the ions in the bacterial wall are of the same order but somewhat lower than those in the bulk electrolyte solution. The occurrence of surface conductance reduces the electrophoretic mobility in electrophoresis studies. If this effect is not taken into account, the ζ-potential will be underestimated, especially at low electrolyte concentrations.

Electron microscopy and energy dispersive X-ray micro-analysis were used to examine the elemental composition of large polyphosphate granules in unfixed and unstained intact cells of Acinetobacter strain 210A. When grown in medium with butyrate, Acinetobacter strain 210A possessed 1 or 2 large granules with a diameter of 0.4 µm besides a relatively large number of small granules. The large granules were composed of phosphorus, magnesium and potassium. A decrease in the Mg/Ca-ratio of the medium from 5.95 to 0.0073 resulted in a decline in the intracellular Mg/Ca-ratio from 15 to 0.56. At a high intracellular Mg/Ca-ratio, magnesium was the dominant counterion in the polyphosphate granule. Calcium became the major cation in the polyphosphate bodies at a low intracellular Mg/Ca-ratio. Omission of Ca2+ or modification of the K/Mg ratio in the medium did not significantly affect the cation composition of the polyphosphate granules. The dissociation constants for Mg- and Ca-polyphosphate were 9.3×10-2 mol/l and 1.5×10-1 mol/l, respectively.

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. “Ferric iron reductase” activity of 9 nmol Fe2+ mg-1 protein min-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe2+-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).

Enzyme measurements were carried out with crude cell-free extracts of the propionate oxidizing coculture of Syntrophobacter wolinii and Desulfovibrio G11. Using cell-free extracts of a pure culture of Desulfovibrio G11 as a blank, most of the enzymes involved in the methylmalonyl-CoA pathway for propionate oxidation, including a propionyl-CoA: oxaloacetate transcarboxylase, were demonstrated in S. wolinii.

A methanogenic bacterium, commonly seen in digested sludge and referred to as the “fat rod” or Methanobacterium soehngenii, has been enriched to a monoculture and is characterized. Cells are gramnegative, non-motile and appear as straight rods with flat ends. They form filaments which can grow to great lengths. The structure of the outer cell envelop is similar to Methanospirillum hungatii. The organism grows on a mineral salt medium with acetate as the only organic component. Acetate is the energy source, and methane is formed exclusively from the methyl group. Acetate and carbon dioxide act as sole carbon source and are assimilated in a molar ratio of about 1.9:1. The reducing equivalents necessary to build biomass from these two precursors are obtained from the total oxidation of some acetate. Hydrogen is not used for methane formation and is not needed for growth. Formate is cleaved into hydrogen and carbon dioxide. Coenzyme M was found to be present at levels of 0.35 nmol per mg of dry cells and F420 amounted to 0.55 µg per mg protein. The mean generation time was 9 days at 33°C.

This article examines the role of virtual water and stresses the need to develop rain-fed agriculture. Food production accounts for over 80 percent of global total water use. Many countries resort to food importation to address domestic water deficits and increasing food demand. This is similar with importing water to produce food locally. The water represented in food imports is called virtual water. A chart is presented that demonstrates average global virtual water import and export. The major sources of virtual water include North America, South America and Oceania.

The methyl-CoM reductase from Methanothrix soehngenii was purified 18-fold to apparent homogeneity with 50% recovery in three steps. The native molecular mass of the enzyme estimated by gel-fitration was 280 kDa. SDS-polyacrylamide gel electrophoresis revealed three protein bands corresponding to Mr 63 900, 41 700 and 30 400 Da. The methyl-coenzyme M reductase constitutes up to 10% of the soluble cell protein. The enzyme has Km apparent values of 23 μM and 2 mM for N-7-mercaptoheptanoylthreonine phosphate (HS- HTP=componentB) and methyl-coenzyme M (CH3?CoM) respectively. At the optimum pH of 7.0 60 nmol of methane were formed per min per mg protein.

In Methanothrix soehngenii acetate is first activated by an acetate thiokinase rather than a phosphotransacetylase. The specific activity of the acetate thiokinase was 5.29 μmol acetate activated min−1 mg−1 protein with a half maximum rate at 0.74 mM acetate and at 0.047 mM CoA. In cell-free extracts a CO-dehydrogenase activity was measured of 3.02 μmol min−1 mg−1 protein with a half maximum rate at 0.44 mM CO and at 0.18 mM methylviologen. NADP and NAD could not replace methylviologen. F420 showed only low activity as electron acceptor.

The interconversion of adenine nucleotides during acetate fermentation was investigated with concentrated cell suspensions of Methanothrix soehngenii. Starved cells contained high levels of AMP (2.2 nmol/mg protein), but had hardly any ADP or ATP. The energy charge of these cells was 0.1. Immediately after the addition of the substrate acetate, the level of ATP increased, reaching a maximum of 1.4 nmol/mg protein, corresponding to an energy charge of 0.7 when half of the acetate was consumed. Once the acetate was depleted, the ATP concentration decreased to its original level of 0.1 nmol/mg protein. As M. soehngenii contained relatively high amounts of AMP, the luciferase system for the determination of ATP gave not always satisfactory results. Therefore a reliable method based on the separation of adenine nucleotides by anion exchange HPLC was used.