Alfons J. M. Stams

The ecology and biotechnology of sulphate-reducing bacteria

Sulphate-reducing bacteria (SRB) are anaerobic microorganisms that use sulphate as a terminal electron acceptor in, for example, the degradation of organic compounds. They are ubiquitous in anoxic habitats, where they have an important role in both the sulphur and carbon cycles. SRB can cause a serious problem for industries, such as the offshore oil industry, because of the production of sulphide, which is highly reactive, corrosive and toxic. However, these organisms can also be beneficial by removing sulphate and heavy metals from waste streams. Although SRB have been studied for more than a century, it is only with the recent emergence of new molecular biological and genomic techniques that we have begun to obtain detailed information on their way of life.

Electron transfer in syntrophic communities of anaerobic bacteria and archaea

Interspecies electron transfer is a key process in methanogenic and sulphate-reducing environments. Bacteria and archaea that live in syntrophic communities take advantage of the metabolic abilities of their syntrophic partner to overcome energy barriers and break down compounds that they cannot digest by themselves. Here, we review the transfer of hydrogen and formate between bacteria and archaea that helps to sustain growth in syntrophic methanogenic communities. We also describe the process of reverse electron transfer, which is a key requirement in obligately syntrophic interactions. Anaerobic methane oxidation coupled to sulphate reduction is also carried out by syntrophic communities of bacteria and archaea but, as we discuss, the exact mechanism of this syntrophic interaction is not yet understood.

Metabolic interactions between anaerobic bacteria in methanogenic environments.

In methanogenic environments organic matter is degraded by associations of fermenting, acetogenic and methanogenic bacteria. Hydrogen and formate consumption, and to some extent also acetate consumption, by methanogens affects the metabolism of the other bacteria. Product formation of fermenting bacteria is shifted to more oxidized products, while acetogenic bacteria are only able to metabolize compounds when methanogens consume hydrogen and formate efficiently. These types of metabolic interaction between anaerobic bacteria is due to the fact that the oxidation of NADH and FADH2 coupled to proton or bicarbonate reduction is thermodynamically only feasible at low hydrogen and formate concentrations. Syntrophic relationships which depend on interspecies hydrogen or formate transfer were described for the degradation of e.g. fatty acids, amino acids and aromatic compounds.

Syntrophism among Prokaryotes

The study of pure cultures in the laboratory has provided an amazingly diverse diorama of metabolic capacities among microorganisms, and has established the basis for our understanding of key transformation processes in nature. Pure culture studies are also prerequisites for research in microbial biochemistry and molecular biology. However, desire to understand how microorganisms act in natural systems requires the realization that microorganisms don’t usually occur as pure cultures out there, but that every single cell has to cooperate or compete with other microor macroorganisms. The pure culture is, with some exceptions such as certain microbes in direct cooperation with higher organisms, a laboratory artifact. Information gained from the study of pure cultures can be transferred only with great caution to an understanding of the behavior of microbes in natural communities. Rather, a detailed analysis of the abiotic and biotic life conditions at the microscale is needed for a correct assessment of the metabolic activities and requirements of a microbe in its natural habitat. In many cases, relationships of bacteria with other organisms may be relatively unimportant, as appears to be the case with most aerobes: they can usually degrade even fairly complex substrates to water and carbon dioxide without any significant cooperation with other organisms. Nutritional cooperation may exist, but may be re stricted to the transfer of minor growth factors, such as vitamins, from one organism to the other. However, we have to realize that this assumption is based on experience gained from pure cultures that were typically enriched and isolated in simple media, and the selection aimed at organisms

Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm.Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently, an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.

Distinctive properties of high hydrogen producing extreme thermophiles, Caldicellulosiruptor saccharolyticus and Thermotoga elfii

Growth and hydrogen production by two extreme thermophiles during sugar fermentation was investigated. In cultures of Caldicellulosiruptor saccharolyticus grown on sucrose and Thermotoga eli grown on glucose stoichiometries of 3:3 mol of hydrogen and 2 mol of acetate per mol C6-sugar unit were obtained. The hydrogen level was about 83% of the theoretical maximum. C. saccharolyticus and T. eli reached maximum cell densities of 1:1 × 10 9 and 0:8 × 10 9 cells=ml, respectively, while their maximum hydrogen production rates were 11.7 and 5:1 mmol=g dry weight=h, respectively. For growth of C. saccharolyticus on sucrose, a biomass yield of 45: 1g DW=mol sucrose and a YATP of 11.3-14.1 were calculated. Replacement of yeast extract by casamino acids, plus proline and vitamins in the medium of C. saccharolyticus resulted in similar yields of hydrogen production on sucrose, but diminished the rate by about 30%. Both yeast extract and tryptone were required by T. eli , and appeared to function as sources of carbon, nitrogen and energy. In the absence of tryptone, T. eli converted 26% of the glucose to another by-product, resulting in a lower yield of hydrogen. Growth of T. eli ceased prior to glucose depletion, but the culture continued to ferment glucose to hydrogen and acetate until all glucose was consumed. ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.

Physiology, Ecology, Phylogeny, and Genomics of Microorganisms Capable of Syntrophic Metabolism

Syntrophic metabolism is diverse in two respects: phylogenetically with microorganisms capable of syntrophic metabolism found in the Deltaproteobacteria and in the low G+C gram‐positive bacteria, and metabolically given the wide variety of compounds that can be syntrophically metabolized. The latter includes saturated fatty acids, unsaturated fatty acids, alcohols, and hydrocarbons. Besides residing in freshwater and marine anoxic sediments and soils, microbes capable of syntrophic metabolism also have been observed in more extreme habitats, including acidic soils, alkaline soils, thermal springs, and permanently cold soils, demonstrating that syntrophy is a widely distributed metabolic process in nature. Recent ecological and physiological studies show that syntrophy plays a far larger role in carbon cycling than was previously thought. The availability of the first complete genome sequences for four model microorganisms capable of syntrophic metabolism provides the genetic framework to begin dissecting the biochemistry of the marginal energy economies and interspecies interactions that are characteristic of the syntrophic lifestyle.

Anaerobic treatment of sulphate-rich wastewaters

Until recently, biological treatment of sulphate-rich wastewater was rather unpopular because of the production of H2S under anaerobic conditions. Gaseous and dissolved sulphides cause physical-chemical (corrosion, odour, increased effluent chemical oxygen demand) or biological (toxicity) constraints, which may lead to process failure. Anaerobic treatment of sulphate-rich wastewater can nevertheless be applied successfully provided a proper treatment strategy is selected. The strategies currently available are discussed in relation to the aim of the treatment: i) removal of organic matter, ii) removal of sulphate or iii) removal of both. Also a whole spectrum of new biotechnological applications (removal of organic chemical oxygen demand, sulphur, nitrogen and heavy metals), recently developed based on a better insight in sulphur transformations, are discussed.

Thermotoga lettingae sp. nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor.

A novel, anaerobic, non-spore-forming, mobile, Gram-negative, thermophilic bacterium, strain TMOT, was isolated from a thermophilic sulfate-reducing bioreactor operated at 65 C with methanol as the sole substrate. The G+C content of the DNA of strain TMOT was 39.2 mol%. The optimum pH, NaCl concentration, and temperature for growth were 7.0, 1.0%, and 65 degrees C, respectively. Strain TMOT was able to degrade methanol to CO2 and H2 in syntrophic culture with Methanothermobacter thermautotrophicus AH or Thermodesulfovibrio yellowstonii. Thiosulfate, elemental sulfur, Fe(III) and anthraquinone-2,6-disulfonate were able to serve as electron acceptors during methanol degradation. In the presence of thiosulfate or elemental sulfur, methanol was converted to CO2 and partly to alanine. In pure culture, strain TMOT was also able to ferment methanol to acetate, CO2 and H2. However, this degradation occurred slower than in syntrophic cultures or in the presence of electron acceptors. Yeast extract was required for growth. Besides growing on methanol, strain TMOT grew by fermentation on a variety of carbohydrates including monomeric and oligomeric sugars, starch and xylan. Acetate, alanine, CO2, H2, and traces of ethanol, lactate and alpha-aminobutyrate were produced during glucose fermentation. Comparison of 16S rDNA genes revealed that strain TMOT is related to Thermotoga subterranea (98%) and Thermotoga elfii (98%). The type strain is TMOT (= DSM 14385T = ATCC BAA-301T). On the basis of the fact that these organisms differ physiologically from strain TMOT, it is proposed that strain TMOT be classified as a new species, within the genus Thermotoga, as Thermotoga lettingae.

Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positiveDesulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a V max of 28 units/mg protein and a K m of 20 μm. The pH and temperature optimum were 8.2 and 52 °C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (E m ) = −440 mV), one [3Fe-4S] cluster (E m = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an E m of −370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.