Influence of the starting microbial nucleus type on the anaerobic granulation dynamics

 The influence of four different granulation precursors, syntroph-enriched methanogenic consortia, Methanosaeta-enriched, Methanosarcina-enriched nuclei and acidogenic flocs, on the time course of complex granule development and the lag time for start-up was investigated in four upflow anaerobic sludge-bed and filter reactors. Although the operational conditions allowed the maintenance of the same specific growth rate of biomass in the four reactors, granulation proceeded rapidly with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei. However, granulation was significantly retarded when acidogenic flocs were used as precursors. The granule mean Sauter diameter increased rapidly in the reactor inoculated with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei and reached, at the end of the experiment, 3.1, 2.7 and 2.4 mm compared to 1.1 mm in that inoculated with acidogenic flocs. This corresponded to a rate of granule size increase of 31, 21, 18 μm/day in syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei, respectively, compared to 7 μm/day in acidogenic flocs. Biomass specific activities (i.e. acidogenic, syntrophic and methanogenic activities) increased stepwise in all reactors with time, especially in those inoculated with syntroph/methanogenic consortia and Methanosaeta nuclei. From these results it appears that syntrophs and Methanosaeta spp. play an important role in the anaerobic granulation process. Received: 25 January 1996 / Received revision: 3 September 1996 / Accepted: 13 September 1996     

Genomic analysis of carbon monoxide utilization and butanol production by Clostridium carboxidivorans strain P7

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7(T) possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO(2) fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7(T) chromosome. The functionality of these pathways was also confirmed by growth of P7(T) on CO and production of CO(2) as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7(T) was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7(T) genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans's natural capacity to produce potential biofuels from syngas.     

Differentiation of Methanosaeta concilii and Methanosarcina barkeri in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.     

The partition matrix: exploring variable phylogenetic signals along nucleotide sequence alignments

The partition matrix is a graphical tool for comparative analysis of nucleotide sequences following alignment. It is particularly useful for investigating the divergent phylogenies of sequence regions undergoing reticulate evolution. A partition matrix is generated by determining the consistency of the parsimoniously informative sites in a set of aligned sequences with the binary partitions inferred from the sequences. Since the linear order of sites is maintained, the matrix can be used to assess whether the distribution of sites either supporting or conflicting with particular partitions changes along the length of the alignment. The usefulness of the matrix in allowing visual identification of differences in evolutionary history among regions depends on the order in which partitions are shown; several suitable ordering schemes are proposed. We demonstrate the use of the partition matrix in interpreting the evolution of the pseudoautosomal boundary region on the sex chromosome of catarrhine primates. Its routine use should help to avoid attempts to derive single phylogenies from sequences whose evolution has been reticulate and to identify the gene conversion or recombination events underlying the reticulation. The method is relatively fast. It is exploratory, and it can form the basis for more formal analysis, which we discuss.      

Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin

Bindin is a gamete recognition protein of sea urchins that mediates species-specific attachment of sperm to an egg-surface receptor during fertilization. Sequences of bindin from closely related urchins show fixed species-specific differences. Within species, highly polymorphic bindin alleles result from point substitution, insertion/deletion, and recombination. Since speciation, positive selection favoring allelic variants has generated diversity in bindin polypeptides. Intraspecific bindin variation can be tolerated by the egg receptor, which suggests functional parallels between this system and other flexible recognition systems, including immune recognition. These results show that polymorphism in mate recognition loci required for rapid evolution of sexual isolation can arise within natural populations.      

Origin of p-cresol in the anaerobic degradation of trinitrotoluene

p-Cresol was repeatedly detected as a trace metabolite in anaerobic slurry reactors treating 2,4,6-trinitrotoluene (TNT)-contaminated soils. This study shows that p-cresol was not a metabolite of the anaerobic degradation of TNT, by using a combination of analytical techniques and 13C-labelled TNT. Instead, p-cresol, an intermediate in the degradation pathway of some amino acids, was shown to be inhibited by TNT and its metabolites. The range and persistence of inhibition to p-cresol microbial degradation decreased with the level of amino-substitution of the derivatives. This explains why p-cresol accumulated within the TNT-treating anaerobic bioslurry, as it could not be further biodegraded in the presence of TNT.     

Detection of intermediate metabolites of benzene biodegradation under microaerophilic conditions

The intermediate metabolites of benzene transformation by a microaerophilic bacterial consortium, adapted to degrade gasoline and benzene at low concentrations of dissolved oxygen (<1 mg l^-1), were identified. The examined range of initial DO concentration, 0.05 to 1 mg l^-1, was considerably lower than the previously reported values believed to be necessary to initiate benzene biodegradation. An extensive transformation of benzene, higher than the theoretical predictions for its aerobic oxidation, was observed. Phenol was identified as the most stable and the major intermediate metabolite which was subsequently transformed into catechol and benzoate. The use of ¹³C-labeled compounds identified benzene as the source of phenol, and phenol as the source of catechol and benzoate, suggesting the involvement of a monooxygenase enzymatic system in biodegradation of benzene at low DO concentrations. A metabolic sequence was proposed to describe the simultaneous detection of catechol and benzoate during the microaerophilic transformation of benzene. The results of this work demonstrate that it is possible to transform benzene, a highly carcinogenic hydrocarbon and a major contaminant of groundwater, to more easily biodegradable compounds in the presence of very small amounts of oxygen.