The algal trophic mode affects the interaction and oil production of a synergistic microalga-yeast consortium.

The use of non-food feedstocks to produce renewable microbial resources can limit our dependence on fossil fuels and lower CO2 emissions. Since microalgae display a virtuous CO2 and O2 exchange with heterotrophs, the microalga Chlamydomonas reinhardtii was combined with the oleaginous yeast Lipomyces starkeyi, known for their production of oil, base material for biodiesel. The coupled growth was shown to be synergistic for biomass and lipid production. The species were truly symbiotic since synergistic growth occurred even when the alga cannot use the organic carbon in the feedstock and in absence of air, thus depending entirely on CO2-O2 exchange. Since addition of acetate as the algal carbon source lowered the performance of the consortium, the microbial system design should take into account algal mixotrophy. The mixed biomass was found be suitable for biodiesel production, and whereas lipid production increased in the consortium, yields should be improved in future studies.

Multiple Rieske/cytb complexes in a single organism.

Most organisms contain a single Rieske/cytb complex. This enzyme can be integrated in any respiratory or photosynthetic electron transfer chain that is quinone-based and sufficiently energy rich to allow for the turnover of three enzymes - a quinol reductase, a Rieske/cytb complex and a terminal oxidase. Despite this universal usability of the enzyme a variety of phylogenetically distant organisms have multiple copies thereof and no reason for this redundancy is obvious. In this review we present an overview of the distribution of multiple copies among species and describe their properties from the scarce experimental results, analysis of their amino acid sequences and genomic context. We discuss the predicted redox properties of the Rieske cluster in relation to the nature of the pool quinone. It appears that acidophilic iron-oxidizing bacteria specialized one of their two copies for reverse electron transfer, archaeal Thermoprotei adapted their three copies to the interaction with different oxidases and several, phylogenetically unrelated species imported a second complex with a putative heme ci that may confer some yet to be determined properties to the complex. These hypothesis and all the more the so far completely unexplained cases call for further studies and we put forward a number of suggestions for future research that we hope to be stimulating for the field. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

The "green" phylogenetic clade of Rieske/cytb complexes.

More than a decade ago, Heliobacteria were recognised to contain a Rieske/cytb complex in which the cytochrome b subunit is split into two separate proteins, a peculiar feature characteristic of the cyanobacterial and plastidic b (6) f complex. The common presence of RCI-type reaction centres further emphasise possible evolutionary links between Heliobacteria, Chlorobiaceae and Cyanobacteria. In this contribution, we further explore the evolutionary relationships among these three phototrophic lineages by both molecular phylogeny and consideration of phylogenetic marker traits of the superfamily of Rieske/cytb complexes. The combination of these two methods suggests the existence of a "green" clade involving many non-phototrophs in addition to the mentioned RCI-type photosynthetic organisms. Structural and functional idiosyncrasies are (re-)interpreted in the framework of evolutionary biology and more specifically evolutionary bioenergetics.

Structure, organization and expression of the genes encoding mitochondrial cytochrome c(1) and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii.

The sequence and organization of the Chlamydomonas reinhardtii genes encoding cytochrome c(1) ( Cyc1) and the Rieske-type iron-sulfur protein ( Isp), two key nucleus-encoded subunits of the mitochondrial cytochrome bc(1) complex, are presented. Southern hybridization analysis indicates that both Cyc1 and Isp are present as single-copy genes in C. reinhardtii. The Cyc1 gene spans 6404 bp and contains six introns, ranging from 178 to 1134 bp in size. The Isp gene spans 1238 bp and contains four smaller introns, ranging in length from 83 to 167 bp. In both genes, the intron/exon junctions follow the GT/AG rule. Internal conserved sequences were identified in only some of the introns in the Cyc1 gene. The levels of expression of Isp and Cyc1 genes are comparable in wild-type C. reinhardtii cells and in a mutant strain carrying a deletion in the mitochondrial gene for cytochrome b (dum-1). Nevertheless, no accumulation of the nucleus-encoded cytochrome c(1) or of core proteins I and II was observed in the membranes of the respiratory mutant. These data show that, in the green alga C. reinhardtii, the subunits of the cytochrome bc(1) complex fail to assemble properly in the absence of cytochrome b.

Response of a soil bacterial community to grassland succession as monitored by 16S rRNA levels of the predominant ribotypes.

The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the alpha subclass of the class Proteobacteria. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes.

Polytomella spp. growth on ethanol. Extracellular pH affects the accumulation of mitochondrial cytochrome c550.

A defined medium with ethanol as sole carbon source was devised for growth of the colorless, unicellular alga Polytomella spp. Cell density on this carbon source was related to extracellular pH. An acidic pH was required for ethanol utilization; best yields were obtained at pH 3.7. Spectroscopic analysis of the cells showed that the concentration of cytochrome c per cell was 40% higher than at pH 6.0; the concentrations of cytochrome a606 (cytochrome c oxidase) and b566 (cytochrome bc1 complex) were the same. A soluble cytochrome c550 was purified from cells grown at pH 3.7 and characterized by peptide sequencing as the 12-kDa cytochrome c550 of the mitochondrial respiratory chain. Immunoblots of total cell proteins showed higher accumulation of cytochrome c550 at pH 3.7 than at pH 6.0. RNA blot analysis gave clear evidence of the abundance of c550 transcript in cells grown at pH 3.7. The amount of mitochondrial proteins obtained from cells grown at pH 3.7 was twofold higher than that of cells grown at pH 6.0. Mitochondria isolated from both cell types readily oxidized succinate, malate or ethanol. The rates of oxygen uptake were 20-25% higher in mitochondria from cells grown at pH 3.7. Cyanide and antimycin A inhibited respiration with succinate up to 95% in both types of mitochondria. The participation of cytochrome c550 in mitochondrial electron transport from succinate to oxygen was shown by spectral measurements.

Searching for predominant soil bacteria: 16S rDNA cloning versus strain cultivation.

The predominant bacteria in Dutch grassland soils, as identified by direct DNA extraction, PCR amplification of 16S rDNA and subsequent cloning and sequencing, were compared to the most abundant culturable bacteria. The 16S rDNAs of the strains from a comprehensive cultivation campaign were compared to some of the predominant cloned sequences by temperature gradient gel electrophoresis (TGGE). Four ribotypes were selected that were found to be abundant in the clone library: two closely related Bacillus-like sequences, a representative from the Verrucomicrobiales cluster and an uncultured member of the Actinobacteria. Using a variety of cultivation approaches a total of 659 pure cultures were isolated. Initially, approximately 8% of all isolates matched any of these ribotypes by same migration speed of their 16S rDNA amplicons on TGGE. However, sequencing analysis of matching isolates indicated that their 16S rDNA sequences were clearly different from the cloned sequences representing the fingerprint bands. Comparing the cultivation approach and the molecular 16S rDNA analysis from the same soil sample, there was no correlation between the collection of cultured strains and the 16S rDNA clone library.

Phylogeny of the main bacterial 16S rRNA sequences in Drentse A grassland soils (The Netherlands).

The main bacteria in peaty, acid grassland soils in the Netherlands were investigated by ribosome isolation, temperature gradient gel electrophoresis, hybridization, cloning, and sequencing. Instead of using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active bacteria. After direct ribosome isolation from soil, a partial amplicon of bacterial 16S rRNA was generated by reverse transcription-PCR. The sequence-specific separation by temperature gradient gel electrophoresis yielded soil-specific fingerprints, which were compared to signals from a clone library of genes coding for 16S rRNA. Cloned 16S rDNA sequences matching with intense bands in the fingerprint were sequenced. The relationships of the sequences to those of cultured organisms of known phylogeny were determined. Most of the amplicons originated from organisms closely related to Bacillus species. Such sequences were also detected by direct dot blot hybridization on soil rRNA: a probe specific for Firmicutes with low G+C content counted for about 50% of all bacterial rRNA. The bacterial activity in Drentse A grassland soil could be estimated by direct dot blot hybridization and sequencing of clones; it was found that about 65% of all the bacterial ribosomes originated from Firmicutes. The most active bacteria apparently were Bacillus species, from which about half of the sequences derived. Other sequences similar to those of gram-positive bacteria were only remotely related to known Firmicutes with a high G+C content. Other sequences were related to Proteobacteria, mainly the alpha subclass.

Detection and quantification of microorganisms in anaerobic bioreactors.

The presence of sulfate in anaerobic reactors can trigger competitive and syntrophic interactions between various groups of microorganisms, such as sulfate reducers, methanogens and acetogens. In order to steer the reactor process in the direction of sulfidogenesis or methanogenesis, it is essential to get insight into the population dynamics of these groups of microorganisms upon changes in the reactor operating conditions. Several methods exist to characterize and quantify the microbial sludge composition. Combining classical microbiological and modern molecular-based sludge characterization methods has proven to be a powerful approach to study the microbial composition of the anaerobic sludge.