Freshwater bacterioplankton richness in oligotrophic lakes depends on nutrient availability rather than on species–area relationships

A central goal in ecology is to grasp the mechanisms that underlie and maintain biodiversity and patterns in its spatial distribution can provide clues about those mechanisms. Here, we investigated what might determine the bacterioplankton richness (BR) in lakes by means of 454 pyrosequencing of the 16S rRNA gene. We further provide a BR estimate based upon a sampling depth and accuracy, which, to our knowledge, are unsurpassed for freshwater bacterioplankton communities. Our examination of 22 669 sequences per lake showed that freshwater BR in fourteen nutrient-poor lakes was positively influenced by nutrient availability. Our study is, thus, consistent with the finding that the supply of available nutrients is a major driver of species richness; a pattern that may well be universally valid to the world of both micro- and macro-organisms. We, furthermore, observed that BR increased with elevated landscape position, most likely as a consequence of differences in nutrient availability. Finally, BR decreased with increasing lake and catchment area that is negative species–area relationships (SARs) were recorded; a finding that re-opens the debate about whether positive SARs can indeed be found in the microbial world and whether positive SARs can in fact be pronounced as one of the few ‘laws'' in ecology.     

Digestibility of whole-crop barley and oat silages in dairy heifers

This study evaluated the digestibility of whole-crop cereal silage (WCCS) made from oats and six-rowed barley harvested at the heading, early milk and early dough stages, and two-rowed barley harvested at the early milk and early dough stages of maturity. The eight WCCSs were fed to 32 Swedish Red heifers in a changeover design over three periods of 28 days each. The heifers were first fed ad libitum for 17 days and then at 0.95 of ad libitum for 11 days of each period. During the last 5 days all faeces and orts were collected to determine the digestibility of the silages. Only the maturity stage effect was significant for the WCCS organic matter (OM) digestibility and the average OM digestibility was higher at the heading stage (698 g/kg) than at early milk (647 g/kg) and early dough (652 g/kg) stages of maturity. For neutral detergent fibre (NDF) digestibility the crop × maturity stage effect was significant. The NDF digestibility decreased from the heading to the early milk stage for both six-rowed barley (746 to 607 g/kg) and oats (698 to 596 g/kg). There was no further significant decrease in NDF digestibility for six-rowed barley at the early dough stage (577 g/kg), but for two-rowed barley it decreased from the early milk (682 g/kg) to the early dough (573 g/kg) stage, and also for oats the NDF digestibility was lowest at the early dough stage (507 g/kg). The decrease in NDF digestibility during maturation was to a large extent compensated by an increase in starch concentration in the crops. The starch digestibility was lower for six-rowed barley at early dough stage (948 g/kg) than at early milk stage (977 g/kg), and was also lower compared with oats (979 g/kg) at early dough stage. The average crude protein (CP) digestibility was higher at the heading (646 g/kg) and the early milk (642 g/kg) stages than at the early dough stage (599 g/kg), and oats had higher average CP digestibility (650 g/kg) than six-rowed (613 g/kg) and two-rowed (624 g/kg) barley. Delaying the harvest of WCCS from the heading to the early milk and dough stage of maturity will decrease the OM digestibility; as a result there is a decreased NDF digestibility.     

Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas)

Even if many Vibrio spp. are endemic to coastal waters, their distribution in northern temperate and boreal waters is poorly studied. To identify environmental factors regulating Vibrio populations in a salinity gradient along the Swedish coastline, we combined Vibrio-specific quantitative competitive PCR with denaturant gradient gel electrophoresis-based genotyping. The total Vibrio abundance ranged from 4 x 10(3) to 9.6 x 10(4) cells liter(-1), with the highest abundances in the more saline waters of the Skagerrak Sea. Several Vibrio populations were present throughout the salinity gradient, with abundances of single populations ranging from 5 x 10(2) to 7 x 10(4) cells liter(-1). Clear differences were observed along the salinity gradient, where three populations dominated the more saline waters of the Skagerrak Sea and two populations containing mainly representatives of V. anguillarum and V. aestuarianus genotypes were abundant in the brackish waters of the Baltic Sea. Our results suggest that this apparent niche separation within the genus Vibrio may also be influenced by alternate factors such as nutrient levels and high abundances of dinoflagellates. A V. cholerae/V. mimicus population was detected in more than 50% of the samples, with abundances exceeding 10(3) cells liter(-1), even in the cold (annual average water temperature of around 5 degrees C) and low-salinity (2 to 4 per thousand) samples from the Bothnian Bay (latitude, 65 degrees N). The unsuspected and widespread occurrence of this population in temperate and boreal coastal waters suggests that potential Vibrio pathogens may also be endemic to cold and brackish waters and hence may represent a previously overlooked health hazard.     

Evaluation of 23S rRNA PCR Primers for Use in Phylogenetic Studies of Bacterial Diversity

The availability of a diverse set of 23S rRNA gene sequences enabled evaluation of the specificity of 39 previously published and 4 newly designed primers specific for bacteria. An extensive clone library constructed using an optimized primer pair resulted in similar gene richness but slightly differing coverage of some phylogenetic groups, compared to a 16S rRNA gene library from the same environmental sample.     

Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform

As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner – with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition.     

Novel primers for 16S rRNA-based archaeal community analyses in environmental samples

Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (< 1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.     

High Ratio of Bacteriochlorophyll Biosynthesis Genes to Chlorophyll Biosynthesis Genes in Bacteria of Humic Lakes

Recent studies highlight the diversity and significance of marine phototrophic microorganisms such as picocyanobacteria, phototrophic picoeukaryotes, and bacteriochlorophyll- and rhodopsin-holding phototrophic bacteria. To assess if freshwater ecosystems also harbor similar phototroph diversity, genes involved in the biosynthesis of bacteriochlorophyll and chlorophyll were targeted to explore oxygenic and aerobic anoxygenic phototroph composition in a wide range of lakes. Partial dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide oxidoreductase (COR) genes in bacteria of seven lakes with contrasting trophic statuses were PCR amplified, cloned, and sequenced. Out of 61 sequences encoding the L subunit of DPOR (L-DPOR), 22 clustered with aerobic anoxygenic photosynthetic bacteria, whereas 39 L-DPOR sequences related to oxygenic phototrophs, like cyanobacteria, were observed. Phylogenetic analysis revealed clear separation of these freshwater L-DPOR genes as well as 11 COR gene sequences from their marine counterparts. Terminal restriction fragment length analysis of L-DPOR genes was used to characterize oxygenic aerobic and anoxygenic photosynthesizing populations in 20 lakes differing in physical and chemical characteristics. Significant differences in L-DPOR community composition were observed between dystrophic lakes and all other systems, where a higher proportion of genes affiliated with aerobic anoxygenic photosynthetic bacteria was observed than in other systems. Our results reveal a significant diversity of phototrophic microorganisms in lakes and suggest niche partitioning of oxygenic and aerobic anoxygenic phototrophs in these systems in response to trophic status and coupled differences in light regime.Recent studies have discovered novel phototrophic organisms and pointed us to their diversity in the oceans (1, 2, 3, 27, 39, 47, 50). Microorganisms such as picocyanobacteria, picoeukaryotes, and bacteriochlorophyll- and rhodopsin-containing bacteria use diverse photopigments to photosynthesize. These organisms represent a significant fraction of marine microbial communities and are likely to be ecologically and biogeochemically significant (1, 2, 13, 27, 28, 35, 39, 47). Several molecular studies based on genes of the puf operon, coding for the bacteriochlorophyll subunits, have shown that Roseobacter and Roseobacter-like bacteria constitute a significant portion of the aerobic anoxygenic photosynthetic bacteria (AAnPB) in marine waters (37). Microscopic counts have revealed that AAnPB contribute 1 to 16% to the total marine bacterioplankton in the euphotic zone and that there are regional and temporal differences in their abundances (see, for example, references 28, 41, and 50). Still, the global significance of this functional group and the role of, for example, AAnPB in the oceanic flow of energy and carbon are controversial (13, 20, 27, 28, 41, 48). Most AAnPB isolated so far have been described as photoorganoheterotrophs that rely primarily on organic substrates for growth but can derive a significant portion of their energy requirements from solar radiation (references 13 and 26 and references therein).AAnPB have been isolated from various freshwater habitats, ranging from cyanobacterial mats (49) to the pelagic zone of oligotrophic lakes (19, 38), but there are so far no studies of freshwater AAnPB diversity and community composition based on culture-independent techniques. A recent survey revealed some first patterns in the distribution of bacteriochlorophyll a-containing cells as well as the concentrations of the pigments in lakes ranging from oligotrophic to eutrophic. Infrared epifluorescence microscopy, fluorescence emission spectroscopy, and high-performance liquid chromatography were used to demonstrate that AAnPB may constitute up to 80% of total bacterial biomass in some low-productive lakes, implying that they are an important component of many lake ecosystems (33). In addition, genes encoding proteins for light harvesting (bacteriochlorophyll pufL and pufM gene clusters) have been identified in fosmid libraries from bacteria of the Delaware River (48) and in a functional gene survey of an Antarctic lake (24).In the present study, we used a specific primer set that amplifies the L subunit of the dark-operative protochlorophyllide oxidoreductase (L-DPOR) and its homologs (nitrogenase and chlorophyllide oxidoreductase [COR]). The dark-operative protochlorophyllide oxidoreductase (DPOR) is encoded by three genes (chlN-bchN, chlB-bchB, and chlL-bchL) and catalyzes the reductive formation of chlorophyllide from protochlorophyllide during biosynthesis of chlorophylls (chl) and bacteriochlorophylls (bch) in the dark (see reference 7 for more detail). Analysis of the deduced amino acid sequences indicated the presence of significant sequence dissimilarity in DPOR between oxygenic and anoxygenic photosynthetic organisms (5, 8, 16, 17, 18). Molecular studies have shown that AAnPB contain only DPOR (15) but that cyanobacteria, algae, and gymnosperms (nonflowering plants) contain both DPOR and a light-dependent protochlorophyllide oxidoreductase (POR) which carries one of the only two known enzymes other than photochemical reaction centers with light-driven catalysis (7).Despite the lack of POR, AAnPB are able to modify their chlorophyllide so that their absorption spectrum is broadened to span from <350 to <1,050 nm (usually 365 to 770 nm) in the UV and near-infrared ranges. This spectral characteristic allows AAnPB to utilize light at a wavelength other than that utilized by chlorophyll-containing cyanobacteria and algae. The first step in a series of modifications is performed by COR, an enzyme that is found in anoxygenic phototrophic bacteria and that transforms chlorophyllide to bacteriochlorophyll (7).In the present study, we assessed the diversity of L-DPOR, COR, and the coamplified homolog nifH genes in bacteria of seven different lakes by using PCR-based clone libraries parallel to a molecular fingerprinting technique to study L-DPOR gene composition in a larger data set (comprising 20 Swedish lakes). By using L-DPOR genes as our target, we simultaneously assessed the compositions of both AAnPB and oxygenic phototrophs in freshwater ecosystems and their distributions along trophic gradients.     

Carbon fluxes of xylose-consuming <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains are affected differently by NADH and NADPH usage in HMF reduction

Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD⁺-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD⁺ deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.