Polyphasic Analyses of Methanogenic Archaeal Communities in Agricultural Biogas Plants

Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescence in situ hybridization (FISH) was carried out to quantify the methanogenic Archaea in the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophic Methanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order, Methanoculleus was found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic family Methanosaetaceae was determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence of Methanosaetaceae in the biogas reactors and high concentrations of total ammonia (sum of NH₃ and NH₄⁺) was observed.During the last decade the production of biogas from organic materials and residues has increased continuously in order to reduce the greenhouse gas emission resulting from the use of fossil energy sources. The energy-bearing substance of biogas is methane, which is produced as an end product of microbial anaerobic degradation of organic substrates, such as energy crops like maize, grains, grasses, or beets. Research for optimization of biogas production from renewable materials was initially focused on the evaluation of substrate eligibility and on the development and optimization of technical systems. However, biogas formation primarily depends on the structure and activity of the microbial community (28).The key microorganisms in the biogas formation process are the methane-generating microorganisms (methanogens). The capacity for methanogenesis is limited to members of the domain Archaea and, within this domain, on the phylum Euryarchaeota. With respect to the main metabolic precursors used, methanogens are usually divided into two groups: the aceticlastic methanogens that strictly metabolize acetate and the hydrogenotrophic methanogens that use H₂ or formate as an electron donor and CO₂ as a carbon source for their metabolism. Besides these major groups, certain methanogens are also able to convert methyl groups, methylamines, or methanol to methane (23, 40). The substrates for the methanogens are provided by several physiological groups of bacteria which degrade organic matter, sometimes in close syntrophic interaction with the methanogens (1).Several studies on the microbial diversity present in lab-scale biogas reactors supplied with renewable raw material (7, 57) have been recently published. However, analyses under laboratory conditions do not necessarily reflect conditions in full-scale reactors (35). Therefore, further research on the methanogenic community in full-scale biogas reactors is crucial.Generally, studies regarding the microbial community structure in full-scale biogas reactors have focused on different systems for wastewater treatment or classical biogas plants based on manure digestion (32, 38, 43). In most systems, approximately 70% of the carbon fixed in methane was derived from acetate. Only minor amounts, up to approximately 30%, were deduced from CO₂ (1, 42). Together with the presence of huge assemblages of Methanosarcina sp., it was assumed by some authors that aceticlastic methanogenesis was the predominant pathway for methane formation. Moreover, as shown by other studies, the relative contribution of H₂/CO₂ versus acetate as metabolic precursors for methanogens can be quite different in other anaerobic environments (10, 33, 37). However, the methanogenic microfloras in full-scale biogas reactors supplied with energy crops as a primary or sole substrate have rarely been studied (35, 37, 45).The aim of this study was to gain insight into the diversity of methane-producing Archaea in six full-scale biogas plants supplied with renewable raw material and different types of liquid manure as substrates. Therefore, a polyphasic approach with three different culture-independent techniques (fluorescence in situ hybridization [FISH], quantitative PCR [Q-PCR], and 16S rRNA gene analysis) to analyze methanogen diversity was carried out to overcome the known limitations of each single approach (15, 46). To analyze potential effects of different process parameters on the methanogenic archaeal community, the reactor performances were correlated with the apparent archaeal diversity.     

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.     

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

The cellular signature of urinary immune cells in Lupus nephritis: new insights into potential biomarkers

IntroductionUrinary T cells represent a reliable noninvasive biomarker for proliferative Lupus nephritis (LN). Little is known about the presence of T cell subsets, B cells and macrophages in the urine although they may further improve the validity of urinary cellular biomarkers for LN.MethodsWe analyzed contemporaneous blood and urine samples of patients with active LN (n = 19), other Systemic Lupus Erythematosus (SLE) patients (n = 79) and urine samples of patients with diabetic nephropathy (DN; n = 14) and anti-neutrophil cytoplasmatic antibody (ANCA) associated vasculitis (AAV; n = 11) by flow cytometry.ResultsNumbers of urinary T cells, B cells and macrophages correlated with disease activity and were significantly higher in the active LN group. Urinary T cells showed excellent distinction of patients with active LN, CD8+ T cells (AUC of ROC = 1.000) and CD4+ T cells (AUC = 0.9969) alike. CD19+ B cells (AUC = 0.7823) and CD14+ macrophages (AUC = 0.9066), as well as the clinical standard proteinuria (AUC = 0.9201), failed to reach these high standards. Patients with DN or AAV also showed increased urinary cell counts, although the CD4/CD8-ratio was significantly lower in SLE compared to in DN (p = 0.0006). Urinary CD4+ T cells of active LN patients proved to be mainly of effector memory phenotype and expressed significantly more CD40L and ki67 than corresponding blood cells. Urinary Treg counts correlated with disease activity.ConclusionsDespite of detectable urinary cell counts for B cells and macrophages, T cells remain the best urinary cellular biomarker for LN. A low CD4/CD8-ratio seems to be characteristic for LN.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0600-y) contains supplementary material, which is available to authorized users.     

Monitoring Lactobacillus plantarum in grass silages with the aid of 16S rDNA-based quantitative real-time PCR assays

Ensiling plant material with the aid of lactic acid bacteria (LAB) is a common agricultural practice for conserving forages independently of the time point of harvest. Despite ensiling being a natural process, it can be improved by the treatment of the harvested forage with starter cultures before storage. Within this context, Lactobacillus plantarum (L. plantarum) is the most frequently used LAB in commercially available starter cultures. In order to enable the monitoring of the population dynamics of L. plantarum in silage, methods for species-specific detection based on the 16S ribosomal DNA (rDNA) sequence were developed by applying a quantitative real-time polymerase chain reaction (QRT-PCR) approach. The QRT-PCR assay was also applied to estimate the development of the L. plantarum population within experimental grass silages. In addition, a multiplex QRT-PCR assay was developed to estimate the amount of L. plantarum 16S rDNA in relation to total bacterial 16S rDNA. This multiplex QRT-PCR assay was applied to monitor the influence of different silage additives on the L. plantarum population.     

Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications

Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.     

Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain

A single episode of ethanol intoxication triggers widespread apoptotic neurodegeneration in the infant rat or mouse brain. The cell death process occurs over a 6-16 h period following ethanol administration, is accompanied by a robust display of caspase-3 enzyme activation, and meets ultrastructural criteria for apoptosis. Two apoptotic pathways (intrinsic and extrinsic) have been described, either of which may culminate in the activation of caspase-3. The intrinsic pathway is regulated by Bax and Bcl-XL and involves Bax-induced mitochondrial dysfunction and release of cytochrome c as antecedent events leading to caspase-3 activation. Activation of caspase-8 is a key event preceding caspase-3 activation in the extrinsic pathway. In the present study, following ethanol administration to infant mice, we found no change in activated caspase-8, which suggests that the extrinsic pathway is not involved in ethanol-induced apoptosis. We also found that ethanol triggers robust caspase-3 activation and apoptotic neurodegeneration in C57BL/6 wildtype mice, but induces neither phenomenon in homozygous Bax-deficient mice. Therefore, it appears that ethanol-induced neuroapoptosis is an intrinsic pathway-mediated phenomenon involving Bax-induced disruption of mitochondrial membranes and cytochrome c release as early events leading to caspase-3 activation.