Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate—one where methanogenesis was allowed and another in which it was completely inhibited with 2‐bromoethane sulfonate. We observed a three‐way syntrophy among ethanol fermenters, acetate‐oxidizing anode‐respiring bacteria (ARB), and a H₂ scavenger. When methanogenesis was allowed, H₂‐oxidizing methanogens were the H₂ scavengers, but when methanogenesis was inhibited, homo‐acetogens became a channel for electron flow from H₂ to current through acetate. We established the presence of homo‐acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo‐acetogens. Both methods documented the presence of the homo‐acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol‐fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H₂‐scavenging syntrophic partners that were either H₂‐oxidizing methanogens or homo‐acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron‐flow distribution and H₂‐scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69–78. © 2009 Wiley Periodicals, Inc.     

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 10⁶ or 10⁹ total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/10⁶-AODC) than in water samples (0.02 to 0.4 PRM/10⁶-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/10⁹-AODC) than from other sites (nondetectable to 0.77 PRM/10⁹-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.     

The stoichiometry of the two photosystems in higher plants revisited

The stoichiometry of Photosystem II (PSII) to Photosystem I (PSI) reaction centres in spinach leaf segments was determined by two methods, each capable of being applied to monitor the presence of both photosystems in a given sample. One method was based on a fast electrochromic (EC) signal, which in the millisecond time scale represents a change in the delocalized electric potential difference across the thylakoid membrane resulting from charge separation in both photosystems. This method was applied to leaf segments, thus avoiding any potential artefacts associated with the isolation of thylakoid membranes. Two variations of this method, suppressing PSII activity by prior photoinactivation (in spinach and poplar leaf segments) or suppressing PSI by photo-oxidation of P700 (the chlorophyll dimer in PSI) with background far-red light (in spinach, poplar and cucumber leaf segments), each gave the separate contribution of each photosystem to the fast EC signal; the PSII/PSI stoichiometry obtained by this method was in the range 1.5-1.9 for the three plant species, and 1.5-1.8 for spinach in particular. A second method, based on electron paramagnetic resonance (EPR), gave values in a comparable range of 1.7-2.1 for spinach. A third method, which consisted of separately determining the content of functional PSII in leaf segments by the oxygen yield per single turnover-flash and that of PSI by photo-oxidation of P700 in thylakoids isolated from the corresponding leaves, gave a PSII/PSI stoichiometry (1.5-1.7) that was consistent with the above values. It is concluded that the ratio of PSII to PSI reaction centres is considerably higher than unity in typical higher plants, in contrast to a surprisingly low PSII/PSI ratio of 0.88, determined by EPR, that was reported for spinach grown in a cabinet under far-red-deficient light in Sweden [Danielsson et al. (2004) Biochim. Biophys. Acta 1608: 53-61]. We suggest that the low PSII/PSI ratio in the Swedish spinach, grown in far-red-deficient light with a lower PSII content, is not due to greater accuracy of the EPR method of measurement, as suggested by the authors, but is rather due to the growth conditions.     

The tissue distribution of porcine 17β-estradiol dehydrogenase and its induction by progesterone

Porcine 17β-estradiol dehydrogenase (EDH) was recently purified and cloned. It catalyzes the NAD⁺-dependent oxidation of estradiol to estrone 360-fold more efficiently than the back reaction with NADPH. The 32 kDa EDH is cut from an 80 kDa primary translation product with a multidomain structure unknown for other hydroxysteroid dehydrogenases. The highest EDH activities and strongest immunoreactions are found in liver (hepatocytes) and kidney (proximal tubuli) followed by uterus (luminal and glandular epithelium), lung (bronchial epithelium). Progesterone treatment of ovariectomized gilts stimulates oxidative EDH activity in uterus, anterior pituitary, skeletal muscle (diaphragm) and kidney. Constitutive levels of EDH activity were seen in the adrenals, the lung and the liver.     

Molecular characterization of mouse 17β-hydroxysteroid dehydrogenase IV

17β-hydroxysteroid dehydrogenases (17β-HSD) catalyze the conversion of estrogens and androgens at the C17 position. The 17β-HSD type I, II, III and IV share less than 25% amino acid similarity. The human and porcine 17β-HSD IV reveal a three-domain structure unknown among other dehydrogenases. The N-terminal domains resemble the short chain alcohol dehydrogenase family while the central parts are related to the C-terminal parts of enzymes involved in peroxisomal β-oxidation of fatty acids and the C-terminal domains are similar to sterol carrier protein 2. We describe the cloning of the mouse 17β-HSD IV cDNA and the expression of its mRNA. A probe derived from the human 17β-HSD IV was used to isolate a 2.5 kb mouse cDNA encoding for a protein of 735 amino acids showing 85 and 81% similarity with human and porcine 17β-HSD IV, respectively. The calculated molecular mass of the mouse enzyme amounts to 79,524 Da. The mRNA for 17β-HSD IV is a single species of about 3 kb, present in a multitude of tissues and expressed at high levels in liver and kidney, and at low levels in brain and spleen. The cloning and molecular characterization of murine, human and porcine 17β-HSD IV adds to the complexity of steroid synthesis and metabolism. The multitude of enzymes acting at C17 might be necessary for a precise control of hormone levels.     

Host adaptive immunity alters gut microbiota

It has long been recognized that the mammalian gut microbiota has a role in the development and activation of the host immune system. Much less is known on how host immunity regulates the gut microbiota. Here we investigated the role of adaptive immunity on the mouse distal gut microbial composition by sequencing 16 S rRNA genes from microbiota of immunodeficient Rag1^−/− mice, versus wild-type mice, under the same housing environment. To detect possible interactions among immunological status, age and variability from anatomical sites, we analyzed samples from the cecum, colon, colonic mucus and feces before and after weaning. High-throughput sequencing showed that Firmicutes, Bacteroidetes and Verrucomicrobia dominated mouse gut bacterial communities. Rag1⁻ mice had a distinct microbiota that was phylogenetically different from wild-type mice. In particular, the bacterium Akkermansia muciniphila was highly enriched in Rag1^−/− mice compared with the wild type. This enrichment was suppressed when Rag1^−/− mice received bone marrows from wild-type mice. The microbial community diversity increased with age, albeit the magnitude depended on Rag1 status. In addition, Rag1^−/− mice had a higher gain in microbiota richness and evenness with increase in age compared with wild-type mice, possibly due to the lack of pressure from the adaptive immune system. Our results suggest that adaptive immunity has a pervasive role in regulating gut microbiota''s composition and diversity.     

Quantifying and monitoring functional photosystem II and the stoichiometry of the two photosystems in leaf segments: approaches and approximations

Given its unique function in light-induced water oxidation and its susceptibility to photoinactivation during photosynthesis, photosystem II (PS II) is often the focus of studies of photosynthetic structure and function, particularly in environmental stress conditions. Here we review four approaches for quantifying or monitoring PS II functionality or the stoichiometry of the two photosystems in leaf segments, scrutinizing the approximations in each approach. (1) Chlorophyll fluorescence parameters are convenient to derive, but the information-rich signal suffers from the localized nature of its detection in leaf tissue. (2) The gross O(2) yield per single-turnover flash in CO(2)-enriched air is a more direct measurement of the functional content, assuming that each functional PS II evolves one O(2) molecule after four flashes. However, the gross O(2) yield per single-turnover flash (multiplied by four) could over-estimate the content of functional PS II if mitochondrial respiration is lower in flash illumination than in darkness. (3) The cumulative delivery of electrons from PS II to P700(+) (oxidized primary donor in PS I) after a flash is added to steady background far-red light is a whole-tissue measurement, such that a single linear correlation with functional PS II applies to leaves of all plant species investigated so far. However, the magnitude obtained in a simple analysis (with the signal normalized to the maximum photo-oxidizable P700 signal), which should equal the ratio of PS II to PS I centers, was too small to match the independently-obtained photosystem stoichiometry. Further, an under-estimation of functional PS II content could occur if some electrons were intercepted before reaching PS I. (4) The electrochromic signal from leaf segments appears to reliably quantify the photosystem stoichiometry, either by progressively photoinactivating PS II or suppressing PS I via photo-oxidation of a known fraction of the P700 with steady far-red light. Together, these approaches have the potential for quantitatively probing PS II in vivo in leaf segments, with prospects for application of the latter two approaches in the field.     

Acclimation of leaves to low light produces large grana: the origin of the predominant attractive force at work

Photosynthetic membrane sacs (thylakoids) of plants form granal stacks interconnected by non-stacked thylakoids, thereby being able to fine-tune (i) photosynthesis, (ii) photoprotection and (iii) acclimation to the environment. Growth in low light leads to the formation of large grana, which sometimes contain as many as 160 thylakoids. The net surface charge of thylakoid membranes is negative, even in low-light-grown plants; so an attractive force is required to overcome the electrostatic repulsion. The theoretical van der Waals attraction is, however, at least 20-fold too small to play the role. We determined the enthalpy change, in the spontaneous stacking of previously unstacked thylakoids in the dark on addition of Mg²⁺, to be zero or marginally positive (endothermic). The Gibbs free-energy change for the spontaneous process is necessarily negative, a requirement that can be met only by an increase in entropy for an endothermic process. We conclude that the dominant attractive force in thylakoid stacking is entropy-driven. Several mechanisms for increasing entropy upon stacking of thylakoid membranes in the dark, particularly in low-light plants, are discussed. In the light, which drives the chloroplast far away from equilibrium, granal stacking accelerates non-cyclic photophosphorylation, possibly enhancing the rate at which entropy is produced.     

Relationship Between Photosystem 2 Electron Transport and Photosynthetic CO2 Assimilation Responses to Irradiance in Young Apple Tree Leaves

The responses to irradiance of photosynthetic CO₂ assimilation and photosystem 2 (PS2) electron transport were simultaneously studied by gas exchange and chlorophyll (Chl) fluorescence measurement in two-year-old apple tree leaves (Malus pumila Mill. cv. Tengmu No.1/Malus hupehensis Rehd). Net photosynthetic rate (P _N) was saturated at photosynthetic photon flux density (PPFD) 600-1 100 (mol m^-2 s^-1, while the PS2 non-cyclic electron transport (P-rate) showed a maximum at PPFD 800 mol m^-2 s^-1. With PPFD increasing, either leaf potential photosynthetic CO₂ assimilation activity (F_d/F_s) and PS2 maximal photochemical activity (F_v/F_m) decreased or the ratio of the inactive PS2 reaction centres (RC) [(F_i – F_o)/(F_m – F_o)] and the slow relaxing non-photochemical Chl fluorescence quenching (q_s) increased from PPFD 1 200 mol m^-2 s^-1, but cyclic electron transport around photosystem 1 (RFp), irradiance induced PS2 RC closure [(F_s – F_o)/F_m – F_o)], and the fast and medium relaxing non-photochemical Chl fluorescence quenching (q_f and q_m) increased remarkably from PPFD 900 (mol m^-2 s^-1. Hence leaf photosynthesis of young apple leaves saturated at PPFD 800 mol m^-2 s^-1 and photoinhibition occurred above PPFD 900 mol m^-2 s^-1. During the photoinhibition at different irradiances, young apple tree leaves could dissipate excess photons mainly by energy quenching and state transition mechanisms at PPFD 900-1 100 mol m^-2 s^-1, but photosynthetic apparatus damage was unavoidable from PPFD 1 200 mol m^-2 s^-1. We propose that Chl fluorescence parameter P-rate is superior to the gas exchange parameter P _N and the Chl fluorescence parameter F_v/F_m as a definition of saturation irradiance and photoinhibition of plant leaves.     

Alterations in the subcellular distribution of 17β-estradiol dehydrogenase in porcine endometrial cells over the course of the estrous cycle

The uteri of German landrace gilts slaughtered at different days of the cycle were processed for immunocytochemistry and biochemical analyses. Plasma was collected for hormone assays. The monoclonal antibody F1 against the structure-bound 17-estradiol dehydrogenase of porcine endometrial epithelium was applied to rehydrated paraffin sections either as a direct, peroxidase-linked probe or in combination with a fluorescing secondary antibody. The oxidation of estradiol was measured in homogenates of tissue powdered in liquid nitrogen. Immunoreactivity was restricted to endometrial epithelium. In the glandular epithelium, faint dots of fluorescence became visible at day 4, which apparently coalesced to spherical structures of 2–4 m diameter at the cell basis between days 11 through 17 before disappearing by day 18. A similar distribution was observed for the oxidation products of diaminobenzidine beginning with a faint uniform staining and followed by the appearance of intensely stained basal bodies persisting until day 17. Essentially the same time course was seen in the luminal epithelium but with a different distribution. Immunoreactive material amassed in the apical region of the cells, but the conspicuous aggregations were absent. Time course and intensities of the immunological responses are matched by the enzymatic activity measured in parallel. Both correlate with the plasma progesterone levels, suggesting an induction of the enzyme by the hormone. An involvement of the cytoskeleton in the sequence of subcellular distribution patterns is discussed.