Enhanced anaerobic digestion of cellulose by intermittent floc-dispersion at 55°C

Summary The thermophilic (55°C) bioconversion of cellulose (-floc) to volatile fatty acids ceased prior to completion at high cellulose concentrations. This was attributable to the formation of large aggregates of partially digested cellulose which resulted in mass transfer limitations. Mechanical re-dispersion of cellulose enhanced the acid production. The total acid concentration increased from 4.18 g/L volatile acids (control) to 6.92 g/L after two blendings.     

The energy balance in farm scale anaerobic digestion of crop residues at 11–37 °C

Crop residues can be used for biogas production in farm scale reactors. Use of a process temperature below mesophilic conditions reduces the need for heating as well as investment and operating costs, although it may also reduce the methane yield. In the present study the effect of temperature on net energy output was studied using sugar beet tops and straw as substrates for two pilot-scale reactors. Digestion was found to be stable down to 11 °C and optimal methane yield was obtained at 30 °C. The methane yield and process performance was studied at 15 °C and 30 °C as organic loading rates were increased. It was found that the highest net energy production would be achieved at 30 °C with a loading rate of 3.3 kg VS m⁻³ day⁻¹. Running a low-cost process at ambient temperatures would give a net energy output of 60% of that obtained at 30 °C.     

Structural Changes in Dengue Virus When Exposed to a Temperature of 37°C

Previous binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed.     

Pathways of methanol conversion in a thermophilic anaerobic (55 °C) sludge consortium

The pathway of methanol conversion by a thermophilic anaerobic consortium was elucidated by recording the fate of carbon in the presence and absence of bicarbonate and specific inhibitors. Results indicated that about 50% of methanol was directly converted to methane by the methylotrophic methanogens and 50% via the intermediates H₂/CO₂ and acetate. The deprivation of inorganic carbon species [(HCO₃^–+CO₂)] in a phosphate-buffered system reduced the rate of methanol conversion. This suggests that bicarbonate is required as an electron (H₂) sink and as a co-substrate for the efficient and complete removal of the chemical oxygen demand. Nuclear magnetic resonance spectroscopy was used to investigate the route of methanol conversion to acetate in bicarbonate-sufficient and bicarbonate-depleted environments. The proportions of [1,2-¹³C]acetate, [1-¹³C]acetate and [2-¹³C]acetate were determined. Methanol was preferentially incorporated into the methyl group of acetate, whereas HCO₃^– was the preferred source of the carboxyl group. A small amount of the added H¹³CO₃^– was reduced to form the methyl group of acetate and a small amount of the added ¹³CH₃OH was oxidised and found in the carboxyl group of acetate when ¹³CH₃OH was converted. The recovery of [¹³C]carboxyl groups in acetate from ¹³CH₃OH was enhanced in bicarbonate-deprived medium. The small amount of label incorporated in the carboxyl group of acetate when ¹³CH₃OH was converted in the presence of bromoethanesulfonic acid indicates that methanol can be oxidised to CO₂ prior to acetate formation. These results indicate that methanol is converted through a common pathway (acetyl-CoA), being on the one hand reduced to the methyl group of acetate and on the other hand oxidised to CO₂, with CO₂ being incorporated into the carboxyl group of acetate.     

The growth of yeasts at 37° C

Summary More than 2,300 strains of 70 species of yeasts have been tested on yeast autolysate agar at 37° C. Of these, all strains of 15 species grew at this elevated temperature while no strains of 13 species grew well. The remaining 42 species, represented by 2 or more strains each, included strains both capable and incapable of growth at 37° C. It is suggested that such species include two groups of strains, one capable of adaptation to growth conditions at elevated temperatures. In sewage-polluted waters such strains may be indicative of fecal pollution.U.S. Department of Health, Education, and Welfare, Bureau of State Services, Public Health Service.     

Evaluation of the hydrolytic–acidogenic step of a two-stage mesophilic anaerobic digestion process of sunflower oil cake

The influence of the hydraulic retention time (HRT) and organic loading rate (OLR) on the performance of the hydrolytic–acidogenic step of a two-stage anaerobic digestion process of sunflower oil cake (SuOC) were assessed. The experiments were performed in laboratory-scale completely stirred tank reactors at mesophilic (35 °C) temperature. Six OLR (ranging from 4 to 9 g VS L⁻¹ d⁻¹) for four HRTs (8, 10, 12 and 15 days) were tested to check the effect of each operational variable. Based on the results obtained, it can be concluded that the hydrolysis yields obtained for all HRTs and OLRs assayed were in the range of 20.5–30.1%. In addition, the acidification degree of the substrate was mainly influenced by the OLR but not by the HRTs, the highest value (83.8%) being achieved for an HRT of 10 days and an OLR of 6 g VS L⁻¹ d⁻¹.     

Effects of Mild Cold Shock (25°C) Followed by Warming Up at 37°C on the Cellular Stress Response

Temperature variations in cells, tissues and organs may occur in a number of circumstances. We report here that reducing temperature of cells in culture to 25°C for 5 days followed by a rewarming to 37°C affects cell biology and induces a cellular stress response. Cell proliferation was almost arrested during mild hypothermia and not restored upon returning to 37°C. The expression of cold shock genes, CIRBP and RBM3, was increased at 25°C and returned to basal level upon rewarming while that of heat shock protein HSP70 was inversely regulated. An activation of pro-apoptotic pathways was evidenced by FACS analysis and increased Bax/Bcl2 and BclX_S/L ratios. Concomitant increased expression of the autophagosome-associated protein LC3II and AKT phosphorylation suggested a simultaneous activation of autophagy and pro-survival pathways. However, a large proportion of cells were dying 24 hours after rewarming. The occurrence of DNA damage was evidenced by the increased phosphorylation of p53 and H2AX, a hallmark of DNA breaks. The latter process, as well as apoptosis, was strongly reduced by the radical oxygen species (ROS) scavenger, N-acetylcysteine, indicating a causal relationship between ROS, DNA damage and cell death during mild cold shock and rewarming. These data bring new insights into the potential deleterious effects of mild hypothermia and rewarming used in various research and therapeutical fields.     

Walking performance and aerobic and anaerobic metabolism of Carcinus maenas (L.) in sea water at 15°C

Walking performance of the shore crab Carcinus maenas (L.) in sea water at 15 °C was assessed. In large crabs there was an inverse relationship between fatigue time and speed; crabs ran for $\text{\$}\text{?}$10 min at 3.2 m·min^?1 and for only 2 min at 14 m·min^?1. There were linear relationships between oxygen consumption and walking speeds for small and large animals walking at up to 4 m·min^?1 Estimates of maximum oxygen consumption were proportional to W^0.13 whereas inactive consumption is proportional to W^0.44 this resulted in aerobic scope (i.e. the difference between inactive and maximal rates of oxygen consumption) remaining almost constant across a weight range of animals whereas the aerobic expansibility (maximal rates/inactive rates) declined from 7- to 4-fold with increasing size. After a 12-h period without handling (settled animals) the animals could immediately become active and reach maximal rates of oxygen consumption similar to those of animals handled 1 h before the experiment. The aerobic expansibility of these settled animals could range from 21 to 8 times their inactive rates of oxygen consumption in small and large animals respectively. After 10 min of exercise oxygen consumption and whole body lactate levels returned to pre-exercise values within 5 to 25 min. The net oxygen debts range from 16 to 64% of the net oxygen consumption increase during exercise in small and large animals respectively.Calculations of the energy gained from lactate accumulation indicated that the net aerobic energy production during walking was supplemented from 4 to 71 % by anaerobic metabolism in small and large animals respectively. With increasing animal size the decline in aerobic expansibility was offset by an increased capacity for lactate production so that the overall maximum energy production during sustained activity remained almost constant at around seven times the inactive rate. The cost of transport (the net increase in oxygen consumption per g per m) falls with increased walking speed and increased animal size.     

Thermodynamically specific gating kinetics of cardiac mammalian K (ATP) + channels in a physiological environment near 37°C

Elementary K⁺ currents through isolated ATP-sensitive K⁺ channels from neonatal rat cardiocytes were recorded to study their temperature dependence between 9°C and 39°C. Elementary current size and, thus, K⁺ permeation through the open pore varied monotonically with temperature with a Q₁₀ of 1.25 corresponding to a low activation energy of 3.9 kcal/mol. Open-state kinetics showed a complicated temperature dependence with Q₁₀ values of up to 2.94. Arrhenius anomalies of _open(1) and _open(2) indicate the occurrence of thermallyinduced perturbations with a dominating influence on channel portions that are involved in gating but are obviously ineffective in altering pore-forming segments. At 39°C, open-state exit reactions were associated with the highest activation energy (O₂ exit reaction: 12.1 kcal/ mol) and the largest amount of entropy. A transition from 19°C to 9°C elucidated a paradoxical kinetic response, shortening of both O-states, irrespective of the absence or presence of cAMP-dependent phosphorylation. Another member of the K⁺ channel family and also a constituent of neonatal rat cardiocyte membranes, 66 pS outwardly-rectifying channels, was found to react predictably since _open increased on cooling. Obviously, cardiac K _(ATP) ⁺ channels do not share this exceptional kinetic responsiveness to a temperature transition from 19°C to 9°C with other K⁺ channels and have a unique sensitivity to thermally-induced perturbations.     

Lipid Formation by Aqueous Fischer-Tropsch-Type Synthesis over a Temperature Range of 100 to 400 °C

The formation of lipid compounds during anaqueous Fischer-Tropsch-type reaction was studied withsolutions of oxalic acid as the carbon and hydrogensource. The reactions were conducted in stainlesssteel vessels by heating the oxalic acid solution atdiscrete temperatures from 100 to 400 °C, atintervals of 50 °C for two days each. Themaximum lipid yield, especially for oxygenatedcompounds, is in the window of 150–250 °C. At atemperature of 100 °C only a trace amount oflipids was detected. At temperatures above150 °C the lipid components ranged from C₁₂to >C₃₃ and included n-alkanols, n-alkanoic acids, n-alkyl formates, n-alkanals, n-alkanones, n-alkanes, andn-alkenes, all with essentially no carbon numberpreference. The n-alkanes increased inconcentration over the oxygenated compounds attemperatures of 200 °C and above, with a slightreduction in their carbon number ranges due tocracking. It was also noted that the n-alkanoicacids increased while n-alkanols decreased withincreasing temperature above 200 °C. Attemperatures above 300 °C synthesis competeswith cracking and reforming reactions. At 400 °Csignificant cracking was observed and polynucleararomatic hydrocarbons and their alkylated homologswere detected. The results of this work suggest thatthe formation of lipid compounds by aqueous FTTreactions proceeds by insertion of a CO group at theterminal end of a carboxylic acid functionality toform n-oxoalkanoic acids, followed by reductionto n-alkanoic acids, to n-alkanals, thento n-alkanols. The n-alkenes areintermediate homologs for n-alkan-2-ones andn-alkanes. This proposed mechanism for aqueousFTT synthesis differs from the surface-catalyzedstepwise FT process (i.e., gaseous) of polymerization of methylene reported in the literature.     

Crotonobetaine reductase fromEscherichia coli — a new inducible enzyme of anaerobic metabolization of L(-)-carnitine

Crotonobetaine reductase fromEscherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, -butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, -butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40–45 °C). The K _m value for crotonobetaine was determined to be 1.1×10^–2M. -Butyrobetaine,^D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For -butyrobetaine (K _i =3×10^–5M) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.     

The growth of dermatophytes at 4° C and 37° C; The relation of this character to others

Summary The ability of the generaEpidermophyton, Microsporon andTrichopyton to grow on some media at 4° C and 37° C was studied. It has been shown that specific differences exist among these fungi in the capability or rapidity of the growth at extreme temperatures.There is high positive correlation among perfect state production, isolation from the soil and growth at 4° C (group of characters A) and between pathogenicity and growth at 37° C (group of characters B). Between the groups A and B of characters exists a slighter negative correlation. Some prognosis about the five characters by certain species of dermatophytes may be given.     

Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.     

Adaptation of Methanogenic Communities to the Cofermentation of Cattle Excreta and Olive Mill Wastes at 37°C and 55°C

The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter⁻¹) and ammonia N concentrations (1.3 g liter⁻¹). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH₄ production (r_Pearson = 0.94; P = 0.02). At 55°C, the rise in temperature and H₂ partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH₄ production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H₂ scavengers while yielding the highest CH₄ production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.Turning residues into energy is a societal and scientific priority due to climate change, fossil fuel exhaustion, and waste accumulation. In 2006, in Europe (EU27), less than 3% of electricity production came from biomass and wastes (11). Biogas plants, which anaerobically treat organic wastes to produce energy, are increasingly promoted in Europe, but their distribution is highly biased (35). While thousands of full- and farm-scale biogas plants are spread over central and northern Europe, anaerobic digestion technology in Mediterranean countries—Portugal, Spain, Italy, Greece, and Turkey—is in its early stages (35). These nations and other circum-Mediterranean countries lead in the production of olive oil and thus in olive mill wastes and wastewaters, which have a huge biogas production potential due to their lipid composition (1). Spain alone generates one-third of the world''s oil production and millions of tons of two-phase olive mill wastes (TPOMW) per year. TPOMW are mostly burned or composted (28), hence releasing methane into the atmosphere. This compels a change in strategy: methane production from TPOMW should be optimized in engineered environments and transformed into energy.TPOMW is a humid residue containing the olive pulp and stone. Its anaerobic digestibility is hampered by its low pH, low ammonia N, and high content in antimicrobial substances (1). However, it has been successfully fermented under laboratory conditions by supplementing it with nutrients and increasing the reactor organic loading rate stepwise (2) or by codigesting it with residues with a high buffering capacity, e.g., cattle excreta (17). These approaches seem to facilitate the adaptation of the methane-producing anaerobic community to the environmental conditions that TPOMW impose.Methanogenic archaea—microbes clustered within five orders of the Euryarchaeota—constitute the last step in the trophic chain of decomposers degrading organic matter in oxygen-free environments (36). Methanogenesis is often the rate-limiting step of anaerobic digestion of organic wastes (3) due to the fast duplication times of bacteria, which generate all substrates for the slow-growing methane-producing archaea. It is also the most sensitive step in processing imbalances (4), likely due to the lack of functional redundancy among methanogens (8). High concentrations of volatile fatty acids, salts, ammonia, and heavy metals can be inhibitory for methanogens (5, 22) and are the most common reasons for reactor failure (3). Our objective was to understand the adaptation of methanogenic communities to TPOMW. We investigated methanogenic diversity and abundance in pilot digesters fed with cattle excreta and after changing their feed to TPOMW or TPOMW plus excreta. We expected that mixing both residues would allow a faster adaptation and more efficient performance of the methanogenic communities in digesting TPOMW. The cofermentation was evaluated at 37°C and 55°C. During an acclimatization period of 28 days, we screened the methanogenic diversity using an in-house-devised phylogenetic microarray, the AnaeroChip (13), and quantified dominant genera by real-time quantitative PCR (qPCR). We have taken primers from the literature, and we present four new sets of genus-specific primers and SYBR green I-optimized assays for quantifying methanogens in anaerobic environments.     

Simultaneous decrease in ammonia and hydrogen sulfide inhibition during the thermophilic anaerobic digestion of protein-rich stillage by biogas recirculation and air supply at 60 °C

We had proposed a novel method to reduce ammonia inhibition during thermophilic anaerobic digestion via recirculation of water-washed biogas into the headspace (R1 system) or liquid phase (R2 system) of reactors. The feasibility of reducing the ratio of recirculated biogas to biogas produced (called the biogas recirculation ratio) was investigated in the present study. Thermophilic anaerobic digestion at 53 °C and 60 °C with a biogas recirculation ratio of 150 facilitated stable digestion performance and biogas production at a higher organic loading rate of 7 g/L/d in the R1 system, while the ammonia removal efficiency increased 1.23-fold when the temperature increased from 53 °C to 60 °C. At 60 °C, the biogas recirculation ratios in the R1 and R2 systems decreased to 50 and 10, and the ammonia absorption rates were 6.1 and 8.3 mmol/L/d, respectively, without decreasing the anaerobic digestion performance. The ammonia absorption rate of 8.3 mmol/L/d in the R2 system was higher than the rate of 7.8 mmol/L/d at the biogas recirculation ratio of 150 in the R1 system. The hydrogen sulfide content in the biogas was reduced to less than 50 ppm by supplying air at 3% of the amount of biogas produced into the reactor.     

Effect of carbon monoxide,hydrogen and sulfate on thermophilic (55°C) hydrogenogenic carbon monoxide conversion in two anaerobic bioreactor sludges

The conversion routes of carbon monoxide (CO) at 55°C by full-scale grown anaerobic sludges treating paper mill and distillery wastewater were elucidated. Inhibition experiments with 2-bromoethanesulfonate (BES) and vancomycin showed that CO conversion was performed by a hydrogenogenic population and that its products, i.e. hydrogen and CO₂, were subsequently used by methanogens, homo-acetogens or sulfate reducers depending on the sludge source and inhibitors supplied. Direct methanogenic CO conversion occurred only at low CO concentrations [partial pressure of CO (P _CO) <0.5 bar (1 bar=10⁵ Pa)] with the paper mill sludge. The presence of hydrogen decreased the CO conversion rates, but did not prevent the depletion of CO to undetectable levels (<400 ppm). Both sludges showed interesting potential for hydrogen production from CO, especially since after 30 min exposure to 95°C, the production of CH₄ at 55°C was negligible. The paper mill sludge was capable of sulfate reduction with hydrogen, tolerating and using high CO concentrations (P _CO>1.6 bar), indicating that CO-rich synthesis gas can be used efficiently as an electron donor for biological sulfate reduction.