Production of volatile fatty acids by fermentation of waste activated sludge pre-treated in full-scale thermal hydrolysis plants

This work focuses on fermentation of pre-treated waste activated sludge (WAS) to generate volatile fatty acids (VFAs). Pre-treatment by high-pressure thermal hydrolysis (HPTH) was shown to aid WAS fermentation. Compared to fermentation of raw WAS, pre-treatment enabled a 2-5x increase in VFA yield (gVFA(COD)gTCOD(-1)) and 4-6x increase in VFA production rate (gVFA(COD) L(-1) d(-1)). Three sludges, pre-treated in full-scale HPTH plants, were fermented. One was from a plant processing a mix of primary sludge and WAS and the other two from plants processing solely WAS. The HPTH plants solubilised suspended matter, evidenced by a 20-30% decrease in suspended solids and an increase of soluble COD : total COD from 0.04 to 0.4. Fermentation of the three sludges yielded similar VFA concentrations (15-20gVFA(COD) L(-1)). The yields were largely independent of retention time (1 d-6 d) and temperature (42°C, 55°C). Also, the product spectrum depended mostly on the composition of the sludge rather than on operating conditions.     

Longterm persistence of proteolytic activities in frass of Blattella germanica increases its allergenic potential

Chromogenic microplate assays in 96 wells were used to determine the stability of enzyme activity in frass of Blattella germanica (Blattodea: Blattellidae). Frass samples were exposed to controlled conditions [temperature 15-35 °C and/or 53-100% relative humidity (RH)] and to household conditions (apartment). Exposure times were 0 (control), 90, 183 and 276 days. Starch digestion and cellulolytic activities decreased during exposure. Non-specific proteolytic activities were affected by changes in selective proteolytic activities. Activities towards AAPpNA and SA(3) pNA strongly increased at 100% RH, indicating the possible influence of microorganisms growing on frass. Activities towards BApNA and ArgpNA decreased with increasing decomposition time, whereas activity towards ZRRpNA was not influenced by exposure time. The largest decrease in activities towards ArgpNA and BApNA occurred at temperatures of 15 °C, 30 °C and 35 °C and at 100% RH. Activities towards BApNA and ZRRpNA were very stable under different temperature and RH conditions; this was confirmed by findings showing that these activities were stable in the experimental apartment. In comparison with the control, activities towards ZRRpNA and BApNA after 276 days decreased by 1% and 19%, respectively. The longterm persistence of proteolytic activities in cockroach frass increases their allergenic hazard potential.     

Effect of digestion temperature and pH on treatment efficiency and evolution of volatile fatty acids during thermophilic aerobic digestion of model high strength agricultural waste

Thermophilic aerobic digestion (TAD) of a model agricultural waste, potato peel slurry, at soluble chemical oxygen demand (COD) load equivalent to approximately 8.0 gl(-1), was carried out under batch conditions at 0.5 vvm aeration rate. Digestions were carried out at temperatures of 45, 50, 55, 60 and 65 degrees C (or left unregulated) without pH control to study the effect of digestion temperatures on TAD. The effects of digestion pH on the process were studied at pH 6.0, 7.0, 8.0, 9.0 and 9.5 (and in unregulated control) all at 55 degrees C. Except for digestion at 65 degrees C, which was inoculated extraneously using culture of Bacillus strearothermophilus all reactions were carried out using the populations indigenous to the waste. During digestion at different temperatures, the removal of soluble COD increased with temperature to reach a peak at 60 degrees C before declining slightly, removal of soluble solid (SS) followed similar pattern and reached peak at 65 degrees C being the highest temperature studied, while the degradation of TSS and TS (TSS + TS) decreased with an increase in temperature. Digestion at pH 7.0 was more efficient than at other pH values. Acetate was the predominant volatile fatty acid (VFA) in all the reactions and accounted for up to 90% of the total. Digestion at 60 degrees C led to the greatest accumulation of acetate, and this coincided with the period of highest oxygen uptake, and rapid consumption of soluble carbohydrate. Iso-valerate was also produced at all pH values. Digestion at 55 degrees C and also at pH 7.0 led to rapid and efficient processes with least accumulation of VFA and should be of interest in full-scale processes whenever it is practicable to regulate the digestion pH and temperature. The result of digestion at unregulated pH indicates that gradual adaptation may be used to achieve efficient treatment at elevated pH values. This would be of interest in full-scale processes where it is not practicable to tightly regulate digestion pH, and where the waste is produced at a pH value much higher than neutral.     

Stability of recombinant bovine interferon-γ antiviral activity in the absence of stabilizing additives

The stability of recombinant bovine interferon-γ (rbIFN-γ) produced by a baculovirus expression system was investigated under different storage conditions: freezing-thawing and storage for 30 days at temperatures of -80, 4, 25, and 37°C. Antiviral activity was not significantly decreased by freeze-thawing at least five times. Furthermore, although not statistically different, antiviral activity gradually decreased as temperature increased. These findings suggest that rbIFN-γ possesses high thermal and freeze-thaw stability.     

Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass

The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.     

Lipase-producing microorganisms from a Kenyan alkaline soda lake

Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55 degrees C. Most of them showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.     

Biological sludge reduction and enhanced nutrient removal in a pilot-scale system with 2-step sludge alkaline fermentation and A2O process

To enhance nutrient removal performance and reduce disposal amount of waste activated sludge (WAS), a pilot-scale continuous system consisting of a 2-step sludge alkaline fermentation process and an A²O reactor was proposed. The feasibility of WAS reducing and resourcing by alkaline fermentation was investigated. Volatile fatty acids (VFA) yield was higher under alkaline condition than that under acidic condition. Through 2-step alkaline fermentation, substantial VFA was accumulated, and then elutriated out continuously from an up-flow column by domestic wastewater. The results showed that 38.2% of sludge was hydrolyzed, 19.7% was finally acidified into VFA, and as high as 42.1% of WAS was reduced. Moreover, after introducing the fermentation liquids with higher proportion of acetic acid and propionic acid into the A²O reactor, the total nitrogen and phosphorus removal efficiencies reached to 80.1% and 90.0%, respectively. Sludge reduction and enhanced nutrient removal could be achieved simultaneously in the proposed system.     

Organic-matter degradative potential of Halomonas glaciei isolated from frazil ice in the Ross Sea (Antarctica)

Halomonas glaciei isolated from frazil ice in the Ross Sea (Antarctica) during austral summer 2003 was phenotypically characterized and its capability of degrading organic matter was tested. We evaluated specific bacterial growth rates (mu) to understand at which temperatures bacterial growth shows a linear and direct relationship with the available substrate (4-22 degrees C) and afterwards we tested H. glaciei growth curves and degradative potential at 0, 10 and 37 degrees C using two different media (one enriched and one depleted in PO(4)). The strain grew exponentially only at 10 degrees C. The fastest hydrolysis rates were expressed by enzymes aimed at polysaccharide degradation (alpha-d-glucosidase, beta-d-glucosidase and beta-d-galactosidase) while alkaline phosphatase and aminopeptidase activities were rather low. Our data suggest a preferential demand for carbon derived from carbohydrates rather than from proteins: ectoenzyme activities transformed into carbon mobilization from organic polymers, showed that the total carbon potentially released from polysaccharides can be almost one order of magnitude higher than the protein carbon mobilization. Principal component analysis of the enzyme affinity separated the six experimental conditions, highlighting how different physical (temperature) and chemical (PO(4) enrichment or depletion) features actively lead to a differentiation in the efficiency of the ectoenzymes produced, resulting in preferential degradation of diverse kinds of organic substrates.     

Long-term effect of inoculum pretreatment on fermentative hydrogen production by repeated batch cultivations: homoacetogenesis and methanogenesis as competitors to hydrogen production

Long-term effects of inoculum pretreatments (heat, acid, loading-shock) on hydrogen production from glucose under different temperatures (37 °C, 55 °C) and initial pH (7 and 5.5) were studied by repeated batch cultivations. Results obtained showed that it was necessary to investigate the long-term effect of inoculum pretreatment on hydrogen production since pretreatments may just temporarily inhibit the hydrogen consuming processes. After long-term cultivation, pretreated inocula did not enhance hydrogen production compared to untreated inocula under mesophilic conditions (initial pH 7 and pH 5.5) and thermophilic conditions (initial pH 7). However, pretreatment could inhibit lactate production and lead to higher hydrogen yield under thermophilic conditions at initial pH 5.5. The results further demonstrated that inoculum pretreatment could not permanently inhibit either methanogenesis or homoacetogenesis, and methanogenesis and homoacetogenesis could only be inhibited by proper control of fermentation pH and temperature. Methanogenic activity could be inhibited at pH lower than 6, both under mesophilic and thermophilic conditions, while homoacetogenic activity could only be inhibited under thermophilic condition at initial pH 5.5. Microbial community analysis showed that pretreatment did not affect the dominant bacteria. The dominant bacteria were Clostridium butyricum related organisms under mesophilic condition (initial pH 7 and 5.5), Thermoanaerobacterium sp. related organisms under thermophilic condition (initial pH 7), and Thermoanaerobacterium thermosaccharolyticum related organisms under thermophilic condition (initial pH 5.5). Results from this study clearly indicated that the long-term effects of inoculum pretreatments on hydrogen production, methanogenesis, homoacetogenesis and dominant bacteria were dependent on fermentation temperature and pH.     

基于正交实验法优化从栀子黄色素中制备藏红花酸的工艺

基于正交实验法,优化从栀子黄色素中提取制备藏红花酸的碱水解工艺,以期可以简单高效地获得高纯度藏红花酸。建立高效液相色谱法(HPLC)测定藏红花酸含量,以藏红花酸的含量和得率为考察指标,采用正交实验法考察碱水解工艺中的料液比、NaOH浓度、水解温度和水解时间对产品中藏红花酸含量和得率的影响。确定栀子黄色素碱水解的最佳条件:料液比1∶6 g/mL、NaOH浓度3 mol/L、水解温度55℃、水解时间60 min。在该条件下制备获得的藏红花酸得率可达15.33%±1.25%;含量可达到97.24%±0.78%。优化后方法步骤简单易行,绿色无污染,一步制得高纯度藏红花酸,适用于工业化生产。     

Hemoglobin interactions with αB crystallin: a direct test of sensitivity to protein instability

As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a) the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.     

The effect of high temperature on the kinetics of lipopolysaccharide (LPS)-induced human monocytes activity in vitro

Human peripheral blood monocytes were stimulated with lipopolysaccharide at different temperatures (34, 37 and 40 °C) and TNF-α, IL-1β and NO production was measured. Levels of TNF-α mRNA and IL-1β mRNA were measured by RT-PCR. Phagocytic activity of LPS-stimulated monocytes in terms of bacterial uptake and intracellular bacterial killing was checked at different conditions in vitro. Early elevation of TNF-α, IL-1β and NO production was found in LPS-stimulated monocytes that incubated at 40 °C followed by cells that incubated at 37 °C and lowest level was detected at 34 °C. Similar results were observed in the phagocytic activity. Expression of TNF-α mRNA and IL-1β mRNA was observed as early as 30 min post exposure to LPS in all studied temperatures and these, decreased sharply after 12 h post exposure to LPS in LPS-stimulated monocytes that incubated at 40 °C only. This report describes the striking effects of incubation temperature on activity of LPS-stimulated monocytes.     

Effect of acclimation on heat-escape temperatures of two aphid species: Implications for estimating behavioral response of insects to climate warming

An aphid usually stays at one feeding site for a long time to achieve its development and reproduction, while high temperatures can make it decide to escape from heat stress. Climate warming increases daily high-temperature both in degree and time. However, it remains unknown whether such heat-escape behavior will be influenced by those daily temperature changes. In this study, a wheat-leaf temperature gradient was created based on field microhabitat temperatures. We defined a parameter, heat-escape temperature (HET) to describe the critical temperature at which an aphid turns back when it walks along the gradient from mild temperature to high temperatures. HET indicates behavioral responses of the aphids to heat stress. Two aphid species, Sitobion avenae and Rhopalosiphum padi, main economic pests in temperate areas were selected as test insects. Detailed studies were conducted on the temperature gradient to reveal effects of acclimation temperature, time, and condition (temperature×time) on HET of both species. Results showed that HET decreased non-linearly (S. avenae: 41.4-38.6°C, R. padi: 41.3-39.4°C), when acclimation temperature increased from 25 to 36°C. For both species, HET declined linearly (S. avenae: 40.1-38.0°C, R. padi: 41.3-38.5°C) as acclimation time increased from 0.5 to 6h at 35°C, whereas HET descended non-linearly with reduction of acclimation time at 10°C. HET for both species acclimated under constantly warm conditions (future daily temperature) were significantly lower than those acclimated under gradually warm conditions (current daily temperature). These results suggest that aphids' heat-escape behavior is significantly influenced by brief thermal history, implying that aphids make decision to avoid heat stress based on the combination of temperature and exposure time and escape before they were hurt by high temperatures under the conditions of climate warming. Avoiding high temperatures may cost a lot of time and resources of aphids and thus potentially reduced growth, development, and reproduction. Changes in insect behaviors caused by ongoing climate warming and their ecological consequences should be more concerned.     

Dilute-acid hydrolysis for fermentation of the Bolivian straw material Paja Brava

Hydrolysis of the straw material Paja Brava, a sturdy grass characteristic for the high plains of Bolivia, was studied in order to find suitable conditions for hydrolysis of the hemicellulose and cellulose parts. Dried Paja Brava material was pre-steamed, impregnated with dilute sulfuric acid (0.5% or 1.0% by wt), and subsequently hydrolyzed in a reactor at temperatures between 170 and 230 degrees C for a reaction time between 3 and 10 min. The highest yield of xylose (indicating efficient hydrolysis of hemicellulose) were found at a temperature of 190 degrees C, and a reaction time of 5-10 min, whereas considerably higher temperatures (230 degrees C) were needed for hydrolysis of cellulose. Fermentability of hemicellulose hydrolyzates was tested using the xylose-fermenting yeast species Pichia stipitis, Candida shehatae and Pachysolen tannophilus. The fermentability of hydrolyzates decreased strongly for hydrolyzates produced at temperatures higher than 200 degrees C.     

Tolerance of the widespread cyanobacterium Nostoc commune to extreme temperature variations (?269 to 105°C), pH and salt stress

Nostoc commune is a widespread colonial cyanobacterium living on bare soils that alternate between frost and thaw, drought and inundation and very low and high temperatures. We collected N. commune from alternating wet and dry limestone pavements in Sweden and tested its photosynthesis and respiration at 20°C after exposure to variations in temperature (-269 to 105°C), pH (2-10) and NaCl (0.02-50 g NaCl kg(-1)). We found that dry field samples and rewetted specimens tolerated exposure beyond that experienced in natural environmental conditions: -269 to 70°C, pH 3-10 and 0-20 g NaCl kg(-1), with only a modest reduction of respiration, photosynthesis and active carbon uptake at 20°C. (14)CO(2) uptake from air declined markedly below zero and above 55°C, but remained positive. Specimens maintained a high metabolism with daily exposure to 6 h of rehydration and 18 h of desiccation at -18 and 20°C, but died at 40°C. The field temperature never exceeded the critical 40°C threshold during the wet periods, but it frequently exceeded this temperature during dry periods when N. commune is already dry and unaffected. We conclude that N. commune has an excellent tolerance to low temperatures, long-term desiccation and recurring cycles of desiccation and rewetting. These traits explain why it is the pioneer species in extremely harsh, nutrient-poor and alternating wet and dry environments.     

Transition from positive to neutral in mutation fixation along with continuing rising fitness in thermal adaptive evolution

It remains to be determined experimentally whether increasing fitness is related to positive selection, while stationary fitness is related to neutral evolution. Long-term laboratory evolution in Escherichia coli was performed under conditions of thermal stress under defined laboratory conditions. The complete cell growth data showed common continuous fitness recovery to every 2°C or 4°C stepwise temperature upshift, finally resulting in an evolved E. coli strain with an improved upper temperature limit as high as 45.9°C after 523 days of serial transfer, equivalent to 7,560 generations, in minimal medium. Two-phase fitness dynamics, a rapid growth recovery phase followed by a gradual increasing growth phase, was clearly observed at diverse temperatures throughout the entire evolutionary process. Whole-genome sequence analysis revealed the transition from positive to neutral in mutation fixation, accompanied with a considerable escalation of spontaneous substitution rate in the late fitness recovery phase. It suggested that continually increasing fitness not always resulted in the reduction of genetic diversity due to the sequential takeovers by fit mutants, but caused the accumulation of a considerable number of mutations that facilitated the neutral evolution.     

Effects of temperature and dietary protein level on hepatic oxidative status of Senegalese sole juveniles (Solea senegalensis)

Effects of 55 and 45% dietary protein levels (55P and 45P diets, respectively) and temperature (12 and 18°C) on hepatic activity of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase (GR), glucose-6-phosphate dehydrogenase and lipid peroxidation (LPO) levels of Solea senegalensis juveniles were studied. Further, effects of acute thermal shocks provoked by a drop (18°C to 12°C) or a rise (12°C to 18°C) of water temperature on sole oxidative state was also evaluated. Dietary protein reduction increased LPO levels though no major alterations were found on antioxidant enzyme activities between dietary treatments. At 12°C GR activity was higher and SOD activity was lower than 18°C but LPO levels were not affected. In both thermal shock cases, LPO levels increased in 55P group, probably due to insufficient antioxidant enzyme activation. In contrast, fish of 45P group under acute exposition to warmer and colder temperature exhibited no substantial changes and a significant decrease on LPO levels, respectively, along with no major changes in antioxidant enzymes. Overall, results suggest that independently of rearing temperatures 45P group was more susceptible to oxidative stress than 55P group. Thermal shock either due to rise or drop of temperature seemed to induce oxidative stress in 55P group.     

Effects of temperature on circadian clock and chronotype: an experimental study on a passerine bird

Daily schedules of many organisms, including birds, are thought to affect fitness. Timing in birds is based on circadian clocks that have a heritable period length, but fitness consequences for individuals in natural environments depend on the scheduling of entrained clocks. This chronotype, i.e., timing of an individual relative to a zeitgeber, results from interactions between the endogenous circadian clock and environmental factors, including light conditions and ambient temperature. To understand contributions of these factors to timing, we studied daily activity patterns of a captive songbird, the great tit (Parus major), under different temperature and light conditions. Birds were kept in a light (L)-dark (D) cycle (12.5?L:11.5 D) at either 8°C or 18°C with ad libitum access to food and water. We assessed chronotype and subsequently tested birds at the same temperature under constant dim light (LL(dim)) to determine period length of their circadian clock. Thermal conditions were then reversed so that period length was measured under both temperatures. We found that under constant dim light conditions individuals lengthened their free-running period at higher temperatures by 5.7?±?2.1?min (p?=?.002). Under LD, birds kept at 18°C started activity later and terminated it much earlier in the day than those kept under 8°C. Overall, chronotype was slightly earlier under higher temperature, and duration of activity was shorter. Furthermore, individuals timed their activities consistently on different days under LD and over the two test series under LL(dim) (repeatability from .38 to .60). Surprisingly, period length and chronotype did not show the correlation that had been previously found in other avian species. Our study shows that body clocks of birds are precise and repeatable, but are, nonetheless, affected by ambient temperature. (Author correspondence: marina.lehmann@uni-konstanz.de ).     

Studies on anaerobic proteolytic bacteria of Leh,India

Five anaerobic proteolytic bacteria were isolated from water bodies of Leh, India, where the ambient temperature varies from –25 to 25 °C. Isolates showed growth at all temperatures ranging from 5 to 37 °C except SPL-4 and SPL-5 which showed no growth at 5 °C. The cultures could grow and produce proteases on various protein substrates and the yield varied with the substrates. Two of the cultures showed the presence of spores. Acetate was the dominant VFA during hydrolysis of protein substrates.     

The role of HRT and low temperature on the acid-phase anaerobic digestion of municipal and industrial wastewaters

Two identical 31 completely mixed reactors with solids recycling capabilities were used to investigate the effects of hydraulic retention time (HRT) and low temperatures on volatile fatty acid (VFA) production. One reactor was fed with a 1:1 ratio of diluted primary sludge and a starch-rich industrial wastewater, while the other was fed with diluted primary sludge alone. The VFA and soluble COD concentrations and specific production rates reached their highest values at 30 h HRT and at 25 degrees C. Further increase in HRT (at 25 degrees C) or decrease in temperature (at an HRT of 30 h) resulted in lower amounts of VFA and COD produced. All parameters related to VFA and COD production were significantly higher in the industrial-municipal reactor than in the municipal-only reactor. The VFA:COD ratios were very high, with values ranging from about 0.8 to 1.0 indicating that hydrolysis was the rate-limiting step. Degradation of proteins (measured by ammonia production) was inhibited by the starch-rich wastewater in the industrial-municipal reactor, while no evidence of inhibition was found in the municipal-only reactor. This study revealed that VFA production was feasible at low temperatures (down to 8 degrees C), particularly in the presence of the industrial waste. Ultimately, the amount VFA produced was adequate, in most cases, to support subsequent biological nutrient removal (BNR) processes.