Split Addition of Nitrogen-Rich Substrate at Thermophilic and Mesophilic Stages of Composting: Effect on Green House Gases Emission and Quality of Compost

Composting as a solution to the increasing generation of municipal solid waste (MSW), also contribute to GHGs emission when not controlled and could lack some basic nutrients, especially nitrogen. This study assessed the split-additions of nitrogen-rich substrate to composting materials and their effect on GHGs emissions as well as the quality of the composts. Nitrogen-rich substrates formulated from pig and goat manure were co-composted with MSW for a 12-weeks period by split adding at mesophilic (˚C) and thermophilic (>50˚C) stages in five different treatments. Representative samples from the compost were taken from each treatment for physicochemical, heavy metals and bacteriological analysis. In-situ CH4, CO2, N2O gas emissions were also analyzed weekly during composting. It was observed that all the treatments showed significant organic matter decomposition, reaching thermophilic temperatures in the first week of composting. The absence affects the suitable agronomic properties. All nitrogen-rich substrate applied at thermophilic stage (Treatment two) recorded the highest N, P and K concentrations of 1.34%, 0.97% and 2.45%, respectively with highest nitrogen retention. In terms of GHG emissions, CO2 was highest at the thermophilic stage when N-rich substrate was added in all treatment, while CH4 was highest in the mesophilic stage with N-rich substrate addition. N2O showed no specific trend in the treatments. Split addition of the N-rich substrate for co-composting of MSW produced compost which is stable, has less nutrient loss and low GHG emissions. Split addition of a nitrogen-rich substrate could be an option for increasing the fertilizer value of MSW compost.

The plasmodesmata-associated β-1,3-glucanase gene GhPdBG regulates fiber development in cotton

Trichomes are specialized structures that originate from epidermal cells of organs in higher plants.The cotton fiber is a unique single-celled trichome that elongates from the seed coat epidermis.Cotton(Gossypium hirsutum)fibers and trichomes are models for cell differentiation.In an attempt to elucidate the intercellular factors that regulate fiber and trichome cell development,we identified a plasmodesmal β-1,3-glucanase gene(designated GhPdBG)controlling the opening and closing of plasmodesmata in cotton fibers.Structural and evolutionary analysis showed haplotypic variation in the promoter region of the GhPdBG gene among 352 cotton accessions,but high conservation in the coding region.GhPdBG was expressed predominantly in cotton fibers and localized to plasmodesmata(PD).Expression patterns of PdBG that corresponded to PD permeability were apparent during fiber development in G.hirsutum and G.barbadense.The PdBG-mediated opening-closure of PD appears to be involved in fiber development and may account for the contrasting fiber traits of these two species.Ectopic expression of GhPdBG revealed that it functions in regulating fiber and trichome length and/or density by modulating plasmodesmatal permeability.This finding suggests that plasmodesmal targeting of GhPdBG,as a switch of intercellular channels,regulates single-celled fiber and trichome development in cotton.

Enzymatic Transformation of Notoginsenoside Fe by β-Glucanase

Aim An industrial enzyme β-glucanase was used to transfortn notoginsenoside Fe for the first time. Methods Notoginsenoside Fe was isolated from the leave saponin of Panax notoginseng （Burk.） Chen FH. The enzymatically transformed compounds were detected by HPLC and two transformed compounds were identified as 20 （S） -protopanaxadiol-20- O- α-L-arabinofuranosyl （ 1→6 ） - β-gluco- pyranoside, ginsenoside-Mc） and 20（S）-protopanaxadiol-20-O-β-D-glucopyranoside compound-K （C-K） respectively on the basis of their ^1H NMR and ^13 C NMR spectral data. Results Based on the enzymolytic kinetic curve, the transformation rate of notoginsenoside Fe reached 95% after 24 h. Conclusion The enzymatic transformation pathway of notoginsenoside Fe by β-glucanase has been proposed as notoginsenoside Fe→ginsenoside Mc→C-K.

Characterization of apparent sulfur oxidation activity of thermophilic archaea in bioleaching of chalcopyrite

The apparent sulfur oxidation activities of four pure thermophilic archaea, Acidianus brierleyi （JCM 8954）, Metallosphaera sedula （YN 23）, Acidianus manzaensis （YN 25） and Sulfolobus metallicus （YN 24） and their mixture in bioleaching chalcopyrite were compared, which were characterized indirectly by the evolution of the cells concentration, pH value and sulfate ions concentration in solution. The results show that the mixed culture contributed significantly to the raising of leaching rate, which suggests that the mixed culture had a higher sulfur oxidation activity than the pure culture. Meanwhile, the results also indicate that the changes of parameters characterizing the sulfur oxidation activity of thermophilic archaea are often influenced by many factors, so it is hard to reflect accurately the specific sulfur oxidation activities among the different sulfur-oxidizing microbes when bioleaching chalcopyrite at different conditions. Accordingly, an efficient method to characterize microbial sulfur oxidation activity appears to be desirable.

Surface species of chalcopyrite during bioleaching by moderately thermophilic bacteria

X-ray diffraction （XRD） and X-ray photoelectron spectroscopy （XPS） analyses were carried out to investigate the surface species and interfacial reactions during bioleaching of chalcopyrite by different strains of moderately thermophilic bacteria （45 °C）. Results show that monosulfide （CuS）, disulfide （S22？）, polysulfide （Sn2？）, elemental sulfur （S0） and sulfate （SO42？） are the main intermediate species on the surface of chalcopyrite during bioleaching byA. caldus,S. thermosulfidooxidans andL. ferriphilum. The low kinetics of dissolution of chalcopyrite inA. caldus can be mainly attributed to the incomplete dissolution of chalcopyrite and the passivation layer of polysulfide. Polysulfide and jarosite should be mainly responsible for the passivation of chalcopyrite in bioleaching byL. ferriphilumorS. thermosulfidooxidans. However, elemental sulfur should not be the main composition of passivation layer of chalcopyrite during bioleaching.

Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.

Effect of moderately thermophilic bacteria on metal extraction and electrochemical characteristics for zinc smelting slag in bioleaching system

The effects of moderately thermophilic bacteria on the extraction of metals from zinc smelting slag and electrochemical characteristics of zinc smelting slag carbon paste electrode in bioleaching process were studied. The results show that the extraction rates of Fe, Cu and Zn from the slag reach 86.7%, 90.3% and 66.7% after adsorbed bacteria sterilize, while those with adsorbed bacteria are 91.9%, 96.0% and 84.5% in conditions of pulp density 2%, pH 1.0, temperature 65 °C and stirring rate 120 r/min, respectively. Some stretching peaks of functional groups from bacterial secretes on the bioleached residue surface, such as 1007 cm-1 and 1193 cm-1, turn up through FI-IR analysis and indirectly reveal the presence of the adsorbed bacteria on the slag particles surface. Besides, the corrosion of zinc smelting slag is enhanced by bacteria according to the characteristics of cyclic voltametry and Tafel curves in bioleaching system.

Research on the Nature of Thermophilic Anaerobic Ethanol Producer Thermo anaerobacter sp DF3 in Petroleum Reservoirs

[Objective] The aim was to study the physiological and biochemical char- acteristics of Thermoanaerobacter sp DF3 in petroleum reservoirs and optimize the culture plan of producing ethanol from xylose. [Method] DF3, an anaerobic bacillus producing ethanol, was isolated from produced liquid from oil layer of Dagang oil field with anaerobic isolation technique. The phylogenetic position was analyzed by physiological and biochemical identification and phylogeny of 16S rDNA sequence. The metabolites were analyzed by gas chromatograph. [Result] The strain DF3 was a strict anaerobic thermophilic bacterium, which was straight in rod shape,and gram negative. Besides, it was 0.42 μmx（1.60-5.20） iJm in length. The strains can be soli- tary,in pairs or string and apical spore usually produced. Its growth temperature was 45-78 and 65 ℃ was the optimum. Many substances could be used as carbon sources, including glucose, xylose, fructose, ribose, mannose, arabinose, sucrose, galactose, lactose, cellobiose, melizitose, raffinose-, and starch. The similarity between strain DF3 and T. pseudoethanolicus achieved 99.7%. The main product of glucose and xylose fermentation was ethanol. After the culture plan was optimized,the final concentration of ethanol was 2.0 g/L. [Conclusion] It was proved through experiments that the strain DF3 was one of the strains with higher activity to produce ethanol at present and it could produce 2.0 g/L ethanol from xylose metabolization at 65 ℃. It has been demonstrated that DF3 is one of the known strains with high-production to produce ethanol,for example, 2.0 g/L ethano at 65℃. Presently, all the high-yield ethanol can be produced from metabolic xylose strains of metabolic xylose were isolat- ed by foreign countries, therefore, isolation of strain DF3 has provided an excellent original strain for studying ethanol production from lignocellulose in China.

CHARACTERISTICS OF THERMOPHILIC ANAEROBIC ACIDOGENESIS OF STARCH WASTEWATER

Acidogenic dissimilation of synthetic starch wastewater (1 000～10 000 mg COD·L -1 ) was studied in a thermophilic (55 ℃) upflow anaerobic sludge blanket (UASB) reactor.The production of volatile fatty acids (VFA) was proportional to the chemical oxygen demand (COD) loading rate.The yield of VFA was around 0.28 g VFA/g COD over the COD loading rate from 1.25 to 30 g COD·L -1 ·d -1 and the hydraulic retention time from 8.8 h to 24 h.Distribution of organic acids,the contents of propionic and butyric acids in the effluent in particular were also dependent on the COD loading rate.The thermophilic UASB reactor showed a stable performance on hydrolysis and acidogenesis of starch as well as suspended solid removal at short hydraulic retention times and high influent pH(10～11),during the operation of 110 d.

Cloning of Endo-β-Glucanase Ⅲ and Expression in Eerevisiae Fermentum

Objective The aim was to construct bioengineering strains that could degrade the cellulosic solid waste. Method The cDNA of endo-β-glucanase III of Trichoderma vi ride AS313711 was cloned by RT-PCR method. After sequenced, this gene was constructed to expression vector pESP-2, and then the plasmid was transformed into competent cell of cerevisiae fermentum by electric shock, the transformant was then obtained. The enzyme activity of this transformant at the different temperatures and pH was measured by DNS method. Result The length of ORF of EG III was 1 257 bp, encoding 418 amino acids, while the deduced molecular weight was 44.1 × 103 kD. Conclusion The enzyme activity of EG III was the highest when it was at PH 4.9 and tempeture was of 60℃. Then the corresponding enzyme activity was about 100%.

Start-up performances of dry anaerobic mesophilic and thermophilic digestions of organic solid wastes

Two dry anaerobic digestions of organic solid wastes were conducted for 6 weeks in a lab-scale batch experiment for investigating the start-up performances under mesophilic and thermophilic conditions. The enzymatic activities, i.e., β-glucosidase, N-α-benzoyl-Largininamide （BAA）-hydrolysing protease, urease and phosphatase activities were analysed. The BAA-hydrolysing protease activity during the first 2-3 weeks was low with low pH, but was enhanced later with the pH increase. β-Glucosidase activity showed the lowest values in weeks 1-2, and recovered with the increase of BAA-hydrolysing protease activity. Acetic acid dominated most of the total VFAs in thermophilic digestion, while propionate and butyrate dominated in mesophilic digestion. Thermophilic digestion was confirmed more feasible for achieving better performance against misbalance, especially during the start-up period in a dry anaerobic digestion process.

Autoheated thermophilic aerobic sludge digestion and metal bioleaching in a two-stage reactor system

A two-stage process has been developed for stabilization of sludge and removal of heavy metals from the secondary activated sludge with high rate of energy and time conservation. The first stage of the process involves autoheated thermophilic aerobic digestion at 55-60℃ inoculated with less-acidophilic thermophilic sulfur-oxidizing microorganisms （ATAD）. The results show that it is possible to maintain the autoheated conditions （55-60℃） in the ATAD reactor up to 24 hr, leading to reduction of 21% total solids （TS）, 27% volatile solids （VS）, 27% suspended solids （SS） and 33% volatile suspended solids （VSS） from the sludge. The sludge pH also decreased from 7 to 4.6 due to the activity of less-acidophilic thermophilic microorganisms. In the second stage operation, the digested sludge （pH 4.6, TS 31.6 g/L） from stage one was subjected to bioleaching in a continuous stirred tank reactor, operated at mean hydraulic retention times （HRTs） of 12, 24 and 36 hr at 30℃. An HRT of 24 hr was found to be sufficient for removal of 70% Cu, 70% Mn, 75% Ni, and 80% Zn from the sludge. In all, 39% VSS, 76% Cu, 78.2% Mn, 79.5% Ni and 84.2% Zn were removed from the sludge in both the stages.

Removal of copper from molybdenite concentrate by mesophilic and extreme thermophilic microorganisms

Mixed mesophilic and extreme thermophilic bioleaching were evaluated to remove copper from the molybdenite concentrate.Bioleaching tests were carried out in shake flasks and in a 50-L bioreactor.The shake flask tests were performed with different inoculum size,solids density,pH.and temperature in order to identify optimum conditions.The highest amount of copper elimination,75%was obtained with extreme thermophilic microorganisms(at 12%inoculation,10%solids,65℃and a pH of 1.5).The highest copper elimination by mesophilic microorganisms was 55%(at 12%inoculation,5%solids,30℃at pH 2).The optimum conditions in shake flask tests were applied to 7 days batch tests in a50-L bioreactor.Extreme thermophilic experiment gave the best copper elimination of 60%(at 12%inoculation,10%solids,65℃and pH 1.5).Mesophilic test removed 50%of the copper(at 12%inoculation,10%solids,35℃at pH 2).

Complete genome sequence and proteomic analysis of a thermophilic bacteriophage BV1

Viruses of thermophiles are of great interest due to their roles in gene transfer, global geochemical cycle and evolution of life on earth. However, the thermophilic bacteriophages have not been studied extensively. In this investigation, a typical bacteriophage BV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k512, which was isolated from an inshore hot spring in Xiamen of China. The BV1 contained a double-stranded linear DNA of 35 055 bp, which encodes 54 open reading frames （ORFs）. Interestingly, eight of the 54 BV1 ORFs shared sequence similarities to genes from human disease-relevant bacteria. Seven proteins of the purified BV1 virions were identified by proteomic analysis. Determination of BV1 functional genomics would facilitate the better understanding of the mechanism for virus-thermophile interaction.

Relatedness between catalytic effect of activated carbon and passivation phenomenon during chalcopyrite bioleaching by mixed thermophilic Archaea culture at 65°C

The relatedness between catalytic effect of activated carbon and passivation phenomenon during chalcopyrite bioleachingby mixed thermophilic Archaea culture(Acidianus brierleyi,Metallosphaera sedula,Acidianus manzaensis and Sulfolobusmetallicus)at65°C was studied.Leaching experiments showed that the addition of activated carbon could significantly promote thedissolution of chalcopyrite for both bioleaching and chemical leaching.The results of synchrotron-based X-ray diffraction,ironL-edge and sulfur K-edge X-ray absorption near edge structure spectroscopy indicated that activated carbon could change thetransition path of electrons through galvanic interactions to form more readily dissolved secondary mineral chalcocite at a low redoxpotential(?400mV)and then enhanced the copper dissolution.Jarosite accumulated immediately in the initial stage of bioleachingwith activated carbon but copper dissolution was not hindered.However,much jarosite precipitated on the surface of chalcopyrite inthe late stage of bioleaching,which might account for the decrease of copper dissolution rate.More elemental sulfur(S0)was alsodetected with additional activated carbon but the mixed thermophilic Archaea culture had a great sulfur oxidation activity,thus S0waseliminated and seemed to have no significant influence on the dissolution of chalcopyrite.

Comparison of leaching of bornite from different regions mediated by mixed moderately thermophilic bacteria

Bioleaching experiments combined with X-ray photoelectron spectroscopy(XPS),X-ray diffraction(XRD)and scanning electron microscopy(SEM)were conducted to investigate three kinds of bornites from different regions leached by moderately thermophilic mixed bacteria of Leptospirillum ferriphilum YSK,Acidithiobacillus caldus D1 and Sulfobacillus thermosulfidooxidans ST.The results of bioleaching experiments showed that the leaching efficiency and the redox potential were significantly increased.The copper extraction efficiencies of three kinds of bornite maintained rapid growth until around the 12th day and no longer increased after the 18th,reaching 83.7%,96.5%and 86.6%,respectively.The XRD results of the leaching residue indicated that three kinds of bornites all produced jarosite in the late stage of leaching,and the leaching residues from of Daye Museum and Yunnan Geological Museum contained a mass of elemental sulfur.XPS analysis and scanning electron microscopy experiments showed that the surface of mineral particles was jarosite and the copper in the leaching residue was almost dissolved.

Bioleaching of chalcopyrite by mixed culture of moderately thermophilic microorganisms

A mixed culture of moderately thermophilic microorganisms was enriched from acid mine drainages(AMDs)samples collected from several sulphide mines in China,and the bioleaching of chalcopyrite was conducted both in shake flask and bioreactor.The results show that in the shake flask,the mixture can tolerate 50 g/L chalcopyrite after being acclimated to gradually increased concentrations of chalcopyrite.The copper extraction increases obviously in bioleaching of chalcopyrite with moderately thermophilic microorganisms supplemented with 0.4 g/L yeast extract at 180 r/min,74% copper can be extracted in the pulp of 50 g/L chalcopyrite after 20 d.Compared with copper extractions of mesophilic culture,unacclimated culture and acclimated culture without addition of yeast extract,that of accliniated culture with addition of yeast extract is increased by 53%,44% and 16%,respectively.In a completely stirred tank reactor,the mass fraction of copper and total iron extraction reach up to 81% and 56%,respectively.The results also indicate that it is necessary to add a large amount of acid to the pulp to extract copper from chalcopyrite effectively.

Enhanced Production of Hybrid Extracellular β-Glucanase by Re-combinant Escherichia coli Using Experimental Design Method

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid ex-tracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L-1. After optimization of the medium, 297.71U·ml-1 of β-glucanase activity in the medium and 352350U·g-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those ob-tained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.

Optimization of cultural conditions for thermostable β-1,3-1,4-glucanase production by Bacillus subtilis ZJF-1A5

The optimization of cultural conditions for β glucanase production by Bacillus subtilis ZJF 1A5 was investigated in flask trials. Temperature had great effect on β glucanase production which maximized at optimal temperature of 37℃ and decreased significantly when temperature was over 37℃.Charge quantity affected β glucanase production significantly. Adding oxygen vector N dodecane or acetic ether benefited β glucanase production, but it depended on the concentration and charge quantity. The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting β glucanase production and the cultivation time span to reach the highest β glucanase activity. The optimal cultural conditions for β glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82%(v/v)), shaking speed 210 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37℃ for 50 h. Repeated experimental results accorded with those predicted by a second order polynomial model. The amount of β glucanase, α amylase and neutral protease produced by B subtilis ZJF 1A5 was associated partially with cell growth. Those three enzymes' activities increased following the cell growth and increased significantly when cells entered the stationary phase.

Adsorption and leaching behaviors of chalcopyrite by two extreme thermophilic archaea

The adsorption and leaching of chalcopyrite by two extreme thermophilic archaea(A.brierleyi and S.metallicus)and their mixture were studied.The results revealed that the chalcopyrite leaching rate of S.metallicus was slightly higher than that of A.brierleyi;the mixed system showed the highest rate.Community structure analysis during the leaching process showed that S.metallicus was maintained in a predominant state.However,the proportion of A.brierleyi in the community increased during leaching.Copper concentrations,which increased faster in the mixed system than in the single-organism systems during later stages,was related to the change of A.brierleyi in the community.Langmuir parameter analysis revealed no competitive adsorption between these two thermophilic archaea.Furthermore,qPCR(quantitative polymerase chain reaction)confirmed that adsorption was promoted between A.brierleyi and S.metallicus during mixed leaching.These findings can improve our understanding of the adsorption behaviors of mixed extreme microbial populations on mineral surfaces.