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.

深海嗜热菌超氧化物歧化酶基因耐盐性研究

为探究深海嗜热菌(Thermophile thermus)中铁离子超氧化物歧化酶Fe-SOD基因(Tt SOD)与植物耐盐性的关系,对该基因的全长序列进行了分析,构建了Tt SOD基因过量表达载体,并将该过量表达载体成功转化烟草中,获得转基因植株。结果表明,该基因全长615 bp,是典型的Fe-SOD。转Tt SOD基因烟草中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性显著提高,而丙二醛(MDA)含量显著降低;转基因烟草的种子萌发率、叶绿素和类胡萝卜素含量显著提高。本研究的结果表明植物过表达Tt SOD基因,提高了转基因烟草耐盐能力,为选育作物耐盐性新品种提供技术参考。

Feasibility study on heap bioleaching of chalcopyrite

Bioleaching of chalcopyrite often encountered the formation of passivation layer, which inhibited the leaching process and resulted in a low leaching rate. This inhibitory effect can be eliminated by thermophilic biole- aching. The industrial test of BioCOP technology based on thermophiles was successfully completed, which confirmed the feasibility of chalcopyrite bioleaching. However, industrial leaching rate of chalcopyrite heap bioleaching is lower. This paper described the development status and industrial test of chalcopyrite heap bioleaching technology. The reasons for the lower efficiency of chalcopyrite heap bioleaching were analyzed. The strategies for successful chalcopyrite heap bioleaching were proposed.

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.

Purification and characterization of a thermostable glutamate dehydrogenase from a thermophilic microorganism from Deception Island, Antarctica

Glutamate dehydrogenase （GDH） catalyzes the oxidative deamination of glutamate to a-ketoglutarate and ammonium ions. Currently the determination of ammonium and glutamate is carried out using a bovine GDH enzyme, which lacks optimal thermostability for long term storage at room temperature. From samples of Deception Island, Antarctica, we obtained the thermophilic bacteria PID 15 belonging to the Bacillus genera with high GDH specific activity. This new enzyme exhibited NAD＋ dependent activity and no activity was observed when NADP＋ was used. This enzyme shows a specific activity of 4.7 U.mg-1 for the oxidative deamination reaction and 15.4 U·mg-1 for the reduction of a-ketoglutarate. This enzyme has an optimum temperature of 65℃ and pH of 8.5 for the oxidative deamination. For the reduction of a-ketoglutarate, the optimum temperature is 60℃, with a pH of 8.0. One of the most important characteristics of this enzyme is its ability to retain more than 60% of its activity when it is incubated for 8 h at 65℃. The enzyme is also able to retain full activity when it is incubated for 48 d at 4℃ and over 80% of its activity when it is incubated at 25℃. Characterization of its kinetics suggests that it primarily catalyzes the formation of α-ketoglutarate. This enzyme has an important biological role in the catabolism of glutamate and may have some interesting biotechnological applications based on its thermostable properties.

Metabolic Engineering of <i>Thermoanaerobacterium thermosaccharolyticum</i>for Increased n-Butanol Production

Thermoanaerobacterium thermosaccharolyticum shows promise as a host for n-butanol production since it natively has the required genes involved in the n-butanol biosynthetic pathway. Overexpression of the natively occurring bcs operon containing the genes thl, hbd, crt, bcd, etfA, and etfB responsible for the formation of butyryl CoA increased the n-butanol production by 180% compared to the wild type from a n-butanol titer of 1.8 mM to 5.1 mM. The deletion of one of the six alcohol dehydrogenase genes confirmed that it was the primary gene responsible for ethanol and n-butanol production from acetyl CoA and butyryl CoA respectively.

Influence of Hot Spring Phages on Community Carbon Metabolism: Win, Lose or Draw?

Abundant virus-like particles were concentrated from large-volume samples from two hot springs. Both addition of viral concentrates and addition of samples induced by addition of mitomycin-C changed patterns of carbon source utilization by hot spring microbial communities. Specific effects of the two treatments depended upon both temperature and incubation period. Increased metabolic capability with greater exposure to free phages, consistent with the view that phages are major lateral transporters of metabolic genes, was observed most clearly in microbes incubated at a temperature lower than that encountered in situ. On the other hand, decreases in the diversity of utilizable C sources upon exposure to phages may have been due to lytic activity in which susceptible bacterial populations were differentially reduced by infective viruses, consistent with the “killing the winner” hypothesis. Treatment of cultures with MC-treated culture extracts, assumed to increase exposure to excised prophages, resulted in higher average metabolic rates after 18 h, but lower rates after 48 h of incubation. With incubation at in situ temperature, this same treatment led to an initial increase in the number of readily utilized C sources, followed by a decrease in community metabolic diversity relative to controls in samples from both hot springs. Thus, treatments designed to increase the interaction between hot spring microbes and either free or newly-excised phages had observable time- and temperature-dependent effects on community metabolism, demonstrating an important, yet complex, ecological role for phages in hot spring waters.

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).

Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88,isolated from Taptapani hotspring of odisha,India

Objective:To screen and isolate an eco-friendly,u thermophilic and potent L-asparaginase producing bacterium,with novel immunological properties that may obviates hypersensitivity reactions.Methods:In the present study baclerial strain isolated for extracellular L-asparaginase production from hotspring,identified by morphological,biochemical and physiological tests followed by t6S rDNA technology and the L-asparaginase production ability was tested by both semi quantitative and quantitative enzymatic assay.Result:The bacterial strain was identified as Bacillus sublilis strain hswx88(GenBank Accession Number JQ237656.1).The extracellular enzyme yielding capacity isolate Bacillus subtilis strain hswx88(23.8 IU/mL)was found to be 1.7 and 14.5 limes higher than the reference organism Pectobacterium carotovorum MTCC 1428(14.2 IU/mL)and Bacillus sp.BCCS 034(1.64 IU/mL).Conclusion:The isolate is eco-friendly and useful to produce bulk quantity of extracellular,thermophilic L-asparaginase for the treatment of various tumor cases and for preparation of acrylamide free fry food preparation.

Mineralization of a Proterozoic Sulfide Black Smoker Chimney and Thermophilous Microorganisms in Eastern Hebei,China

A sulfide black smoker chimney exists in the Gaobanhe seabed exhalation massive sulfide deposit in the Xingiong-Kuancheng secondary fault basin of the Proterozoic Yaniiao rift trough in Hebei Province, taking the shape of mounds, individually about 2-3 cm high. Abundant fossils of thermophilous bacteria and algae in perfect preservation are found in the ore surrounding the black smoker chimney. Scanning electron microscopy （SEM） and molecular biomarker studies on the microorganismai ore fabric show that the microorganism in the sulfide ore is in fact a sedimentary product of probiotic bacteria and algae. In the special food chain based on black smoker chimney at ancient seabed- thermophilous bacteria, the thermophilous bacteria and algae reproduce in large quantity. Intermittently erupting of fluid from the chimney creats conditions for formation of sulfide deposit. In the process of exhalation action of hot fluid, thermophilous bacteria and algae grow and reproduce around the sulfide black smoker chimney, absorbing mineralizing substances brought by the fluid. Massive sulfide deposits are formed in this process of absorption of seabed black smoker chimney exhalation-mineralizing fluid pulsation-thermophilous microorganism.

Isolation and molecular identification of cellulolytic bacteria from Dig Rostam hot spring and study of their cellulase activity

Cellulose is the main structural component of lignocellulosic wastes that can be converted to sugars and biofuels by cellulase.Due to wide applications of this enzyme in various industries around the world,cellulase is considered as the third industrial enzyme.The ability of thermophilic bacteria in the production of heat-stable cellulases has made them valuable tools in biotechnology.The aim of this study was isolation and molecular identification of cellulolytic thermophile bacteria from Dig Rostam hot spring and investigating their cellulase activity.Samples were taken from water and sediments of this hot spring,and cellulolytic bacteria were enriched in media containing cellulose as the only carbon source.The bacteria were incubated at 60℃,and single colonies were then isolated on solid media.Congo red assay was used as a quick test for the qualitative screening of cellulase activity.According to these qualitative results,four colonies named CDB1,CDB2,CDB3,and CDB4 were isolated,and their growth curve and some other characteristics were determined by biochemical assays.Moreover,endoglucanase,exoglucanase,and FPase activities of the isolates were investigated quantitatively.Results indicated that CDB1 exhibited the highest endoglucanase(0.096 U/mL)and exoglucanase(0.156 U/mL)activities among other isolates.16S rDNA partial sequencing indicated that CDB1 had 99%similarity to the genus Anoxybacillus,and the other isolates showed the highest similarity to the genus Geobacillus.The cellulase gene of CDB1 isolate with the highest cellulase activity was also cloned,and its sequence is reported for the first time.Further studies on this thermophilic enzyme might be useful for industrial applications.

Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum

Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the elucidation of further aspects of nitrogen metabolism. To circumvent this, Thermosynechococcus elongatus BP-1 was selected as a surrogate host and its nirA gene was substituted by the homologous gene of P. laminosum. This process, based on Long Flanking Homology Polymerase Chain Reaction and the natural competence of T. elongatus BP-1, required an intermediate T. elongatus BP-1 ΔnirA::kat mutant, which carries a gene encoding a thermostable kanamycin nucleotidyl transferase in place of nirA_Te. In the presence of nirA_Pl, nirA defective mutants of T. elongatus BP-1 recovered the ability to grow with nitrate as the sole nitrogen source, and showed a phenotype similar to that observed in wild-type cells. The procedure could be useful to substitute other genes from T. elongatus BP-1 with the homologues from P. laminosum in order to study this particular operon. Furthermore, it may be used as a general tool to explore phenotypic changes due to the exchange of a single gene between cyanobacteria.

A culture-dependent survey of thermophilic bacteria from hot springs in Xiamen area in China

Microbes are believed to play important roles in ecosystem function in many environments.The hot springs of Xiamen Island are close to the Xiamen Sea,and may have some characteristics different from those of inland hot springs.Microbes living in the hot springs of Xiamen may have new characteristics.However,little is known about microbial communities of hot springs close to the Xiamen Sea.A culture-dependent survey of microbial population in the Xiamen hot springs was performed by using an approach combining total cellular protein profile identification and 16S rRNA gene sequencing.A total of 328 isolates of bacteria were obtained from liquid and sediment samples from the Xiamen hot springs,including neutrophilic thermophilic bacteria and moderately thermophilic acidophiles.Neutrophilic thermophilic bacteria,which grow at a temperature range of 55-90℃ including Rhodothermus marinus（Strain 1）,Thermus thermophilus（Strain 2）,Thermus thiopara（Strain 3）,Geobacillus stearothermophilus（Strain 4）,Geobacillus thermoleovorans（Strain 5）,and Pseudomonas pseudoalcaligenes（Strain 6）,were recovered by 2216E plates.Moderately thermophilic acidophiles,which can grow at temperatures above 50℃ and a pH range of 1.8-3.5 such as Alicyclobacillus acidoterrestris（Strain 8）,Sulfobacillus acidophilus（Strain 9）,and Sulfobacillus thermosulfidooxidans（Strain 10）,were isolated on selective solid medium containing sulfur and Fe2＋.Among these strains,Rhodothermus marinus,Thermus thermophilus and Geobacillus stearothermophilus are not only thermophiles,but also halophiles.One bacterium strain（Strain 6） shared 99% nucleotide sequence homology with Pseudomonas pseudoalcaligenes on the 16S rRNA gene sequence,but was quite different from Pseudomonas pseudoalcaligenes in biological characteristics,suggesting that it may represent a novel thermophilic species.Results indicated that various species of neutrophilic thermophiles and moderately thermophilic acidophiles were widely distributed in the Xiamen hot springs and that Rhodothermus marinus and Thermus thermophilus dominated the cultivable microbial community.

Improving Sensitivity of Enzyme to Organophosphorous Compounds by Combining Experiment and Theory Methods

In this paper,we compared the sensitivities of AFEST（a thermophilic esterase from the archaea Archaeoglobus fulgidus） and acetylcholinesterase（AChE） towards five organophosphorus compounds（OPs） by means of molecular docking,and found that only the docking energy between AFEST and dichlorvos is lower than that between AChE and dichlorvos.Via the docking model of AFEST and dichlorvos,Arg43 was found to play an important role in the interaction between AFEST and dichlorvos by means of stabilizing the complex.Then mutant R43S was constructed,the IC 50（the concentration required to reduce virus-induced cytopathicity by 50% is estimated as 50% inhibitory concentration） of which to dichlorvos was lower than that of the wild type AFEST by a factor of 1.56,indicating the enhanced sensitivity of mutant R43S to dichlorvos.Combining of theory with experiment,we have obtained important structure-function information of AFEST,which will be helpful to the further studies of esterase.

Recent Advances in Microbial Degradation of 4-hydroxybenzoate-a Review

4-Hydroxybenzoate （4HBA） is a naturally occurring aromatic compound, as a key intermediate metabolite not only for natural products but also for arti- ficial products. There are four metabolic pathways for 4HBA： protocatechuate cleavage pathway; catechol cleavage pathway; anaerobic degradation pathway in anaerobes ; and gentisate cleavage pathway. The last pathway including a NIH shift reaction remains to be elucidated. In this review we emphasized on the NIH shift reaction involved in the 4HBA degrdation. The key enzymes of each 4HBA metabolic pathway also were introduced. Finnaly, we described the thermophilic Bacillus sp. B1 strain which was capable of degrading varous aromatic compounds including gHBA, and presented a direction for the research of NIH shift reaction.

Degradation of Phenolic Compounds in Creosote Treated Wood Waste by A Mixed Microbial Culture Augmented with Cellulolytic- Thermophilic Actinomaycets <i>Thermobifida fusca</i>

Creosote is used for preservation of railway ties and timbers, electric utility poles, marine and foundation piling, fences and garden furniture. Creosote-treated wood waste may cause potential contamination of soil and water if they are not disposed properly. Creosote contains over 300 organic compounds including polycyclic aromatic hydrocarbons, phenolic compounds and heterocyclic organic compounds, many of which are toxic to human and can cause damage to kidney, liver, eyes and skin. The feasibility of using a composting technique inoculated with the cellulose degrading actinomycetesThermobifidafusca as a mesophilic/thermophilic bioremediation option to degrade phenolic compounds in creosote treated wood waste was evaluated. The temperature profile of bioremediation process clearly identified mesophilic and thermophilic phases in both experiments. Different degradation rates were observed in the mesophilic and thermophilic phases. Fluctuations of pH was observed in both experiment as the result of the breakdown of organic nitrogen to ammonium in the first week and the formation of organic acids and the loss of ammonium with the exhaust gases in the latter stage. The moisture content decreased in both trials because of the net loss of water with the exhaust gas. Both experiments achieved similar reductions in total carbon and TKN, volatile solids and phenolic compounds, cellulose and lignin indicating similar levels of microbial activities during the composting process. The stability and maturity of the final products were also similar. The inoculation of the cellulolytic-thermophilicactinomycetesThermobifidafusca did not manifest observable differences in degrading cellulose, lignin and phenolic compounds compared with the control.

Carbon utilization profile of a thermophilic fungus, <i>Thermomyces lanuginosus</i>using phenotypic microarray

The thermophilic filamentous fungus, Thermomyces lanuginosus produces the largest amount of xylanase reported. In addition to this, it expresses large amount of other enzymes that have been used industrially or have academic interest. Thus, this fungus has a potential to be applied for biomass conversion to produce biofuel or other applications. In this study, the Biolog system was used to characterize the utilisation and growth of T. lanuginosus on 95 carbon sources. The carbohydrates based compounds, both single sugars and oligosaccharide, showed the best utilisation profile, with the pentose sugar xylose inducing the highest growth, followed by trehelose, raffinose, D-mannose turanose fructose and glucose. Among oligosaccharides, sucrose had the highest mycelium formation followed by stachyose, maltose, maltotriose, glycogen and dextrin. Interestingly the fungus also grew well on cellobiose suggesting that this fungus can produce cellulose hydrolysing proteins. D-alanine was the best amino acid to promote fungal growth while the effect of other amino acids tested was similar to the control. These results demonstrate the ability of this fungus to grow relatively well on most plant based compounds thus making this fungus a possible candidate for plant biomass conversion which can be applied to a number of biotechnological applications including biofuel production.

Comparison of ASBR and CSTR reactor for hydrogen production from palm oil mill effluent under thermophilic condition

Hydrogen production from palm oil mill effluent (POME) by Thermoanaerobacterium thermosaccharolyticum PSU-2 was investigated both in batch and continuous reactors using anaerobic sequencing batch reactor (ASBR) and continuous stirred tank reactor (CSTR). The hydrogen production determined from batch experiment of POME at an inoculum size of 0%, 10%, 20% and 30% (v/v) was 161, 201, 246 and 296 mL H2/g-COD with COD removal efficiency of 21%, 23%, 23% and 23%, respectively. Continuous hydrogen production was start-up with 30% (v/v) inoculum in both ASBR and CSTR reactors and more than 30% COD removal could be obtained at HRT of 4 days, corresponding to OLR of 11.3 g COD/ L·day. Similar hydrogen production rates of 2.05 and2.16 LH2/L. day were obtained from ASBR and CSTR, respectively. COD removal efficiency of ASBR was 37.7%, while it was 44.8% for CSTR. However, ASBR was stable in term of alkalinity, while the CSTR was stable in term of hydrogen production, soluble metabolites concentration and alkalinity. Therefore, the CSTR was found to be more stable in hydrogen production than ASBR under the same OLR.