Hydrolysis of newspaper polysaccharides under sulfate reducing and methane producing conditions

The initial decomposition rates of cellulose and hemicellulose were measured using toluene to specifically inhibit the microbial uptake of hydrolysis products during the degradation of newspaper under sulfate reducing and methane producing conditions. The amount of glucose and xylose accumulation in the first 2 weeks of incubation period was higher in the sulfate reducing condition compared to the methane producing condition. It was estimated that 28 and 6% of initially loaded cellulose in the sulfate reducing condition and the methane producing condition was hydrolyzed, respectively. Accordingly, the newspaper-cellulose hydrolysis rate constant was estimated to be 6.7 times higher in sulfate reducing condition than in methane producing condition. Based on the glucose accumulation patterns, when sulfate reducing bacteria (SRB) were inhibited by anthraquinone and molybdate (Na₂MoO₄), it may be suggested that SRB might have contributed to the hydrolysis of cellulose, while their effect on the hydrolysis of hemicellulose could not be elucidated.     

Development of an enzymatic assay for the determination of cellulose bioavailability in municipal solid waste

As there is a constant need to assess the biodegradation potential of refuse disposed of in landfills, we have developed a method to evaluate the biodegradability of cellulosic compounds (cellulose and hemicellulose) in municipal solid waste. This test is based on the quantification of monosaccharides released after the hydrolysis of solid waste samples with an optimised enzyme preparation containing commercially available cellulases and hemicellulases. We show that the amounts of monosaccharides could be related to the biodegradability of the cellulosic material contained in the samples. This enzymatic cellulose degradation test was assayed on 37 samples originating from three Belgian landfills and collected at different depths. As results correlated well with those obtained with a classical biochemical methane potential assay, this new and rapid test is sufficiently reliable to evaluate cellulose bioavailability in waste samples.     

The effects of certain antibiotics on biogas production in the anaerobic digestion of pig waste slurry

Antibiotics commonly used in the treatment of pigs - amoxicillin trihydrate, oxytetracycline hydrochloride and thiamphenicol were added at different concentrations to aliquots of pig waste slurry plus anaerobic sludge in serum bottles. The biogas production and methane concentration in the headspace were monitored to determine the effect of the antibiotics on the anaerobic process. With thiamphenicol significant differences in methane production were found for concentrations of 80 and 160 mg l(-1) slurry. Compared to the control, only minor differences in methane production were noted in the bottles to which amoxicillin (60 and 120 mg l(-1)) had been added. Methane production was about the same for the bottles with different oxytetracycline concentrations (125 and 250 mg l(-1)) and for the control.     

添加剂对水稻秸秆实地混合厌氧发酵产甲烷系统的作用

为提高水稻秸秆利用效率,改进水稻秸秆实地混合厌氧发酵产甲烷技术,本研究开展了添加剂在混合厌氧发酵系统的应用研究。试验选取水稻秸秆和猪粪作为发酵原料,通过分别添加不同的浓度的吐温20和腐植酸,测定甲烷的产气量和浓度、秸秆的降解和土壤肥力的变化,以揭示添加剂类型及其浓度对水稻田实地甲烷生产系统的影响。结果表明:添加剂的掺入并未影响产气的动态变化趋势,但显著地促进产气和提高产气浓度,整体效果由高到低为腐植酸吐温20对照,其中经腐植酸处理的产气量和产气浓度对照相比分别提高了50. 73%和24. 55%。添加剂的掺入有利于水稻秸秆纤维素和半纤维素的降解,但对木质素没有显著影响;其中以0. 15 g/L腐植酸和0. 30 g/L吐温20的降解率最高,相较于对照其纤维素降解率均提高了22. 11%,半纤维素降解率分别提高了107. 13%和98. 39%。添加剂的掺入能显著增加土壤肥力,以0. 15 g/L腐植酸和0. 30 g/L吐温20处理水平的效果最优,相较于对照,其土壤有机质分别增加了29. 63%和23. 72%,全氮分别增加了52. 32%和42. 38%,全磷分别增加了83. 33%和57. 14%。     

Comparative performance of enzymatic and combined alkaline-enzymatic pretreatments on methane production from ensiled sorghum forage

This study investigated the effect of enzymatic and combined alkaline-enzymatic pretreatments on chemical composition and methane production from ensiled sorghum forage. Four commercial enzymatic preparations were tested and the two yielding the highest sugars release were added to evaluate any hydrolytic effect on both untreated and alkaline pretreated samples. In the combined alkaline-enzymatic pretreatment trials, the highest sugar release was found with Primafast and BGL preparations (added at a final concentration 0.12 and 0.20 mL/g TS, respectively), with a total monomeric content of 12 and 6.5 g/L. Fibre composition analysis confirmed that the combined alkaline-enzymatic pretreatment led to cellulose (up to 32 %) and hemicelluloses (up to 56 %) solubilisation, compared to the enzymatic pretreatment alone. BMP tests were performed on both untreated and pretreated samples, and time courses of methane production were fitted. Both enzymatic and combined alkaline-enzymatic pretreatment led to a methane production increase (304 and 362 mL CH₄/g VS), compared to that of untreated sorghum (265 mL CH₄/g VS), as  +15 and  +37 %, respectively. Moreover, higher specific methane production rates, compared to that of untreated sorghum (20.31 mL CH₄/g VS/d), were obtained by applying the enzymatic and combined alkaline-enzymatic pretreatment (33.94 and 31.65 mL CH₄/g VS/d), respectively.     

Bacterial community changes in TCE biodegradation detected in microcosm experiments

Laboratory microcosm experiments were set up to model biodegradation of trichloroethylene (TCE). Groundwater samples from two contaminated sites were taken, one of them with low (70 mg L⁻¹), the other with high sulfate (685 mg L⁻¹) concentration. In order to assess the biodegradative potential of natural microbiota, supplementary substrates (whey or molasses) were added to the bottles. At day 54, 98, 155, and 318, chemical and bacteriological parameters (i.e., Dehalococcoides test) were investigated. Terminal restriction fragment length polymorphism (T-RFLP) based diversity assessments were carried out to observe the bacterial community changes. Whey and molasses enhanced degradation at different rates. In the case of samples with high sulfate content and amended with whey, no ethylene, ethane, or methane was generated. Both ethylene and methane production was detected in samples of low sulfate content with added whey. The results of Dehalococcoides tests were positive for all control and amended samples. Based on T-RFLP analysis, the bacterial communities of high sulfate concentration groundwater microcosms amended with molasses or whey were very similar, while the communities of groundwater samples with low sulfate concentration were different when supplemented with whey or molasses. The rRNA and rDNA based investigations suggest that the proportions of the active microbes and the microbes present in the microcosms differ.     

Prevention of formation of acid drainage from high-sulfur coal refuse by inhibition of iron- and sulfur-oxidizing microorganisms. I. Preliminary experiments in controlled shaken flasks

Changes of pH and sulfate concentration in high-sulfur coal refuse slurries are used as measurements of microbial pyrite oxidation in the laboratory. Sodium lauryl sulfate (SLS), alkylbenzene sulfonate (ABS), benzoic acid (BZ) and combinations of SLS plus BZ and ABS plus BZ effectively inhibited formation of sulfate and acid when added in concentrations greater than 50 mg/L to inoculated 20 or 30% coal refuse slurries. Here 25 mg/L concentrations of SLS, ABS, and ABS + BZ stimulated acid production. Formic, hexanoic, oxalic, propionic, and pyruvic acids at 0.1% concentrations were also effective inhibitors. Four different lignin sulfonates were only slightly effective inhibitors at 0.1% concentrations. It was concluded that acid formation resulting from microbial oxidation in high-sulfur coal refuse can be inhibited.     

Bacterial community changes in TCE biodegradation detected in microcosm experiments

Laboratory microcosm experiments were set up to model biodegradation of trichloroethylene (TCE). Groundwater samples from two contaminated sites were taken, one of them with low (70 mg L⁻¹), the other with high sulfate (685 mg L⁻¹) concentration. In order to assess the biodegradative potential of natural microbiota, supplementary substrates (whey or molasses) were added to the bottles. At day 54, 98, 155, and 318, chemical and bacteriological parameters (i.e., Dehalococcoides test) were investigated. Terminal restriction fragment length polymorphism (T-RFLP) based diversity assessments were carried out to observe the bacterial community changes. Whey and molasses enhanced degradation at different rates. In the case of samples with high sulfate content and amended with whey, no ethylene, ethane, or methane was generated. Both ethylene and methane production was detected in samples of low sulfate content with added whey. The results of Dehalococcoides tests were positive for all control and amended samples. Based on T-RFLP analysis, the bacterial communities of high sulfate concentration groundwater microcosms amended with molasses or whey were very similar, while the communities of groundwater samples with low sulfate concentration were different when supplemented with whey or molasses. The rRNA and rDNA based investigations suggest that the proportions of the active microbes and the microbes present in the microcosms differ.     

Effect of carbon monoxide on fermentation of fiber, starch, and amino acids by mixed rumen microorganisms in vitro.

When 1 atm (101.3 kPa) of carbon monoxide was added to mixed rumen bacterial incubations containing timothy hay, methane production was inhibited by 88% without an increase in hydrogen. The molar ratio of propionate to acetate increased from 0.83 to 1.53, extracellular ammonia declined from 5.2 to 2.4 mM, and hemicellulose and cellulose digestions were inhibited by 40 and 27%, respectively. Even low levels of carbon monoxide (less than 0.1 atm [10.13 kPa]) significantly changed the products of fermentation. With starch, methane production was once again inhibited, but the magnitude of starch fermentation was unaffected. Decrease in acetate was accompanied by an equal molar increase in lactate. Ammonia production from the amino acid source, Trypticase, declined 20% as carbon monoxide was increased to 1.0 atm, and 93% of this decrease was explained by a selective inhibition of branched-chain amino acid fermentation.     

Effect of carbon monoxide on fermentation of fiber, starch, and amino acids by mixed rumen microorganisms in vitro

When 1 atm (101.3 kPa) of carbon monoxide was added to mixed rumen bacterial incubations containing timothy hay, methane production was inhibited by 88% without an increase in hydrogen. The molar ratio of propionate to acetate increased from 0.83 to 1.53, extracellular ammonia declined from 5.2 to 2.4 mM, and hemicellulose and cellulose digestions were inhibited by 40 and 27%, respectively. Even low levels of carbon monoxide (less than 0.1 atm [10.13 kPa]) significantly changed the products of fermentation. With starch, methane production was once again inhibited, but the magnitude of starch fermentation was unaffected. Decrease in acetate was accompanied by an equal molar increase in lactate. Ammonia production from the amino acid source, Trypticase, declined 20% as carbon monoxide was increased to 1.0 atm, and 93% of this decrease was explained by a selective inhibition of branched-chain amino acid fermentation.     

Biochemical methane potential and biodegradability of complex organic substrates

The biomethane potential and biodegradability of an array of substrates with highly heterogeneous characteristics, including mono- and co-digestion samples with dairy manure, was determined using the biochemical methane potential (BMP) assay. In addition, the ability of two theoretical methods to estimate the biomethane potential of substrates and the influence of biodegradability was evaluated. The results of about 175 individual BMP assays indicate that substrates rich in lipids and easily-degradable carbohydrates yield the highest methane potential, while more recalcitrant substrates with a high lignocellulosic fraction have the lowest. Co-digestion of dairy manure with easily-degradable substrates increases the specific methane yields when compared to manure-only digestion. Additionally, biomethane potential of some co-digestion mixtures suggested synergistic activity. Evaluated theoretical methods consistently over-estimated experimentally-obtained methane yields when substrate biodegradability was not accounted. Upon correcting the results of theoretical methods with observed biodegradability data, an agreement greater than 90% was achieved.     

Wet oxidation pretreatment for the increase in anaerobic biodegradability of newspaper waste

Wet oxidation was investigated for its process performance on methane fermentation of newspaper waste. The mechanisms of solubilization of newspaper waste were investigated using the following criteria: destruction of total COD (TCOD), production of soluble COD (SCOD), production of volatile fatty acids, production of soluble carbohydrates, production of soluble lignin derivatives (SLD), production of furan (F) and destruction of lignin and cellulose. Wet oxidation was carried out at 170, 190, and 210 degrees C, with a retention time of 1 h. The highest removal efficiencies of TCOD and cellulose were achieved at 210 degrees C, approximately 40% and 69% were destroyed, respectively. On the other hand, highest lignin removal efficiency was achieved at 190 degrees C in which approximately 65% was removed. Batch methane fermentation tests were performed in 2-l glass bottles filled with the wet oxidized newspaper samples. Methane fermentation of newspaper pretreated at 190 degrees C gave the highest CH(4) conversion efficiency (59% of the initial TCOD was recovered as CH(4) gas). Anaerobic cellulose removals varied from 74% to 88%.     

Solid-state anaerobic digestion of spent wheat straw from horse stall

The spent wheat straw from horse stall bedding has lower cellulose and hemicellulose contents, but higher volatile fatty acid content than raw wheat straw. Biogas production from solid-state anaerobic digestion (SS-AD) of spent wheat straw and raw wheat straw was compared in this study. The SS-AD tests were conducted at 22% total solids (TS) content using inoculum from a liquid AD system at three feedstock-to-inoculum (F/I) ratios of 2.0, 4.0, and 6.0. Daily methane yields of spent wheat straw peaked 8 and 3 days earlier than those of raw wheat straw at F/I ratios of 2.0 and 4.0, respectively. The highest methane yield of 150.0 L/kg volatile solids (VS) was obtained from spent wheat straw at an F/I ratio of 4.0, which was 56.2% higher than that of raw wheat straw. The corresponding cellulose and hemicellulose degradation of spent wheat straw was 24.1% and 49.4% higher than those of raw wheat straw, respectively.     

Can Biomethane Potential (BMP) Be Predicted from Other Variables Such As Biochemical Composition in Lignocellulosic Biomass and Related Organic Residues?

The potential of methane production by anaerobic digestion of lignocellulosic biomass depends not only on the availability of the resources in the considered territory, but also on their physico-chemical characteristics. Relevant methods of characterization are, therefore, needed to select and possibly combine the most appropriate biomass substrates in order to optimize energy recovery through anaerobic digestion processes. The objective of the present study was to determine whether biomethane potential of such substrates could be predicted from a limited number of variables more rapidly or determined more easily. A set of 36 biomass substrates and organic residues from a variety of origins was analyzed for total and easily hydrosoluble organic matter fractions (volatile solid, VS and soluble chemical oxygen demand, SCOD), neutral detergent soluble fraction (SOL), hemicelluloses (HEM), cellulose (CELL), and lignin-like residual fractions (RES). Bioreactivity of all samples was also measured by experimental assays (biochemical oxygen demand, BOD and biochemical methane potential, BMP). The whole set of data thereby obtained was analyzed statistically considering one dependent variable (BMP), and six independent variables (SCOD, SOL, HEM, CELL, RES, and BOD). Partial least square (PLS) analysis revealed very clearly a positive correlation between BMP and BOD, which were both anti-correlated with RES. On the other hand, no correlations were observed between BMP, SCOD, HEM, and CELL contents. PLS analysis showed that BMP was significantly correlated to the six independent variables. The most influential variables were found to be RES and BOD, and a polynomial model was successfully validated for the prediction of BMP from RES and BOD.     

Hydrolytic enzyme activity in landfilled refuse

Extracellular hydrolytic enzyme activity was assayed in 28 refuse samples excavated from 14 bore holes in Fresh Kills Landfill, Staten Island, N. Y. Esterases, proteases and amylases were present in all of the samples. Enzyme screening assays utilizing the API-ZYM test system showed the incidence of enzymes in the order: specific phosphatases > esterases > glycosyl hydrolases. Measurement of cellulase by the cellulose-azure test detected activity in two out of 28 samples. Analysis for cellulase activity using the cellulose-azure test on refuse samples from landfills in Naples, Florida, and Tucson, Arizona, also showed a limited distribution of cellulases. Mineralization of [¹⁴C]cellulose, an independent measure of cellulase activity, ranged from < 5 to 23% in a 4-week incubation, which supports a highly variable cellulolytic activity in landfilled refuse. Correspondence to: A. C. Palmisano     

工业纤维素诱导里氏木霉RUT C-30产葡聚糖酶的条件

为了研究工业纤维素诱导里氏木霉RUT C-30产葡聚糖酶的最佳条件,根据单因素实验结果,以工业纤维素、(NH4)2SO4和生物素为实验因素,滤纸酶活为响应值,进行中心组合设计,建立一个二次多项式数学模型,进行响应面优化,寻找最优产酶结果.经过优化,选出工业纤维素、(NH4)2SO4和生物素的添加量分别为39.485 g/L、6.232 g/L和249.872 μg/L,最高的滤纸比酶活为6.298 U/mL,实验验证,滤纸比酶活为6.118 U/mL,与预测值相差了2.86%.     

Effects of Prechilling and Sequential Washing on Enumeration of Microorganisms from Refuse

Techniques were evaluated for formation of a liquid inoculum from shredded municipal refuse, including chilling the refuse at 4°C prior to blending and multiple washing and blending cycles. The average count of cellulolytic bacteria from six different detachment treatments was 5.1 × 10⁴ cells per g (dry weight) of refuse with a range of 0.7 × 10⁴ to 12.7 × 10⁴ cells per g (dry weight). The liquid obtained from blending the refuse in phosphate buffer followed by hand squeezing was the selected detachment procedure. The inoculum formation procedure was validated by the addition of ruminal cellulolytic bacteria to refuse and recovery of the cellulolytic bacteria by most-probable-number enumerations. The ratio of measured to expected cell counts among tests in which different volumes of ruminal fluid were added to refuse ranged from 2.7 to 14.4. There was no evidence of anaerobic cellulolytic fungi in a refuse sample.     

Quantification and monosaccharide composition of hemicelluloses from different plant functional types

Hemicelluloses are the second most abundant polysaccharide in nature after cellulose. So far, the chemical heterogeneity of cell-wall hemicelluloses and the relatively large sample-volume required in existing methods represent major obstacles for large-scale, cross-species analyses of this important plant compound. Here, we apply a new micro-extraction method to analyse hemicelluloses and the ratio of ‘cellulose and lignin’ to hemicelluloses in different tissues of 28 plant species comprising four plant functional types (broad-leaved trees, conifers, grasses and herbs). For this study, the fiber analysis after Van Soest was modified to enable the simultaneous quantitative and qualitative measurements of hemicelluloses in small sample volumes. Total hemicellulose concentrations differed markedly among functional types and tissues with highest concentration in sapwood of broad-leaved trees (31% d.m. in Fraxinus excelsior) and lowest concentration between 10 and 15% d.m. in leaves and bark of woody species as well as in roots of herbs. As for total hemicellulose concentrations, plant functional types and tissues exhibited characteristic ratios between the sum of cellulose plus lignin and hemicelluloses, with very high ratios (>4) in bark of trees and low ratios (<2) in all investigated leaves. Additional HPLC analyses of hydrolysed hemicelluloses showed xylose to be the dominant hemicellulose monosaccharide in tissues of broad-leaved trees, grasses and herbs while coniferous species showed higher amounts of arabinose, galactose and mannose. Overall, the micro-extraction method permitted for the simultaneous determination of hemicelluloses of various tissues and plant functional types which exhibited characteristic hemicellulose concentrations and monosaccharide patterns.     

Determination of cellulose and apparent hemicellulose in plant tissue by gas-liquid chromatography

A GLC method is described by which the neutral aldoses and cellulose can be measured in whole and digested plant tissue. Apparent hemicellulose is measured by difference. Accuracy of the method is unaffected by protein in concentrations up to 75%.     

三株高效秸秆纤维素降解真菌的筛选及其降解效果

【目的】利用多种筛选方法,获得高效秸秆纤维素降解真菌,并研究其秸秆纤维素的降解能力。【方法】采用滤纸片孔洞法、滤纸条降解法、羧甲基纤维素钠(CMC-Na)水解圈测定法、秸秆失重法、纤维素分解率测定法、胞外酶活测定法等常规秸秆纤维素降解菌的筛选方法。【结果】筛选到3株具有较强纤维素降解能力的真菌菌株,经初步鉴定菌株98MJ为草酸青霉(Penicillium oxalicum)、菌株W3为木霉(Trichoderma sp.)、菌株W4为扩张青霉(Penicillium expansum)。菌株W4具有非常强的秸秆纤维素降解能力,10d内对秸秆的降解率可达56.3%,对纤维素、半纤维素和木质素的分解率分别为59.06%、78.75%和33.79%。菌株W4的胞外纤维素酶活力在14.25-49.75U/mL之间。【结论】筛选获得3株高效秸秆纤维素降解真菌菌株,其中菌株W4的纤维素酶活高于已报道的菌株,是一株十分具有研究开发潜力的纤维素酶生产菌株。