The influence of lignin content and temperature on the biodegradation of lignocellulose in composting conditions

The aim of this research was to study the influence of lignin content and composting temperature on the biodegradation of lignin-containing pulp and paper products in a controlled composting test (European standard prEN 14046). Lignin reduced the biodegradation of the samples, and there was a linear correlation between the lignin content and the biodegradation of pulp and paper products at 58°C. The influence of incubation temperature (35, 50 and 58°C) on biodegradation was studied using bleached kraft paper containing 0.2 wt% lignin and mechanical pulp (stone-ground wood) containing 24–27 wt% lignin. Mechanical pulp biodegraded better at lower temperatures, while kraft paper biodegraded well at all three temperatures. Microbial activity was evaluated by measuring CO₂ evolution and the change in ATP content, and fungal biomass by measuring the ergosterol content during the composting experiments. Kraft paper strongly increased microbial activity during the controlled composting test, but the activity returned to the background level at the end of the composting test. The proportion of sample carbon converted to microbial biomass carbon was considerably higher at lower incubation temperatures. Changes in microbial community structure during biodegradation of mechanical pulp and kraft paper at 50°C were studied by the PCR-based technique denaturing gradient gel electrophoresis. Changes in the microbial community were observed during the intensive degradation phase of kraft paper. Electronic Publication     

Microbial degradation and humification of the lawn care pesticide 2,4-dichlorophenoxyacetic acid during the composting of yard trimmings.

The fate of the widely used lawn care herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) during the composting of yard trimmings consisting of primarily leaves and grass is an important unexplored question. In this study, we determined the extent of 2,4-D mineralization, incorporation into humic matter, volatilization, and sorption during the composting of yard trimmings. Yard trimmings (2:1 [wt/wt] leaves-grass) were amended with 14C-ring-labeled 2,4-D (17 mg/kg of dry weight) and composted in a temperature-controlled laboratory scale compost system. During composting, thermophilic microbes were numerically dominant, reaching a maximum of 2 x 10(11)/g. At the end of composting, 46% of the organic matter (OM) present in the yard trimmings was lost and the compost was stable, with an oxygen uptake rate of 0.09 mg of O2 per g of OM per h, and was well humified (humification index, 0.39). Mineralization of the OM temporally paralleled mineralization of 2,4-D. In the final compost, 47% of the added 2,4-D carbon was mineralized, about 23% was complexed with high-molecular-weight humic acids, and about 20% was not extractable (humin fraction). Less than 1% of the added 14C was present in water expressed from the finished compost, suggesting a low potential for leaching of 2,4-D. Very little volatilization of 2,4-D occurred during composting. It is of interest that our results indicate active mineralization of 2,4-D at composting temperatures of 60 degrees C because microbial 2,4-D degradation at thermophilic temperatures has not been previously documented.     

废次烟叶提取液源木质素降解菌Bacillus subtilis SM降解特性

【目的】目前造纸法再造烟叶工艺已经成为我国重要的废烟叶处理和利用方式,该工艺中烟梗中高木质素的降解是个挑战性的需解决问题。从废次烟叶提取液(Tobacco waste extract,TWE)中筛选木质素的降解微生物用来直接处理烟梗或烟末提取液,可实现对木质素含量的调控。【方法】将废次烟叶提取液(TWE)浓缩液中分离出的Bacillus subtilis SM接种到以Kraft木质素为唯一碳源的无机盐培养基中,在pH 7.0、30°C培养基中培养4 d来检测菌株对木质素的降解效果。通过HPLC、TOC、GPC和色度来表征SM对木质素的降解,并采用烟梗无机盐培养基在pH 7.0、30°C培养4 d检测SM对烟梗木质素的降解。【结果】HPLC结果显示SM在以木质素磺酸钠为唯一碳源的无机盐培养基中可全部降解分子质量为534.5的木质素磺酸钠,而对Kraft木质素降解不明显,仅观察到组分的变化。脱色结果显示脱色率达到40.7%,但在对Kraft木质素矿化方面矿化率只能达到5.4%。SM在烟梗无机盐培养基中可使烟梗失重率分别达到50%以上(对照组为18.9%),烟梗中木质素含量减少了70%左右。【结论】来源于废次烟叶提取液(TWE)的Bacillus subtilis SM能够以Kraft木质素为唯一碳源生长,也能够有效降解烟梗中的木质素,可应用于烟草废弃物原料中木质素的降解。     

Persistence of Mycobacterium avium subsp. paratuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure

Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis (Mycobacterium paratuberculosis). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated M. paratuberculosis, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55 degrees C, manure packing at 25 degrees C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with M. paratuberculosis at 10(6) CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of M. paratuberculosis was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO2, while no significant change in carbon content was observed in the liquid slurry. Escherichia coli, Salmonella, and Listeria were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55 degrees C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days. M. paratuberculosis was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56, M. paratuberculosis was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However, M. paratuberculosis DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.     

Pyrene mineralization capacity increases with compost maturity

Experiments were conducted to determine the effects of composting or simple addition of compost to the mineralization of n-hexadecane, pyrene and benzo(a)pyrene in soil. Soil (contaminated or clean) was composted with maple leaves and alfalfa. Samples from different composting phases were spiked with radiolabeled and cold n-hexadecane, pyrene or benzo(a)pyrene, placed in aerated microcosms at different temperatures, and monitored for mineralization. It was determined that neither composting nor the addition of compost had any effect on n-alkane or benzo(a)pyrene mineralization. In contrast, the pyrene mineralization rate increased dramatically with the amount of time that soil had been composted. Highest pyrene mineralization rates and extents (more than 60% after 20 days) were obtained when pyrene was in contact with composted soil from the curing stage. Neither thermophiles (55 °C) nor fungi were responsible for pyrene mineralization.     

Thermophilic anaerobic biodegradation of [C]lignin, [C]cellulose, and [C]lignocellulose preparations

Thermophilic (55 degrees C) anaerobic enrichment cultures were incubated with [C-lignin]lignocellulose, [C-polysaccharide]lignocellulose, and kraft [C]lignin prepared from slash pine, Pinus elliottii, and C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

Production of manganese peroxidase and organic acids and mineralization of 14C-labelled lignin (14C-DHP) during solid-state fermentation of wheat straw with the white rot fungus nematoloma frowardii

The basidiomycetous fungus Nematoloma frowardii produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic 14C-ring-labelled lignin (14C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as 14CO2, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the 14C-DHP to 14CO2 (which accounted for up to 8% of the initial radioactivity added) and 14C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).     

Development of models for predicting carbon mineralization and associated phytotoxicity in compost-amended soil

Phytotoxicity of compost-amended soil is related to carbon mineralization associated with compost decomposition. The objective of this research was to determine if compost carbon mineralization potential, estimated using compost respiration rate measurements, could be combined with carbon mineralization kinetic models to predict phytotoxicity of compost-amended soil. First-order, second-order, and Monod kinetic models that include compost carbon mineralization potential, compost amendment rate, incubation time, and temperature were developed and compared for their ability to predict carbon mineralization kinetics. Experiments utilized two soil types amended with 0%, 5%, and 50% (v/v) food waste and green waste composts, incubated at 20 degrees C, 25 degrees C, 30 degrees C, 35 degrees C, and 45 degrees C for model development and under a diurnal temperature cycle from 20 degrees C to 30 degrees C for model validation. For most cases, a first-order model had an equivalent or better fit to the data than the other models. Mineralizable carbon estimated using the first-order model was significantly correlated to the probability of phytotoxicity in compost-amended soil.     

Thermophilic Anaerobic Biodegradation of [14C]Lignin, [14C]Cellulose, and [14C]Lignocellulose Preparations

Thermophilic (55°C) anaerobic enrichment cultures were incubated with [¹⁴C-lignin]lignocellulose, [¹⁴C-polysaccharide]lignocellulose, and kraft [¹⁴C]lignin prepared from slash pine, Pinus elliottii, and ¹⁴C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

Ash in composting of source-separated catering waste

Our earlier experiments in small composters (220 l) indicated the favourable effect of ash from co-incineration of sorted dry waste on the composting of catering waste. The aim of this new study was to clarify further, at a scale of 10 m3, the feasibility of using similar ash as an additive in composting. Source-separated catering waste was mixed with bulking agent (peat and wood chips) and fuel ash from a small (4 MW) district heating power plant. Three compost mixes (CM) were obtained: CM I with 0%, CM II with 10% and CM III with 20 wt.% of fuel ash. These three different mixes were composted in a 10-m3 drum composter as three parallel experiments for 2 weeks each, from January to April 2000. After drum composting, masses were placed according to mixing proportions in separate curing piles. The catering waste fed to the drum was cold, sometimes icy. Even then the temperature rapidly increased to over 50 degrees C. In CM III, the temperature rose as high as 80 degrees C, and after the first week of composting the temperature was about 20 degrees C higher in the CMs II and III than in the CM I. It also improved the oxygen concentrations at the feeding end of the drum and obviously prevented the formation of H2S. No odour problems arose during the composting. Addition of ash increased the heavy metal contents of the composting masses, but the compost was suitable for cultivation or green area construction. Ash clearly decreased the loss of total nitrogen in a time span of 2 years. The lower amounts of nitrogen mean that the amounts applied per hectare can be greater than for normal composts. Measured by mineralization, the breaking down of the organic matter was more rapid in the CM III than in the CM I. Humic acid increased steadily during first 12 months composting, from the initial 39 mg/g organic matter to 115 and 137 mg/g in CMs II and III. Measured by temperature, mineralization and humification the addition of ash appeared to boost the composting. Ash had also other beneficial effects on composting it improved the availability of oxygen in compost mass during the drum composting phase and reduced the formation of odorous gases, especially H2S.     

Transformation of organic matter during co-composting of pig manure with sawdust

Co-composting of pig manure with sawdust was studied in order to characterize the organic transformation during the process, using both chemical and spectroscopic methods. Humic acids (HA) and fulvic acids (FA) were fractionated from immature and mature pig manure compost, and characterized. After 63 days of composting, the ratio of total organic carbon and soluble organic carbon decreased to a satisfactory low level and the solid and soluble C/N ratios decreased rapidly for the first 35 days before attaining a constant value, indicating compost maturity. Humification could be responsible for the increase in humic acid proportion during composting. The increase in the aromatic bonds after composting, as indicated by the reduction of C/H and C/O ratios of HA and FA, resulted in a more stabilized product. A substantial increase in high molecular weight compounds along with a small increase in low molecular weight compounds was found in mature compost. Moreover the HA also had more complex organic compounds at this stage. Fluorescence spectral analysis showed an increase in the maximum wavelength of HA associated with the contents of aromatic structures in solution. A decrease in relative absorbance of HA at 1160 cm(-1), 2950 cm(-1) and 2850 cm(-1) was seen in the FTIR spectra indicating the decomposition of complex organic constituents, into simpler ones. Increase in the aromatic compounds with higher stability could account for the relative increase in the absorbance of HA at 1650 cm(-1) and 1250 cm(-1) of the mature compost. The composition of FA was not much altered, indicating most of the degradation of organic matter occurred in HA. Data from organic carbon, C/N ratio, elemental analysis, E(4)/E(6) ratio, gel chromatography, fluorescence and FTIR spectra indicated an increase in polycondensed structures and the presence of more stable organic matter in the mature compost.     

Photochemical mineralization of synthetic lignin in lake water indicates enhanced turnover of aromatic organic matter under solar radiation

The degradation of ¹⁴C-[ring]-labelled syntheticlignin (¹⁴C-DHP) and dissolved organic carbon(DOC) from lake water were studied simultaneously.¹⁴C-DHP was incubated in humic lake water (colour173 mg Pt l^-1) for 7 d in the dark or under solarradiation. In the dark <0.4% of the introduced¹⁴C-DHP label and 4% of the indigenous DOC weremineralized, indicating that the ¹⁴C-labelledaromatic rings of DHP and the humic DOC weremicrobiologically recalcitrant. Under solar radiation(116 MJ m^-2), 17–21% of the ¹⁴C-labelledcarbons in DHP and 18–23% of the indigenous DOC weremineralized in 7 d. Simultaneously the watersolubility of ¹⁴C-DHP increased. Solar radiationconverted the aromatic cores of synthetic lignin toCO₂ and soluble organic photoproducts. Theresults suggest that solar radiation plays a key rolein the decomposition of natural polyaromatic matter.     

Nitrogen loss in chicken litter compost as affected by carbon to nitrogen ratio and turning frequency

The study was undertaken to investigate the effects of carbon to nitrogen (C:N) ratio and turning frequency (TF) on the loss of total nitrogen (TN) during composting of chicken litter (a mixture of chicken manure, waste feed, feathers and sawdust) with a view to producing good quality compost. Carbon to nitrogen ratios of 20:1, 25:1 and 30:1 and TF of 2, 4 and 6 days were experimented. The initial physico-chemical properties of the litter were determined. During the composting process, moisture level in the piles was periodically replenished to 55% and the temperature, pH and TN of the chicken litter were periodically monitored. Also, the dry matter (DM), total carbon (TC), total phosphorus (P) and total potassium (K) were examined at the end of composting. The results showed that both C:N ratio and TF had significant (p < or = 0.05) effect on pile temperature, pH changes, TN, TC, P and K losses while DM was only affected (p < or = 0.05) by C:N ratio. All treatments reached maturation at about 87 days as indicated by the decline of pile temperatures to near ambient temperature. Losses of TN, which were largely attributed to volatilization of ammonia (NH3), were highest within the first 28 days when the pile temperatures and pH values were above 33 degrees C and 7.7, respectively. Moisture loss increased as C:N ratio and TF increased. In conclusion, the treatment with a combination of 4 days TF and C:N ratio 25:1 (T4R25) had the minimum TN loss (70.73% of the initial TN) and this indicated the most efficient combination.     

Respiration profiles in monitoring the composting of by-products from the olive oil agro-industry

The composting of olive press cake (OPC) repeatedly mixed either with olive mill wastewater (OPC+OMW) or with tap water (OPC+W) was studied using the thermogradient respirometer, an apparatus that determines the respiration rates from a substrate over a wide range of different temperatures (respiratory profile). The composting processes took place over a period of five months during which nine moistenings of the OPC were performed with the respective liquids. The composting resulted in detoxification of the materials used in both treatments, as indicated by seed germination tests. However, the repeated applications of OMW resulted in recurring thermophilic phases (following each application) and in greater pH and conductivity increases in the final product, as compared to water applications. Respiration measurements performed at 35 degrees C were good indicators of the mean metabolic potential in the compost piles (the mean respiration derived from the whole respiration profile over a wide range of environmental temperatures). However, respiration measurements at higher temperatures (48.5 degrees C) were better indicators of the respiration activity occurring in situ. Following the initial thermophilic phase, the respiration potential of the composts at high temperatures (42-63 degrees C) increased drastically compared to their respiration potential at lower temperatures (17-42 degrees C) indicating the establishment of a thermophilic microflora. Subsequently, only the periodic new substrate-C applications in the form of OMW resulted in increased ratios of low temperature-to-high temperature respiration potential. These ratios decreased again following the respective thermophilic phase that each new OMW application had induced.     

Study of lignin biotransformation by Aspergillus fumigatus and white-rot fungi using (14)C-labeled and unlabeled kraft lignins

The biodegradation of lignin by fungi was studied in shake flasks using (14)C-labeled kraft lignin and in a deep-tank fermentor using unlabeled kraft lignin. Among the fungi screened, A. fumigatus-isolated in our laboratories-was most potent in lignin biotransformation. Dialysis-type fermentation, designed to study possible accumulation of low MW lignin-derived products, showed no such accumulation. Recalcitrant carbohydrates like mi-crocrystalline cellulose supported higher lignolytic activity than easily metabolized carbohydrates like cellobiose. An assay developed to distinguish between CO(2) evolved from lignin and carbohydrate substrates demonstrated no stoichiometric correlation between the metabolism of the two cosubstrates. The submerged fermentations with unlabeled lignin are difficult to monitor since chemical assays do not give accurate and true results. Lignolytic efficiencies that allowed monitoring of such fermentations were defined. Degraded lignins were analyzed for structural modifications. A. fumigatus was clearly superior to C. versicolor in all aspects of lignin degradation; A. fumigatus brought about substantial demethoxylation and dehydroxylation, whereas C. versicolor degraded lignins closely resembled undegraded kraft lignin. There was a good agreement among the different indices of lignin degradation, namely, (14)CO evolution, OCH(3) loss, OH loss, and monomer and dimer yield after permanganate oxidation.     

Microbial indicators for sawdust and bark compost stability and humification processes

To what extent some microbial index ratios are suitable for use as early criteria for the level of compost stability during aerobic composting of coniferous sawdust and bark at mesophilic conditions was studied. Evolution of the specific respiration activity (CO₂-C/biomass C) and the ratios between some groups of microorganisms were followed as a function of composting time. The specific respiration activity was found to be an early and most reliable indicator of compost stability. The peroxidase and polyphenoloxidase enzyme activity during composting, as well as the composition of newly-formed humus substances were studied. The duration of composting increased the quality of newly-formed humus substances (C_h.a.:C_f.a ratio; Ca-complexed humic acid and resistance of organo-mineral complexes). The quality of humus substances could be used to assess compost stability. However, the results can be applied only under defined conditions.     

Determining thermal inactivation of Escherichia coli O157:H7 in fresh compost by simulating early phases of the composting process

A three-strain mixture of Escherichia coli O157:H7 was inoculated into fresh dairy compost (ca. 10(7) CFU/g) with 40 or 50% moisture and was placed in an environmental chamber (ca. 70% humidity) that was programmed to ramp from room temperature to selected composting temperatures in 2 and 5 days to simulate the early composting phase. The surviving E. coli O157:H7 population was analyzed by direct plating and enrichment. Optimal and suboptimal compost mixes, with carbon/nitrogen (C/N) ratios of 25:1 and 16:1, respectively, were compared in this study. In the optimal compost mix, E. coli O157:H7 survived for 72, 48, and 24 h in compost with 40% moisture and for 72, 24, and 24 h with 50% moisture at 50, 55, and 60°C, respectively, following 2 days of come-up time (rate of heating up). However, in the suboptimal compost mix, the pathogen survived for 288, 72, and 48 h in compost with 40% moisture and for 240, 72, 24 h in compost with 50% moisture at the same temperatures, respectively. Pathogen survival was longer, with 5 days of come-up time compared with 2 days of come-up. Overall, E. coli O157:H7 was inactivated faster in the compost with 50% moisture than in the compost with 40% at 55 and 60°C. Both moisture and come-up time were significant factors affecting Weibull model parameters. Our results suggest that slow come-up time at the beginning of composting can extend pathogen survival during composting. Additionally, both the C/N ratio and the initial moisture level in the compost mix affect the rate of pathogen inactivation as well.     

Compositional Changes in Compost during Composting and Growth of Agaricus bisporus

Samples from conventional compost taken at various stages of composting and mushroom (Agaricus bisporus) growth were analyzed for changes in 80% ethanol and water extracts, monosaccharides in acid hydrolysates of polysaccharides, lignin concentration, and lignin structural features. Variable amounts of extraneous inorganic solids in the form of fine sandy particles were removed by sedimentation of the samples in a carbon tetrachloride-dibromomethane mixture. During composting, about two-thirds of the initial wall polysaccharides were consumed by compost microorganisms, and only 17% of the total polysaccharides were used during mushroom production. The relative lignin content of composts as measured by the acetyl bromide procedure increased, both during composting and mushroom growth, and the chemical structure of lignin was altered by condensation and oxidation reactions.     

The fate of a genetically modified Pseudomonas strain and its transgene during the composting of poultry manure

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 x 1010 CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 degrees C or above for one or more days. In contrast, in the compost microcosms incubated at 23 degrees C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 degrees C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes.     

Production of Manganese Peroxidase and Organic Acids and Mineralization of 14C-Labelled Lignin (14C-DHP) during Solid-State Fermentation of Wheat Straw with the White Rot Fungus Nematoloma frowardii

The basidiomycetous fungus Nematoloma frowardii produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic ¹⁴C-ring-labelled lignin (¹⁴C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as ¹⁴CO₂, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the ¹⁴C-DHP to ¹⁴CO₂ (which accounted for up to 8% of the initial radioactivity added) and ¹⁴C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).