Extracellular secretion of noncatalytic plant cell wall-binding proteins by the cellulolytic thermophile Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (Ka) values of 5.2 × 10(6) to 44 × 10(6) M(-1) among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.

Source of nitrous oxide emissions during the cow manure composting process as revealed by isotopomer analysis of and amoA abundance in betaproteobacterial ammonia-oxidizing bacteria.

A molecular analysis of betaproteobacterial ammonia oxidizers and a N(2)O isotopomer analysis were conducted to study the sources of N(2)O emissions during the cow manure composting process. Much NO(2)(-)-N and NO(3)(-)-N and the Nitrosomonas europaea-like amoA gene were detected at the surface, especially at the top of the composting pile, suggesting that these ammonia-oxidizing bacteria (AOB) significantly contribute to the nitrification which occurs at the surface layer of compost piles. However, the (15)N site preference within the asymmetric N(2)O molecule (SP = delta(15)N(alpha) - delta(15)N(beta), where (15)N(alpha) and (15)N(beta) represent the (15)N/(14)N ratios at the center and end sites of the nitrogen atoms, respectively) indicated that the source of N(2)O emissions just after the compost was turned originated mainly from the denitrification process. Based on these results, the reduction of accumulated NO(2)(-)-N or NO(3)(-)-N after turning was identified as the main source of N(2)O emissions. The site preference and bulk delta(15)N results also indicate that the rate of N(2)O reduction was relatively low, and an increased value for the site preference indicates that the nitrification which occurred mainly in the surface layer of the pile partially contributed to N(2)O emissions between the turnings.

The impact of using mature compost on nitrous oxide emission and the denitrifier community in the cattle manure composting process.

The diversity and dynamics of the denitrifying genes (nirS, nirK, and nosZ) encoding nitrite reductase and nitrous oxide (N(2)O) reductase in the dairy cattle manure composting process were investigated. A mixture of dried grass with a cattle manure compost pile and a mature compost-added pile were used, and denaturing gradient gel electrophoresis was used for denitrifier community analysis. The diversity of nirK and nosZ genes significantly changed in the initial stage of composting. These variations might have been induced by the high temperature. The diversity of nirK was constant after the initial variation. On the other hand, the diversity of nosZ changed in the latter half of the process, a change which might have been induced by the accumulation of nitrate and nitrite. The nirS gene fragments could not be detected. The use of mature compost that contains nitrate and nitrite promoted the N(2)O emission and significantly affected the variation of nosZ diversity in the initial stage of composting, but did not affect the variation of nirK diversity. Many Pseudomonas-like nirK and nosZ gene fragments were detected in the stage in which N(2)O was actively emitted.

Effect of covering composting piles with mature compost on ammonia emission and microbial community structure of composting process.

To control ammonia (NH(3)) volatilization from the dairy cattle (Bos taurus) manure composting process, a compost pile was covered with mature compost and the gas emissions evaluated using the dynamic chamber system. The peak of NH(3) volatilization observed immediately after piling up of the compost was reduced from 196 to 62 mg/m(3) by covering the compost pile with mature compost. The accumulation of NH(4)-N to the covered mature compost was also observed. Covering and mixing the compost with mature compost had no effect on the microbial community structure. However, over time the microbial community structure changed because of a decrease in easily degradable organic compounds in the compost piles. The availability of volatile fatty acids (VFA) was considered to be important for microbial community structure in the compost. After the VFA had disappeared, the NO(3)-N concentration increased and the cellulose degrading bacteria such as Cytophaga increased in number.

Microbiology of nitrogen cycle in animal manure compost.

Composting is the major technology in the treatment of animal manure and is a source of nitrous oxide, a greenhouse gas. Although the microbiological processes of both nitrification and denitrification are involved in composting, the key players in these pathways have not been well identified. Recent molecular microbiological methodologies have revealed the presence of dominant Bacillus species in the degradation of organic material or betaproteobacterial ammonia-oxidizing bacteria on nitrification on the surface, and have also revealed the mechanism of nitrous oxide emission in this complicated process to some extent. Some bacteria, archaea or fungi still would be considered potential key players, and the contribution of some pathways, such as nitrifier denitrification or heterotrophic nitrification, might be involved in composting. This review article discusses these potential microbial players in nitrification-denitrification within the composting pile and highlights the relevant unknowns through recent activities that focus on the nitrogen cycle within the animal manure composting process.

Characterization and spatial distribution of bacterial communities within passively aerated cattle manure composting piles.

The bacterial communities in the core, bottom, top, middle-surface, and lower-surface full-scale passively aerated cattle manure compost was investigated using DGGE of PCR-amplified 16S rRNA sequences. Some Bacillus species and strictly anaerobic thermophilic Clostridium species were dominant only in the core and bottom zones. In contrast, bands belonging to mesophilic bacteria such as Bacteroidetes, Clostoridia,alpha and gamma-proteobacteria were detected in surface zones, even in the initial thermophilic stage of the process. Our results clearly show the spatial distribution of the microbial community within full-scale composting piles, which indicates N or C conversion by zone-specific bacterial communities were occurring in each zone of the pile.

Key odor components responsible for the impact on olfactory sense during swine feces composting.

This study aimed to identify the major odor contributing components produced during swine feces composting which have an impact on the olfactory senses. A total of 64 gas samples collected at different stages of composting were analyzed by both a gas chromatograph and human panel test using the triangle odor bag method. Multiple regression analysis of representative odor substances present in the outlet gas was carried out employing the odor index (OI) as the dependent variable and the odor unit as the independent variable. The recorded changes in OI indicated that turning was an important event during odor evolution, and that the odor emission during the thermophilic phase should be the main target for odor abatement. The model incorporating ammonia, methyl mercaptan and dimethyl sulfide as independent variables confirmed the value of the OI (R(2)=0.70). These compounds were identified to be the key odor components significantly determining the OI.