Catalytic decomposition of cellulose under biological conditions

1. The catalytic decomposition of undegraded cellulose in the form of cotton fibres is described with hydrogen peroxide at 0·4–0·04% (w/v) concentration in the presence of ferrous salts at pH3–5. 2. Complete solubilization of 5mg. of cotton fibres occurred in about 7 days in the presence of 0·4% hydrogen peroxide and 0·2mm-ferrous sulphate at the optimum pH4·2–4·3. 3. With 0·4% hydrogen peroxide the most rapid decomposition of cellulose was confined to ferrous sulphate concentrations of approx. 2–0·02mm. If the concentrations of the reagents were decreased in proportion extensive breakdown occurred but much more slowly. 4. In the primary stages of breakdown cotton fibres were disintegrated to very short fibres. These were subsequently solubilized, but there was little accumulation of soluble material. Organic matter was lost from solution as the reaction progressed. 5. Other naturally occurring cellulose-containing materials, such as grass, straw, hay and sawdust, were also disintegrated and solubilized by hydrogen peroxide and ferrous sulphate.     

Insights into functional modulation of catalytic RNA activity

RNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.     

Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions

Summary Composting was investigated as a bioremediation technology for clean-up of sediments contaminated with explosives and propellants. Two field demonstrations were conducted, the first using 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and N-methyl-N,2,4,6-tetranitroaniline (tetryl) contaminated sediment, and the second using nitrocellulose (NC) contaminated soil. Tests were conducted in thermophilic and mesophilic aerated static piles. Extractable TNT was reduced from 11840 mg/kg to 3 mg/kg, and NC from 13090 mg/kg to 16 mg/kg under thermophilic conditions. Under mesophilic conditions, TNT was reduced from 11 190 mg/kg to 50 mg/kg. The thermophilic and mesophilic half-lives were 11.9 and 21.9 days for TNT, 17.3 and 30.1 days for RDX, and 22.8 and 42.0 days for HMX, respectively. Known nitroaromatic transformation products increased in concentration over the first several weeks of the test period, but decreased to low concentrations thereafter.     

Insights into new techniques for high resolution functional MRI

Non-invasive functional magnetic resonance imaging (fMRI) has opened a unique window into human and animal brain function, with a spatial resolution of a few millimeters and a temporal resolution of a few seconds. To further improve the current technical limitations of fMRI, various post-processing and data acquisition schemes were developed. Improved fMRI methods include variations of a conventional fMRI technique, mapping a single physiological parameter such as cerebral blood flow or cerebral blood volume, and direct mapping of neural activity. Advances in fMRI techniques allow scientists to map submillimeter columnar and laminar functional structures and to detect tens of millisecond neural activity in certain specific tasks.     

Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose

Sixteen batch experiments were performed to evaluate the stability, kinetics, and metabolic paths of heat-shocked digester (HSD) sludge that transforms microcrystalline cellulose into hydrogen. Highly reproducible kinetic and metabolic data confirmed that HSD sludge could stably convert microcrystalline cellulose to hydrogen and volatile fatty acids (VFA) and induce metabolic shift to produce alcohols. We concluded that clostridia predominated the hydrogen-producing bacteria in the HSD sludge. Throughout this study the hydrogen percentage in the headspace of the digesters was greater than 50% and no methanogenesis was observed. The results emphasize that hydrogen significantly inhibited the hydrogen-producing activity of sludge when initial microcrystalline cellulose concentrations exceeded 25.0 g/L. A further 25 batch experiments performed with full factorial design incorporating multivariate analysis suggested that the ability of the sludge to convert cellulose into hydrogen was influenced mainly by the ratio of initial cellulose concentration (So) to initial sludge density (Xo), but not by interaction between the variables. The hydrogen-producing activity depended highly on interaction of So x (So/Xo). Through response surface analysis it was found that a maximum hydrogen yield of 3.2 mmol/g cellulose occurred at So = 40 g/L and So/Xo = 8 g cellulose/g VSS. A high specific rate of 18 mmol/(g VSS-d) occurred at So = 28 g/L and So/Xo = 9 g cellulose/g VSS. These experimental results suggest that high hydrogen generation from cellulose was accompanied by low So/Xo.     

Mechanism for the depolymerization of cellulose under alkaline conditions

The mechanism for the hydroxyl-radical-induced depolymerization of cellulose under alkaline conditions in air was investigated using density functional theory at the B3LYP/6-31+G(d,p) level as well as electron transfer theory. The pathway for the depolymerization of cellulose was obtained theoretically and H abstraction from the C(3) atom of the pyran ring during the cleavage of the glucosidic bond was found to be the rate-limiting step due to its high energy barrier (16.81 kcal/mol) and low reaction rate constant (4.623?×?10⁴ mol L^?1 s^?1). Calculations of the electron transfer between O₂ and the saccharide radical performed with the HARLEM software package revealed that following the H abstraction, the oxygen molecule approaches C(2) on the saccharide radical and obtains an electron from the radical, even though no bond forms between the oxygen molecule and the radical. The rate constant for electron transfer could be as high as 1.572?×?10¹¹ s^?1. Furthermore, an enol intermediate is obtained during the final stage of the depolymerization.     

On hydrolysis of cellulose and nitrocellulose under sulfate-reducing conditions

Hydrolysis of cellulose and nitrocellulose in the presence of sulfate-reducing bacterium Desulfovibrio vulgaris 1388 was studied. The cellulolytic activity was found in culture medium after D. vulgaris growth (1.45 ± 0.04 nmol/mg protein/min). In the presence of cellulose or nitrocellulose the activity accounted for 4.82 ± 0.23 and 2.35 ± 0.11 nmol/mg protein/min, respectively. The initial rates of cellulose decomposition were measured using toluene to inhibit the microbial uptake of hydrolysis product—glucose. It was established that 7.6% of initially added cellulose was hydrolyzed in 3 weeks. The highest rate of glucose accumulation was observed on day 10 (2.13 μmol glucose/g dry-wt cellulose/h). At the same time only 3.3% of nitrocellulose was hydrolyzed, since nitro-groups of polymer exerted negative influence on the hydrolysis process. It is supposed that nonspecific extracellular hydrolases participate in the polymers hydrolysis.     

Insights into plant metabolic networks from steady-state metabolic flux analysis

Steady-state metabolic flux analysis (MFA) is an experimental approach that allows the measurement of multiple fluxes in the core network of primary carbon metabolism. It is based on isotopic labelling experiments, and although well established in the analysis of micro-organisms, and some mammalian systems, the extension of the method to plant cells has been challenging because of the extensive subcellular compartmentation of the metabolic network. Despite this difficulty there has been substantial progress in developing robust protocols for the analysis of heterotrophic plant metabolism by steady-state MFA, and flux maps have now been published that reflect the metabolic phenotypes of excised root tips, developing embryos and cotyledons, hairy root cultures, and cell suspensions under a variety of physiological conditions. There has been a steady improvement in the quality, extent and statistical reliability of these analyses, and new information is emerging on the performance of the plant metabolic network and the contributions of specific pathways.     

Petroleum coke supplementation for enhanced biogas production and phosphate removal under mesophilic conditions

The use of carbon-based conductive materials has been shown to lead to an increase in biogas and methane yields during anaerobic digestion (AD). The effect of these additives on AD using synthetic substrates has been extensively studied, yet their significance for wastewater sludge digestion has not been adequately investigated. Therefore, the aim of this research was to optimize the concentration of petroleum coke (PC) that is a waste by-product of oil refineries, for the anaerobic digestion of wastewater sludge and investigation of phosphate removal in the AD process in the mesophilic temperature range. According to the results of the experiments, supplementing reactors with PC could significantly improve biogas and methane production. Supplementation of reactors with 1.5 g/L PC led to 23.40 ± 0.26% and 42.55 ± 3.97% increase in biogas production and methane generation, respectively. Moreover, the average volatile solids (VS), phosphate, and chemical oxygen demand (COD) removals were 43.43 ± 0.73, 46.74 ± 0.77%, and 60.40 ± 0.38%, respectively.     

Quantitative colorimetric measurement of cellulose degradation under microbial culture conditions

We have developed a simple, rapid, quantitative colorimetric assay to measure cellulose degradation based on the absorbance shift of Congo red dye bound to soluble cellulose. We term this assay “Congo Red Analysis of Cellulose Concentration,” or “CRACC.” CRACC can be performed directly in culture media, including rich and defined media containing monosaccharides or disaccharides (such as glucose and cellobiose). We show example experiments from our laboratory that demonstrate the utility of CRACC in probing enzyme kinetics, quantifying cellulase secretion, and assessing the physiology of cellulolytic organisms. CRACC complements existing methods to assay cellulose degradation, and we discuss its utility for a variety of applications.     

Insights into the organization of biochemical regulatory networks using graph theory analyses

Graph theory has been a valuable mathematical modeling tool to gain insights into the topological organization of biochemical networks. There are two types of insights that may be obtained by graph theory analyses. The first provides an overview of the global organization of biochemical networks; the second uses prior knowledge to place results from multivariate experiments, such as microarray data sets, in the context of known pathways and networks to infer regulation. Using graph analyses, biochemical networks are found to be scale-free and small-world, indicating that these networks contain hubs, which are proteins that interact with many other molecules. These hubs may interact with many different types of proteins at the same time and location or at different times and locations, resulting in diverse biological responses. Groups of components in networks are organized in recurring patterns termed network motifs such as feedback and feed-forward loops. Graph analysis revealed that negative feedback loops are less common and are present mostly in proximity to the membrane, whereas positive feedback loops are highly nested in an architecture that promotes dynamical stability. Cell signaling networks have multiple pathways from some input receptors and few from others. Such topology is reminiscent of a classification system. Signaling networks display a bow-tie structure indicative of funneling information from extracellular signals and then dispatching information from a few specific central intracellular signaling nexuses. These insights show that graph theory is a valuable tool for gaining an understanding of global regulatory features of biochemical networks.     

Engineering microbes for direct fermentation of cellulose to bioethanol

Consolidated bioprocessing (CBP) by micro-organisms is desired for efficient conversion of lignocellulosic biomass to bioethanol fuels. Potential candidates have been discovered, including cellulolytic bacteria and filamentous fungi. Genetic and metabolic manipulation of these organisms further promotes their fermentation capacities and the ethanol tolerance. In addition, Saccharomyces cerevisiae and several other yeasts were genetically modified to express recombinant cellulases in media or display them on the cell surface for CBP of cellulose. To compensate the insufficient capacity of a single strain, various microbial consortia have also been developed. In this article, we reviewed the recent advances in CBP microbes and focused on the efforts in strain improvement employing genetic engineering.     

Monofermentation of grass silage under mesophilic conditions: measurements and mathematical modeling with ADM 1

In this paper experimental data from grass fermentation and simulation results with the Anaerobic Digestion Model (ADM) No. 1 are described. Two laboratory reactors were operated under mesophilic conditions with volumetric loading rates in between 0.3 and 2.5 kg(VS)/(m(3) x d). Two different kinds of grass silage were used as substrates, resulting in an average specific biogas production of 600 L/kg(VS). The ADM 1 was calibrated both manually and with the help of a Genetic Algorithm in Matlab/Simulink. Results from calibration indicate that the NH3 inhibition constant used to model the inhibition of acetate uptake is three to five times higher compared with digested activated sludge. The hydrogen inhibition constants applied for propionate and valerate/butyrate uptake are around two orders of magnitude lower than for sludge digestion.     

Anaerobic digestion of whole stillage from dry-grind corn ethanol plant under mesophilic and thermophilic conditions

Anaerobic digestion of whole stillage from a dry-grind corn-based ethanol plant was evaluated by batch and continuous-flow digesters under thermophilic and mesophilic conditions. At whole corn stillage concentrations of 6348 to 50,786 mg total chemical oxygen demand (TCOD)/L, at standard temperature (0 °C) and pressure (1 atm), preliminary biochemical methane potential assays produced 88 ± 8 L (49 ± 5 L CH₄) and 96 ± 19 L (65 ± 14 L CH₄) biogas per L stillage from mesophilic and thermophilic digesters, respectively. Continuous-flow studies for the full-strength stillage (TCOD = 254 g/L) at organic loadings of 4.25, 6.30 and 9.05 g TCOD/L days indicated unstable performance for the thermophilic digester. Among the sludge retention times (SRTs) of 60, 45 and 30 days tested, the mesophilic digestion was successful only at 60 days-SRT which does not represent a practical operation time for a large scale bioethanol plant. Future laboratory studies will focus on different reactor configurations to reduce the SRT needed in the digesters.     

Single step conversion of cellulose to ethanol by a mesophilic coculture

Summary A coculture consisting of two mesophilic anaerobes, produced about 0.8 mole of ethanol per mole of cellulose from a variety of cellulosic materials. The non-cellulolytic member of this coculture, Clostridium saccharolyticum sp. nov. converted glucose and xylose to ethanol and acetic acid in ratios over 4 to 1.     

Insights into Maize LEA proteins: from proteomics to functional approaches

LEA (late embryogenesis abundant) proteins participate in plant stress tolerance responses, but the mechanisms by which protection occurs are not fully understood. In the present work the unfolded proteins from maize dry embryos were analyzed by mass spectrometry. Twenty embryo proteins were identified, and among them 13 corresponded to LEA-type proteins. We selected three major LEA proteins, Emb564, Rab17 and Mlg3, belonging to groups 1, 2 and 3, respectively, and we undertook a comparative study in order to highlight differences among them. The post-translational modifications of native proteins were analyzed and the anti-aggregation properties of recombinant Emb564, Rab17 and Mgl3 proteins were evaluated in vitro. In addition, the protective effects of the LEA proteins were assessed in living cells under stress in Escherichia coli cells and in Nicotiana bentamiana leaves agroinfiltrated with fluorescent LEA-green fluorescent protein (GFP) fusions. Protein visualization by confocal microscopy indicated that cells expressing Mg3-GFP showed reduced cell shrinkage effects during dehydration and that Rab17-GFP co-localized to leaf oil bodies after heat shock. Overall, the results highlight differences and suggest functional diversity among maize LEA groups.