Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

Pretreatments to enhance the digestibility of lignocellulosic biomass

Biofuels produced from various lignocellulosic materials, such as wood, agricultural, or forest residues, have the potential to be a valuable substitute for, or complement to, gasoline. Many physicochemical structural and compositional factors hinder the hydrolysis of cellulose present in biomass to sugars and other organic compounds that can later be converted to fuels. The goal of pretreatment is to make the cellulose accessible to hydrolysis for conversion to fuels. Various pretreatment techniques change the physical and chemical structure of the lignocellulosic biomass and improve hydrolysis rates. During the past few years a large number of pretreatment methods have been developed, including alkali treatment, ammonia explosion, and others. Many methods have been shown to result in high sugar yields, above 90% of the theoretical yield for lignocellulosic biomasses such as woods, grasses, corn, and so on. In this review, we discuss the various pretreatment process methods and the recent literature that...

http://ucce.ucdavis.edu/files/datastore/234-1388.pdf

Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production

THE JOURNAL OF BIOLOGICAL CHEMISTRY: The Biochemical Behavior of LeadL I. Influence of Calcium, Phosphorus, and Vitamin D on Lead in Blood and Bone

Poplar wood was treated with peracetic acid, KOH, and ball milling to produce 147 model lignocelluloses with a broad spectrum of lignin contents, acetyl contents, and crystallinity indices (CrIs), respectively. An empirical model was identified that describes the roles of these three properties in enzymatic hydrolysis. Lignin content and CrI have the greatest impact on biomass digestibility, whereas acetyl content has a minor impact. The digestibility of several lime-treated biomass samples agreed with the empirical model. Lime treatment removes all acetyl groups and a moderate amount of lignin and increases CrI slightly; lignin removal is the dominant benefit from lime treatment.

Fundamental factors affecting biomass enzymatic reactivity.

Comparison of pretreatment methods on the basis of available surface area

Two different types of extracellular protease activity were identified in the culture fluid of Phanerochaete chrysosporium wild-type BKM-F grown in submerged batch culture on N-limited media. The first activity, which appears to be inherent to the active growth phase, displayed a maximum on day 2 and decreased to a very low level on day 4. The second activity, which appeared at day 8 following the peak of ligninase activity, seems to be characteristic of late secondary metabolism and is stimulated by carbon starvation. Cultures started with half the amount of glucose of other cultures showed a remarkably earlier development of secondary activity. In contrast, the fed-batch addition of glucose started when ligninase activity was at a maximum (day 6) completely repressed secondary protease activity and enhanced ligninase production. The addition of exogenous veratryl alcohol increased the level of secondary protease activity, whereas the oxygen supply pattern significantly affected both the time course and the level of overall proteolytic activity. The addition of phenylmethylsulfonyl fluoride to growing cultures (0, 1, or 6 days) diminished overall protease activity, while it significantly enhanced ligninase activity. In all cases, the time courses of protease and ligninase activities were negatively correlated, indicating that protease activity promotes the decline of ligninase activity in batch culture.

Effect of Environmental Conditions on Extracellular Protease Activity in Lignolytic Cultures of Phanerochaete chrysosporium.

The effect of the oxygen supply pattern on the onset and development of the lignolytic enzyme system of Phanerochaete chrysosporium was studied in submerged culture employing the serum bottle approach. Periodic or continuous flushing through the head phase, and continuous bubbling through the liquid phase with either oxygen (O2) or air were applied. The nature of the O2 supply had a crucial regulatory effect not only on the formation of lignin-degrading peroxidases but also on their decay and on the production of extracellular protease activity and polysaccharides. Continuous oxygenation or aeration increased the glucose consumption rate, extracellular protease activity and polysaccharides. Gassing with air, whether continuous or periodic, sustained Mn-peroxidase activity while ligninase was undetectable. Continuous O2 supply speeded up ligninase decay, displaying a sharper maximum, while a broader maximum and slower decay of ligninase activity were observed when supplying periodic O2. Cultures initially grown with free exposure to air displayed a higher but sharper ligninase activity maximum when shifted to continuous rather than periodic O2 supply. In general, the higher levels of either polysaccharides or protease activity corresponded to the lower levels and faster decay of ligninase and Mn-peroxidase activities.

Effect of oxygenation conditions on submerged cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium. Images

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethylsulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation.

Hans E. Grethlein

Papers

Comparison of pretreatment methods on the basis of available surface area

Effect of Environmental Conditions on Extracellular Protease Activity in Lignolytic Cultures of Phanerochaete chrysosporium.

Effect of oxygenation conditions on submerged cultures of Phanerochaete chrysosporium.

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions.