Photoproduction of H2 from Cellulose by an Anaerobic Bacterial Coculture

Cellulomonas sp. strain ATCC 21399 is a facultatively anaerobic, cellulose-degrading microorganism that does not evolve hydrogen but produces organic acids during cellulose fermentation. Rhodopseudomonas capsulata cannot utilize cellulose, but grows photoheterotrophically under anaerobic conditions on organic acids or sugars. This report describes an anaerobic coculture of the Cellulomonas strain with wild-type R. capsulata or a mutant strain lacking uptake hydrogenase, which photoevolves molecular hydrogen by the nitrogenase system of R. capsulata with cellulose as the sole carbon source. In coculture, the hydrogenase-negative mutant produced 4.6 to 6.2 mol of H₂ per mol of glucose equivalent, compared with 1.2 to 4.3 mol for the wild type.     

Effects of cellulose on growth,enzyme production,and ultrastructure of a Cellulomonas species

Cellulomonas sp. (NRCC 2406) was grown on complex medium (peptone-tryptone-yeast extract) alone, or with the addition of different celluloses (solka floc, avicel, CF 11 cellulose or Whatman No. 1 filter paper) and/or glucose. Cultures growing on the complex medium without cellulose produced low levels of endo- and exo-cellulases and very little -glucosidase. Adding cellulose stimulated growth, as measured by cellular protein or by viable counts, and also stimulated production of cellulases. Adding glucose in the prescene of cellulose inhibited growth and cellulose breakdown. Glucose also inhibited attachment of growing cells to cellulose fibres. Electron microscope studies showed that Cellulomonas sp. adhered to the cellulose fibers. In the presence of cellulose in the media, the cells developed a thicker outer layer which probably helps in the adhesion process.Abbreviations PTYE peptone, tryptone, yeast extract medium - DNS dinitrosalicylic acid - CMC carboxymethyl cellulose - cfu/ml colony-forming units per ml     

Growth of cellulolytic bacteria on sugarcane bagasse

The growth behavior of Cellulomonas has been examined in fermentation system using alkali pretreated sugarcane bagasse. During the batch operation diauxic growth was found which would not seem to be explained by catabolic repression. The relative variation of cellulose and hemicellulose during the fermentation process suggests the initial utilization of easily degradable substrate, i.e., hemicellulose and amorphous cellulose, until their concentration becomes limiting, followed by utilization of the crystalline cellulose. The conversion of substrate was 70% with a yield of 0.355 g of biomass per gram of bagasse feed.     

Growth and enzyme production of Cellulomonas sp. ATCC 21399 on microcrystalline cellulose. Effects of increasing concentration of a mineral medium

Summary The kinetics and production of different extracellular enzyme activities were studied during growth of Cellulomonas sp. ATCC 21399 on 2% Avicel with different concentrations of M9 mineral medium. The lag phase and the doubling time increased with increasing ionic strength of the medium. The highest cell density was obtained during growth at 5 x M9 mineral medium and Cellulomonas grew well at this high salinity. The enzyme activities against carboxymethylcellulose and xylan increased with increasing concentration of M9 medium up to 5 x M9. By contrast, activities against microcrystalline cellulose (Avicel), galactomannan and amylose decreased with increasing concentration of M9 medium. The extracellular proteinase activity increased with increasing concentration of M9 medium, and it is possible that the lability of the cellulolytic and amylolytic enzymes may be due to their susceptibility to proteolytic inactivation by the extracellular proteinases.     

Microcalorimetric study of cellulose degradation by Cellulomonas uda ATCC 21399

A newly designed batch calorimeter was used to investigate the degradability of some celluloses having varying degrees of crystallinity. The PTC of an aerobic culture of Cellulomonas uda ATCC 21399 obtained revealed a diauxic growth which is attributed to the presence of hemicellulose contaminating Avicel and MN300 cellulose. The microcrystalline celluloses used were not completely utilized, whereas amorphous cellulose was easily metabolized, indicating that under the growth conditions used here, the physical structure of cellulose strongly influenced its microbial degradability. An equivalent growth yield of ca. 0.44 g/g was found with all the substrates used. The heat evolved by metabolism of 1 g cellulose was -5.86 kJ/g, a value similar to that obtained with glucose culture. The growth rate was the only variable parameter. The data obtained showed as expected that the hydrolysis product of cellulose was consumed in the same way as that of glucose and that the only limiting factor to the biodegradability of cellulose was the breakdown of the polymeric substrate. It is concluded that data obtained with glucose metabolism can be used to evaluate the extent of cellulose degradation.     

Identification of the major anaerobic end products ofCellulomonas sp. (ATCC 21399)

Summary A novel approach of aerobic growth followed by anaerobic growth was used to identify the anaerobic end products of the facultative organismCellulomonas sp. (ATCC 21399) utilizing cellulose as the substrate. The organism was found to produce an equimolar mixture of ethanol and acetic acid as the two carbon end products.     

Utilization of carbohydrates by Thermomonospora sp. Grown on glucose,cellobiose, and cellulose

Thermomonospora sp. was grown on glucose, cellobiose, and in order to study its growth characteristics with different carbohydrate substrates and to assess the validity of some of the assumptions made in a previously proposed model for the cellulose fermentation with this microorganism. It was observed that the nitrogen and protein contents of the cells are essentially constant during the fermentation and independent of the carbon source when glucose or cellobiose are utilized. Under oxygen starvation conditions it was shown that unidentification organic compound(s) accumulate(s) in the culture broth. Culture fluorescence was shown to be an excellent variable for monitoring and control of the fermentation process. This microorganism showed a preference for crystalline cellulose (Avicel) as substrate although it grows readily on a more amorphous cellulose (Solka Floc). The production of extra cellular protein is shown to be growth related. Data were obtained confirming the decrease in the number of active adsorption sites as the cause for the decrease in the cellulose digestion rate. It is suggested that a future model should account for the time change of surface characteristics of the cellulose particles.     

Use of a Cellulase-Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose

A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30°C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet.     

New isolate of Streptomyces sp. with novel thermoalkalotolerant cellulases

A Streptomyces sp. was isolated that produced novel thermoalkalotolerant cellulase activity after growth on crystalline cellulose at 50°C. Three major components of the cellulases (CMCase, Avicelase and cellobiase) were produced with maximal activities (11.8, 7.8 and 3.9 IU/ml) and maximum specific activities 357, 276 and 118 IU/mg protein, respectively, after 120 h growth. Maximum CMCase activity was between 50 and 60°C measured over 3 h. The enzyme also retained 88% of its maximum activity at 70°C and pH 5, and 80% of the activity at pH 10 and 50°C when assayed after 1 h. After incubation at 40°C for 1 h with commercial detergent (Tide) at pH 11, 95% activity was retained. The enzyme mixture produced glucose from crystalline cellulose.     

Regulation of xylanase activity in cellulomonas sp. IIbc

The regulation mechanism governing the xylanolytic activity in the strain Cellulomonas sp. IIbc was studied. High levels of activity were detected during the cultivation on cellulose as the only carbon source. No activity was produced with glucose, xylose or cellobiose cultures, but in the last one, the synthesis was de-repressed when the sugar concentration dropped to 0.2%. The activity was not inhibited by glucose, cellobiose and xylose up to 1% concentration. A basal constitutive synthesis was detected in nutrient broth cultures. At the same time, xylose and cellobiose acted as inducers of the xylanase activity.     

Evidence for substrate binding of a recombinant thermostable xylanase originating from Rhodothermus marinus

The xyn1 encoded 5 domain xylanase from the thermophilic bacterium Rhodothermus marinus binds specifically to xylan, β-glucan and amorphous but not crystalline cellulose. Our results show that the binding is mediated by the full length xylanase, but not by the catalytic domain only. Based on similarities concerning both predicted secondary structure and binding specificity found with one cellulose binding domain of CenC from Cellulomonas fimi, we suggest that the binding is mediated by the two N-terminally repeated domains.     

The curdlan-type exopolysaccharide produced by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> KU forms part of an extracellular glycocalyx involved in cellulose degradation

The genus Cellulomonas is comprised of a group of Gram-positive, soil bacteria capable of utilizing cellulose as their sole source of carbon and energy. Cellulomonas flavigena KU was originally isolated from leaf litter and subsequently shown to produce large quantities of a curdlan-type (-1,3-glucan) exopolysaccharide (EPS) when provided with an excess of glucose or other soluble carbon-source. We report here that curdlan EPS is also produced by Cellulomonas flavigena KU when growing on microcrystalline cellulose in mineral salts-yeast extract media. Microscopic examination of such cultures shows an adherent biofilm matrix composed of cells, curdlan EPS, and numerous surface structures resembling cellulosome complexes. Those Cellulomonas species that produce curdlan EPS are all non-motile and adhere to cellulose as it is broken down into soluble sugars. These observations suggest two very different approaches towards the complex process of cellulose degradation within the genus Cellulomonas.     

Selection of hypercellulylytic derepressed mutants of Cellulomonas sp.

Summary Mutants from Cellulomonas sp.IIbc were obtained combined treatment of UV light and N-methyl-N-nitrosoguanidine. T The selection criterion for the screening of catabolite-repression-resistant mutants was based on the formation of clear zones around the bacterial colonies in medium containing 0.5% Walseth cellulose and 0.5% glucose. Mutants produced not only clear zones in significantly lower times than the parent strain, but also exhibited higher specific growth rates and cellulolytic activity when grown on bagasse pith. The cellulase-derepressed character of the mutants was demonstrated by the presence of cellulolytic activity in cultures grown in the presence of high levels of glucose. These results raise the possibility of enhancing the productivity of bacterial degradation of lignocellulosic substrates for single cell protein production. Offprint requests to: F. Alea     

A comparative investigation of various cellulase assay procedures

The cellulolytic activity of crude enzyme preparations from different cellulolytic fungi (namely Trichoderma viride, Trichoderma Koningii, Fusarium solani, Sporotrichum pulverulentum, Sporotrichum thermophile) was assayed comparatively with several common analytical procedures described in the literature. The investigation was carried out with the objective of evaluating, with raw culture filtrates, the different cellulase tests in relation to their specificity for endo- and exo-cellulase action as well as to allow comparisons to be made between results from different research groups using different methods. (1)Cellulase activity was tested viscometrically as well as chemically (determination of reducing end groups) with different carboxymethylcelluloses as substrates. Essentially constant ratios between both kinds of activities were obtained, indicating that they are directly related. Nevertheless, international units of activity, calculated from viscometric measurements (glycosidic bonds broken per unit time) were considerably lower than international units deduced from the increase in reducing power (glucose equivalents liberated per unit time), this discrepancy most likely accounted for by the predominant influence of the exo-cellulase component in cellulase tests based on the determination of reducing eng groups. (2) By estimating cellulase activity with insoluble cellulosic substrates no direct relationship could be established with the above-described activities except in the case where the cellulose was amorphous. The ratio profile between activities thus obtained and endo-cellulase activities determined viscometrically shows that some enzyme preparations (such as those from both Trichoderma sp.) are clearly more active than others against crystalline cellulose reflecting quantitative differences in enzyme composition. Nevertheless, for a biological understanding of cellulolysis. analytical procedures using crystalline celluloses are not adequate for specifically monitoring exo-cellulase activity in crude enzyme solutions for essentially two reasons: (a) they are not sufficiently sensitive to detect small changes in enzyme activity during the early phase of growth, and (b) exo-cellulase activity in crude enzyme solutions also depends on the endo-cellulase activity present.     

Formation of Glycerol Dehydrogenase by Microorganisms

The distribution of glycerol dehydrogenase activity was studied with cell-free extracts of bacteria, yeasts, molds and actinomycetes. High activity was found in 4 strains of bacteria and in 3 strains of molds. The enzymes of bacteria were dependent on NAD⁺ and those of molds were dependent on NADP⁺. An isolated gram-positive bacterium, which showed the high activity, was identified as Cellulomonas sp. NT3060. The total and specific activities were associated with growth of this strain and reached the maximum at the early stationary phase. Significant high level activity was detected in cell-free extracts from glycerol and glucose media.     

Nitrogen Fixation Associated with Development and Localization of Mixed Populations of Cellulomonas sp. and Azospirillum brasilense Grown on Cellulose or Wheat Straw

Mixed cultures of Cellulomonas sp. and Azospirillum brasilense were grown with straw or cellulose as the carbon source under conditions favoring the fixation of atmospheric nitrogen. Rapid increases in cell numbers, up to 10⁹ cells per g of substrate, were evident after 4 and 5 days of incubation at 30°C for cellulose and straw, respectively. Nitrogen fixation (detected by acetylene reduction measured on parallel cultures) commenced after 2 and 4 days of incubation for straw and cellulose, respectively, and continued for the duration of the experiment. Pure cultures of Cellulomonas sp. showed an increase in cell numbers, but CO₂ production was low, and acetylene reduction was not detected on either cellulose or straw. Pure cultures of A. brasilense on cellulose showed an initial increase in cell numbers (10⁷ cells per g of substrate) over 4 days, followed by a decline presumably caused by the exhaustion of available carbon substrate. On straw, A. brasilense increased to 10⁹ cells per g of substrate over 5 days and then declined slowly; this growth was accompanied by acetylene reduction. Scanning electron micrographs of straw incubated with a mixed culture under the above conditions for 8 days showed cells of both species in close proximity to each other. Evidence was furnished that the close spatial relationship of cells from the two species facilitated the mutually beneficial association between them and thus increased the efficiency with which the products of straw breakdown were used for nitrogen fixation.     

Single cell protein from bagasse pith by a mixed bacterial culture

A spontaneous association of Cellulomomas sp. with another bacterial strain was studied for its capabilities for single cell protein (SCP) production from bagasse pith. The associated strain was identified as Pseudomonas sp. and further characterized for its physiological properties. The effect of the initial proportions of both strains, the way of propagation, and the effect of pH on the growth of the mixed culture on bagasse pith was studied. Separate propagation of both strains before the fermentation step (“controlled mixed culture”), a range of proportions Cellulomonas-Pseudomonas from 4:1 to 100: 1, and pH 7.0, were found to be the most appropriate conditions of growth. A mutualistic symbiotic relationship was demonstrated to take place between both strains during the mixed growth on bagasse pith, the Cellulomonas supplying the carbon source (glucose produced from bagasse degradation) to the Pseudomonas, and the latter producing the vitamin supplements necessary for the Cellulomonas growth, allowing the growth of the mixed culture in a minimal medium, without any growth factor supplement. Fed-batch cultivation of the mixed culture on this substrate was successful, giving rise to high biomass production (19.4 g/l), thus increasing the productivity of the system. Due to its improved productivity, high biomass production, inexpensiveness of the culture medium, (without any vitamin supplement), and good stability, this culture presents economical advantages and constitutes an attractive choice for lignocellulosic substrate utilization.     

Glycogen, a cytoplasmic reserve polysaccharide of Cellulomonas sp. (DSM20108): its identification, carbon source-dependent accumulation, and degradation during starvation

During batch cultivation on complete medium or mineral salts medium with different carbon sources, Cellulomonas accumulates glycogen. The intracellular concentration of glycogen increases with increasing C/N ratio and reaches a maximum value of about 0.22 mg per mg dry weight. Under conditions of carbon starvation this polymer is degraded. Furthermore, Cellulomonas grows well on glycogen if administered as sole source of carbon and energy. The results show that glycogen is an additional energy and carbon storage compound in Cellulomonas sp. which also accumulates trehalose.     

Comparison of Two Cellulomonas Strains and Their Interaction with Azospirillum brasilense in Degradation of Wheat Straw and Associated Nitrogen Fixation

A mutant strain of Cellulomonas sp. CS1-17 was compared with Cellulomonas gelida 2480 as the cellulolytic component of a mixed culture which was responsible for the breakdown of wheat straw to support asymbiotic nitrogen fixation by Azospirillum brasilense Sp7 (ATCC 29145). Cellulomonas sp. strain CSI-17 was more efficient than was C. gelida in cellulose breakdown at lower oxygen concentrations and, in mixed culture with A. brasilense, it supported higher nitrogenase activity (C₂H₂ reduction) and nitrogen fixation with straw as the carbon source. Based on gravimetric determinations of straw breakdown and total N determinations, the efficiency of nitrogen fixation was 72 and 63 mg of N per g of straw utilized for the mixtures containing Cellulomonas sp. and C. gelida, respectively. Both Cellulomonas spp. and Azospirillum spp. exhibited a wide range of pH tolerance. When introduced into sterilized soil, the Cellulomonas sp.-Azospirillum brasilense association was more effective in nitrogen fixation at a pH of 7.0 than at the native soil pH (5.6). This was also true of the indigenous diazotrophic microflora of this soil. The potential implications of this work to the field situation are discussed.     

Differential Expression of Xylanases and Endoglucanases in the Hybrid Derived from Intergeneric Protoplast Fusion between a Cellulomonas sp. and Bacillus subtilis

A stable hybrid obtained by protoplast fusion between a Cellulomonas sp. and Bacillus subtilis exhibits an altered pattern of enzyme induction with different cellulosic substrates. Unlike in the Cellulomonas sp., xylanase was induced in the hybrid organism specifically by xylan, and endoglucanase was induced by carboxymethyl cellulose. The amount and specific activity of xylanase produced by the hybrid were more than those produced by the Cellulomonas sp. β-Glucosidase which is cell bound or intracellular in the Cellulomonas sp. was secreted by the hybrid organism, and relative amounts of extracellular β-glucosidase were high. Furthermore, this extracellular β-glucosidase activity was dependent on the nature of the cellulosic substrate. Endoglucanases synthesized in the hybrid differed in their electrophoretic mobilities as compared with the parental enzymes.