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.     

The Genome Sequences of Cellulomonas fimi and “Cellvibrio gilvus” Reveal the Cellulolytic Strategies of Two Facultative Anaerobes,Transfer of “Cellvibrio gilvus” to the Genus Cellulomonas,and Proposal of Cellulomonas gilvus sp. nov

Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484^T. For comparative purposes, we also sequenced the genome of the aerobic cellulolytic “Cellvibrio gilvus” ATCC 13127^T. An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that “Cellvibrio gilvus” belongs to the genus Cellulomonas. We thus propose to assign “Cellvibrio gilvus” to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482^T showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.     

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.     

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.     

Nutrient optimization for cellulase biosynthesis by a newly isolatedCellulomonas sp.

Summary A cellulolytic bacterium has been isolated from soil and identified as aCellulomonas sp. Optimization of media components and environmental factors led to a 17-fold increase in cellulase activity within 40 h. The trace elements Zn²⁺, Co²⁺ and Cu²⁺ caused severe inhibition, which could be reduced by the addition of EDTA. The enzymes endoglucanase, exoglucanase and xylanase were always present irrespective of the carbon source used.

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Resumen Se ha aíslado e identificado comoCellulomonas sp. una bacteria cellulolítica del suelo. Mediante la optimización de los componentes del medio de crecimiento y de los factores ambientales se consiguió un incremento de 17 veces de la actividad cellulásica en 40 horas. Los elementos traza Zn²⁺, Co²⁺, y Cu²⁺ causaron una severa inhibición que pudo reducirse gracias a la adición de EDTA. Independientemente de la fuente de carbono utilizada los enzimas endoglucanasa, exoglucanasa y xilanasa se encontraron siempre presentes en el medio.

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Résumé Une bactérie cellulolytique a été isolée à partir du sol et identifiée commeCellulomonas sp. L'activité cellulase en 40 h a été accrue 17 fois par optimisation des constituants du milieu et des conditions de culture. Des traces de Zn²⁺, Co²⁺, et Cu²⁺ déterminent une sévère inhibition, qui peut être réduite par l'addition d'EDTA. Les enzymes endoglucanase, exoglucanase et xylanase sont toujours présentes, quelque soit la source de carbone utilisée.

     

Methods for Intense Aeration, Growth, Storage, and Replication of Bacterial Strains in Microtiter Plates

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter⁻¹ h⁻¹, corresponding to a mass transfer coefficient of 188 h⁻¹) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter⁻¹ during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.     

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.     

Screening Wheat Genotypes for High Callus Induction and Regeneration Capability from Immature Embryo Cultures

Selecting the explant genotypes is crucial step in in vitro culture and Agrobacterium-mediated transformation system due to its host range specificity. Immature embryos of five winter and three spring wheat (Triticum aestivum L) cultivars were evaluated for tissue culture response in three callus initiation media. MS medium containing 2,4-0 (2 mg ml^-1) plus B5 vitamins (MSB5), MS medium containing 2,4-0 (1 mg ml^-1) with no vitamins (MS1GC) or MS medium containing picloram (2.2 mg ml^-1) and 2,4-0 (0.5 mg ml^-1) plus MS vitamins (CM4C) were used for callus initiation. Percentage of callus induction varied widely with the genotype and initiation medium used, with values ranging from 5.7% to 100%. Embryogenic capacity of genotypes was evaluated by number of somatic embryos formed from cultured immature embryos. Bob White (spring) and NE92458 (winter) were equal and most embryogenic; Pronghorn and 2137 (both winter) were the poorest. CM4C medium was found to be the best medium for initiating embryogenic callus among three culture media tested. A standard regeneration procedure was used. The genotypes with the highest regeneration efficiencies were Bob White, Fielder and NE92458, (1.8, 1.4 and 1.6 plantslexplant, respectively).     

Microcalorimetric study of aerobic growth of Cellulomonas sp. 21399 on various carbohydrates

Summary Microcalorimetry was used to study the energetic aerobic growth of Cellulomonas sp. 21399 on glucose, cellobiose and amorphous and crystalline cellulose. The thermochemical aspect of growth on glucose was established with regard to the anabolic contribution. The results obtained allowed the use of glucose as a reference substrate for cellulose degradation. The experimental enthalpy change and the maximum catabolic activity, calculated from the maximum power evolved by the culture, were, respectively,-1079 kJ/mol and 0.85 mmol glucose per hour per dry weight of cells. The growth response on amorphous cellulose was equivalent to that demonstrated on glucose. However, on crystalline cellulose media, Cellulomonas sp. 21399 exhibited eight times less power and the quantity of heat evolved during growth showed that 50% of the cellulose was degraded. Quantitative results and the shape of power-time curves achieved indicate that the structural features of cellulose strongly influence its microbial degradability.     

Use of a combined Cellulomonas and Trichoderma cellulase preparation for cellulose saccharification

Summary An enzyme preparation from a mutant strain of Cellulomonas CS1-17 acts synergistically with low levels of Trichoderma reesei cellulase in saccharification of alkali-pretreated sugar cane bagasse and in assays of Filter Paper activity. Supplementation of the Cellulomonas preparation with 0.1 or 0.25 FPU.ml^- of T. reesei cellulase provides a preparation approximately equivalent to one using T. reesei alone at 1 FPU.ml^-1.     

Modeling the batch bacteriocin production system by lactic acid bacteria by using modified three-dimensional Lotka–Volterra equations

Different batch cultures of Lactococcus lactis CECT 539, a nisin-producing strain, were carried out in culture media prepared with whey and mussel processing wastes. From these cultures, a reasonable system of differential equations, similar to the three-dimensional Lotka–Volterra two predators-one prey model, was set up to describe, for the first time, the relationship between the absolute rates of growth, pH drop and nisin production.Thus, the nisin production system was described as a three-species (pH, biomass and nisin) ecosystem. In this case, both nisin and biomass production were considered as two pH-dependent species that compete for the nitrogen source. Excellent agreement (R² values ≥0.9885) resulted between model predictions and the experimental data, and significant values for all the model parameters were obtained. The developed model was demonstrated (R² values ≥0.9874) for five batch cultivations of the strains L. lactis CECT 539 in MRS broth and Lactobacillus sakei LB 706 (sakacin A producer), Pediococcus acidilactici LB42-923 (pediocin AcH producer), L. lactis ATCC 11454 (nisin producer) and Leuconostoc carnosum Lm1 (leuconocin Lcm1 producer) in TGE broth. These results suggest that the batch bacteriocin production system in these culture media can be successfully described by using the Lotka–Volterra approach.     

S9A Serine Protease Engender Antigenic Gluten Catabolic Competence to the Human Gut Microbe

The human gut microbiome has a significant role in host physiology; however its role in gluten catabolism is debatable. Present study explores the role of human gut microbes in gluten catabolism and a native human gut microbe Cellulomonas sp. HM71 was identified. SSU rDNA analysis has described human gut microbiome structure and also confirmed the permanent residentship of Cellulomonas sp. HM71. Catabolic potential of Cellulomonas sp. HM71 to cleave antigenic gluten peptides indicates presence of candidate gene encoding biocatalytic machinery. Genome analysis has identified the presence of gene encoding S9A serine protease family—prolyl endopeptidase, with Ser591, Asp664 and His685 signature residues. Cellulomonas sp. HM71 prolyl endopeptidase activity was found optimal at pH 7.0 and 37 °C with a K_M of 35.53 μmol and specifically cleaves at proline residue. Current study describes the gluten catabolism potential of Cellulomonas sp. HM71 depicting possible role of human gut microbes in gluten catabolism to confer resistance mechanisms for the onset of celiac diseases in populations with gluten diet.     

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.     

Evidence for a role for calcium in immunosuppression by tumor cells in vitro.

The anti-SRBC response of normal, syngeneic splenocytes in the presence of cells from various tumors (Moloney leukemia spleen cells and methylcholanthrene-induced rhabdomyosarcoma cells (MC)) was tested in vitro in different culture media: RPMI 1640, BME with Hanks balanced salt solution (MEM), and CMRL 1066. The tumor-associated cells expressed an immunosuppressive effect, the degree of which varied with the culture medium used. Whereas spleen cells cultured in RPMI in the presence of tumor-associated cells were highly inhibited in their response to SRBC, those cultured in MEM were not. A full 5 to 10 times more tumor cells were required to achieve the same degree of immunosuppression in MEM. There appeared to be a correlation between the degree of immunosuppression obtained and the Ca²⁺ concentration of the medium. Thus the immunosuppressive effect of tumor-associated cells was greatest in cultures with RPMI 1640 (0.4 mM Ca²⁺), lesser in MEM (1.27 mM Ca²⁺), and least in CMRL 1066 (1.8 mM Ca²⁺). Furthermore, if the Ca²⁺ content of RPMI 1640 was increased to 1.4 mM Ca²⁺ by the addition of CaCl₂, the percent suppression to the anti-SRBC response in vitro mediated by the addition of tumor cells decreased to the level found in MEM. Increasing the Mg²⁺ content of RPMI had no effect on tumor-mediated immunosuppression. Tumor cell replication and RNA synthesis were comparable in all media tested, regardless of Ca²⁺ concentration. In view of the increasing evidence for a role for Ca²⁺ in lymphocyte activation, we postulate herein that the Ca²⁺ content of the medium plays a role in the manifestation of immunosuppression by tumorassociated cells in vitro.     

Optimization of culture conditions for the direct regeneration of Kappaphycus alvarezii (Rhodophyta,Solieriaceae)

Cultivation of seaweeds on a commercial scale requires a large number of propagules with desirable phenotypic traits which include high growth rates and resistance to diseases. Seaweed micropropagation can be considered as one of the best methods to provide a large amount of seedlings for commercial cultivation. This study was carried out to optimize the parameters known to affect the growth of Kappaphycus alvarezii in vitro and subsequently improve the production of seedlings through micropropagation. Suitability of media, concentration of phytoregulators, types and concentration of fertilizers, culture density, light intensity, interval of aeration activity, salinity, and pH were found to be critical factors for the growth of K. alvarezii. The optimum condition for direct regeneration of K. alvarezii in a culture vessel was found to be cultivation of explants in Provasoli's enriched seawater (PES) media supplemented with 2.5 mg L^?1 6-benzylaminopurine (BAP), 1.0 mg L^?1 indole-3-acetic acid (IAA), and 3.0 mg L^?1 natural seaweed extract (NSE) with culture density of 0.4 %?w/v, under light intensity of 75 μmol photons m^?2 s^?1, continuous aeration of 30.0 L h^?1, salinity of 30.0 ppt, and pH 7.5. An airlift photobioreactor was constructed for the mass propagation of K. alvarezii explants with optimum culture conditions obtained from the study. The optimum growth rates of the K. alvarezii explants in culture vessels (5.5 % day^?1) and photobioreactor (6.5 % day^?1) were found to be higher than the growth rate observed in field trials in the open sea (3.5 % day^?1). The information compiled during the course of this study will be of utility to commercial seaweed cultivators.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

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.     

Conditions for fat production by a recombinant strain of yeast

A defect in fatty acid synthetase of Saccharomyces cerevisiae was complemented by intergeneric transformation. A transformation strain, 63a, was selected for further study after preliminary screening of recombinants for lipogenic properties. Fat accumulation by the 63a strain in an aerated, stirred fermentor was affected by the carbon to nitrogen ratio, rate of aeration, pH and various supplementations of culture media. In batch fermentations, fat productivity under optimal conditions in complex media was 4 to 5 gl⁻¹·d⁻¹. Analysis of triacylglycerols, which comprised 85% of total lipids, showed 36.5% palmitate, 19.2% stearate, 35.2% oleate and 9.1% linoleate. The defatted residue contained 32.6% crude protein and 47.8% carbohydrate on a dry weight basis.     

Culture Media Statistical Optimization for Biomass Production of a Ligninolytic Fungus for Future Rice Straw Degradation

The main objective of this study was to optimize a culture media for low scale biomass production of Pleurotus spp. Future applications of this optimization will be implemented for “in situ” rice straw degradation, increase soil nutrients availability, and lower residue and rice culture management costs. Soil samples were taken from different points in six important rice production cities in Colombia. For carbon and nitrogen source selection a factorial 4² design was carried out. The Plackett-Burman design permitted to detect carbon, nitrogen and inducer effects on fungus growth (response variable for all designs). This optimization was carried out by a Box-Behnken design. Finally a re-optimization assay for glucose concentration was performed by means of a One Factor design. Only 4/33 (12 %) isolates showed and important laccase or manganese peroxidase activity compared to Pleurotus ostreatus (HPB/P3). We obtained an increased biomass production in Pleurotus spp. (T1.1.) with glucose, followed by rice husk. Rice straw was considered an inducing agent for lignin degradation. Glucose was a significant component with positive effects, whereas Tween 80 and pH had negative effects. On the contrary, rice husk, yeast extract and CaCl₂ were not significant components for increase the biomass production. Final media composition consisted of glucose 25 g L^?1, yeast extract 5 g L^?1, Tween 80 0.38 % (v/v), Rice husk 10 g L^?1, CaCl₂ 1 g L^?1, and pH 4.88 ± 0.2. The Box-Behnken polynomial prediction resulted to be lower than the experimental validation of the model (6.59 vs. 6.91 Log₁₀ CFU ml^?1 respectively).     

<Emphasis Type="Italic">Cellulomonas macrotermitis</Emphasis> sp. nov., a chitinolytic and cellulolytic bacterium isolated from the hindgut of a fungus-growing termite

To investigate the symbiotic roles of the gut microbiota in the fungus-growing termite Macrotermes barneyi, a novel strain with chitinolytic and cellulolytic activity, designated strain an-chi-1^T, was isolated from the hindgut of M. barneyi. Strain an-chi-1^T grows optimally at 28–30 °C, pH 8.0 in PYG medium. On the basis of 16S rRNA gene sequence analysis, this isolate belongs to the genus Cellulomonas with high sequence similarity to Cellulomonas iranensis (99.4%), followed by Cellulomonas flavigena (98.4%), Cellulomonas phragmiteti (97.4%), Cellulomonas oligotrophica (97.2%) and Cellulomonas terrae (97.0%). The DNA–DNA relatedness between an-chi-1^T and the type strains of C. iranensis and C. flavigena DSM20109^T are 35.4% and 23.7%, respectively. The major cellular fatty acids are anteiso-C_15:0 and C_14:0. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylinositol dimannosides and one unidentified phospholipid. The cell-wall sugar is ribose. The peptidoglycan contains glutamic acid, aspartic acid and alanine. The DNA G+C content is 67.3 mol%. Based on its distinctive phenotypic, phylogenetic, and chemotaxonomic characteristics, an-chi-1^T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas macrotermitis sp. nov. is proposed. The type strain is an-chi-1^T (= JCM 31923^T = CICC 24195^T).