Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.     

Chitinolytic enzymes of the rumen ciliate <Emphasis Type="Italic">Eudiplodinium maggii</Emphasis>

The ability of rumen ciliates to digest chitin is clearly recognized. We investigated the chitinolytic system of the rumen ciliate Eudiplodinium maggii. The ciliates were grown in a selectively faunated sheep. They were isolated from the rumen and purified by sedimentation. A crude enzyme preparation was prepared following incubation of ciliates with antibiotics. This was done in order to reduce their contamination with intracellular bacteria. The activity of particular enzymes was examined by quantification of the products released from specific substrates. It was stated that the optimum conditions for the detected activities varied between 4.5 and 5.5 pH, and 45 and 55 °C. β-N-Acetylglucosaminidase was found as an enzyme of the highest activity (4.2 μmol/l released product per mg protein per h). The activities of endochitinase and exochitinase were almost two times lower than that of β-N-acetylglucosaminidase. Zymographic studies revealed the presence of two endochitinases, two exochitinases and two β-N-acetylglucosaminidases in the examined preparation.     

Cellulase stimulation during biodegradation of lignocellulosic residues at increased biomass loading

Microbial degradation of lignocellulosic biomass is primarily affected by the composition and structure of biomass, as well as enzyme activities that are influenced by the presence of in-process degradation products. This study focuses on the latter, and demonstrates that cellulase activity of Neurospora discreta is stimulated in the presence of in-process soluble lignin degradation products. Two types of biomass - cocopeat and sugarcane bagasse, with contrasting lignin content and cellulose structure were tested at two biomass loadings each. At the higher biomass loading, cocopeat showed the highest amount of hydrolyzed cellulose and cellulase activity, despite its low cellulose content and recalcitrant cellulose structure. A strong positive correlation was revealed between the amount of in-process degraded lignin and cellulase activity, indicating a stimulatory effect on cellulase, which contradicts most previous literature. Furthermore, the causal relationship between the amount of degraded lignin and cellulase activity was established in a model system of commercial cellulase and standard soluble lignin. This work could pave the way for using biomass loading as a process lever to enhance cellulose hydrolysis in microbial conversion of lignocellulosic biomass.     

Effect of rumen ciliates on the digestion of different carbohydrates in sheep. I.--Utilization of cell wall carbohydrates (cellulose and hemicellulose) and of starch

Two diets rich in cell-wall carbohydrates or starch were given to 10 rumen-fistulated sheep; two sheep were defaunated and the others were inoculated with Polyplastron multivesiculatum (P) or Entodinium sp. (E), or both (P + E), or with conventional fauna. Ciliate biomass was greater when the animals were fed a high starch diet than when the diet was rich in cell-wall carbohydrates (table 2). With both diets, the Entodinium genus in the mixed fauna sampling predominated. We showed that Polyplastron was directly involved in cell-wall carbohydrate breakdown, while Entodinium capacity to digest cellulose remained low. We noted that with a diet rich in cellulose and hemicellulose, bacterial cellulolytic activity was improved by the presence of ciliates in the rumen but was decreased with the "starch" diet (table 3). The greater VFA concentration observed in the faunated animals expressed ciliate effect on the fermentations as well as activation of bacterial metabolism. With a high starch diet, the Entodinium sp. ciliates may have a buffering effect on the pH values in the rumen by limiting bacterial fermentation after food intake and by prolonging starch digestion during the day (table 4). The composition of the VFA mixture was modified by ciliate inoculation. The molar proportion of butyric acid always increased, while that of acetic and propionic acids evolved differently according to the diets and the ciliates (table 4). The higher ammonia concentration in the rumen liquor observed in faunated animals (table 4) could be explained either by the breakdown of both feed and bacterial proteins ingested by ciliates or by a lower ammonia nitrogen incorporation by fewer bacteria. Statistical analyses were used to explain the specific effect of P and E and also the interactions between them and between each of them and the diets.     

OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum

The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.     

Protein-extracted lucerne fibres as a carbon and energy source for cellulase production by Thermomonospora curvata

Protein-extracted lucerne fibre (PELF) was evaluated as a carbon/energy source for cellulase production by the thermophilic actinomycete, Thermomonospora curvata. Shake cultures with lucerne fibre produced only one-half the cellulase obtained from growth on cotton fibre (the most inductive substrate). Control of pH at 8·0 under fermenter conditions lowered the rate of PELF carbohydrate solubilization during early growth and raised saccharifying cellulase production by about 70% to a maximum of 0·48 filter paper units/ml.     

Role of anaerobic ciliates in planktonic food webs: abundance, feeding, and impact on bacteria in the field

We studied the dynamics of two populations of anaerobic ciliates, Plagiopyla sp. and Metopus sp., and of their potential prey, heterotrophic and phototrophic purple bacteria, in Lake Cisó throughout a 1-year cycle. The abundance of both ciliates was very low (less than 2 individuals per ml). During mixing, Plagiopyla ciliates exhibited high clearance rates (about 100 nl ciliate h), its integrated abundance increased with a net doubling time of 47 days, and its potential doubling times, as calculated from the number of bacteria consumed, ranged between 5 and 8 days. During stratification, the activity of Plagiopyla ciliates was reduced and the population decreased; this was related to the higher amounts of sulfide present. The impact of predation by the Plagiopyla population on bacterioplankton was found to be insignificant, less than 0.1% of bacterial biomass consumed per day. Thus, anaerobic ciliates cannot control the bacterioplankton in Lake Cisó because of both the low abundance over the period studied and the low feeding rates during certain periods. A review of available field studies suggests that this conclusion can be extrapolated to most other anoxic systems.     

Growth kinetics of Colpoda steinii on Escherichia coli.

Colpoda steinii was grown in two-stage continuous cultures with Escherichia coli as prey species. The concentration of prey and the ciliate mean cell volume, dry weight, and number per milliliter were determined at known growth rates. Steady states were reached in the second-stage continuous cultures at all growth rates. Although changes occurred in mean cell size of the ciliates and in the number per milliliter at various growth rates, the yield of protozoan biomass per unit of prey consumed was constant at all growth rates. The data were compared with several equations proposed to describe the kinetics of protozoan growth as a function of prey density.     

Growth kinetics of Colpoda steinii on Escherichia coli.

Colpoda steinii was grown in two-stage continuous cultures with Escherichia coli as prey species. The concentration of prey and the ciliate mean cell volume, dry weight, and number per milliliter were determined at known growth rates. Steady states were reached in the second-stage continuous cultures at all growth rates. Although changes occurred in mean cell size of the ciliates and in the number per milliliter at various growth rates, the yield of protozoan biomass per unit of prey consumed was constant at all growth rates. The data were compared with several equations proposed to describe the kinetics of protozoan growth as a function of prey density.     

Influence of ciliate protozoa on biochemical changes and hydrolytic enzyme profile in the rumen ecosystem

AIMS: To assess the effect of presence or absence of rumen protozoa on fermentation characteristics and enzyme profile in growing lambs. METHODS AND RESULTS: Weaner lambs (G1, G2, G3, G4, G5 and G6 groups) were defaunated by oral administration of sodium laurel sulphate (at 8 g 100 kg(-1) body weight). The lambs of G4, G5 and G6 groups were refaunated. The roughage and concentrate ratio in the diet of G1 and G4, G2 and G5, and G3 and G6 were 50:50 (R1), 65:35 (R2) and 80:20 (R3), respectively. Daily dry matter intake was similar in defaunated and faunated lambs. However, digestibility of organic matter (OM), cellulose and gross energy were lower in defaunated lambs while crude protein (CP) digestibility was similar in both defaunated and faunated lambs. The rumen pH and NH3-N were lower (P < 0.01) while TVFA, total-N and TCA-ppt-N were higher (P < 0.01), in defaunated lambs. Ruminal activity of carboxymethyl cellulase was lower (P < 0.01) in defaunated lambs and amylase, xylanase, protease and urease were similar in faunated and defaunated lambs. Nutrient utilization, rumen metabolites and ciliate protozoal count were higher, whereas digestibility of fibre fractions was lower in high rather than low concentrate fed lambs. The rumen protozoa present before defaunation were B-type and the protozoa which re-established on refaunation were also B-type. CONCLUSIONS: Absence of ciliate protozoa decreased nutrient digestibility and increased ruminal TVFA and total-N with lower NH3-N concentration, indicating better energy and protein utilization in defaunated lambs. SIGNIFICANCE AND IMPACT OF THE STUDY: Defaunation improved energy and protein utilization in lambs.     

Degradation of 2,4,6-trichlorophenol (2,4,6-TCP) by co-immobilization of anaerobic and aerobic microbial communities in an upflow reactor under air-limited conditions

The co-immobilization and the culture of anaerobic and aerobic communities was tested for the mineralization of 2,4,6-trichlorophenol (2,4,6-TCP). At first, the anaerobic microorganisms (aggregated into granules) were cultivated in an upflow anaerobic sludge blanket (UASB) reactor, in a continuous mode, with glucose, propionate, acetate (COD loading rate = 0.5-2.0 g COD/l per day, ratio 1:1:1) and 2,4,6-TCP (2,4,6-TCP loading rate = 25-278 micromol/l per day) as substrates. 2,4,6-TCP was degraded into 2,4-DCP and 4-CP, but it was not mineralized because of the low degradation rates of 4-CP. Furthermore, the highest loading rates of 2,4,6-TCP (>126 micromol/l per day) caused the inhibition of the strains degrading the propionate. The granules were therefore tested in association with the aerobic community. They were immobilized in kappa-carrageenan/gelatin [2% (w/w) of each polymer] gel beads and cultivated in a reactor, on their own (to test the influence of the gel), and then with the aerobic community, under anaerobic and air-limited conditions, respectively. The results showed that (1) the gel did not influence the activity of the granules, (2) the anaerobic and aerobic communities could be easily co-immobilized in gel beads and cultivated in a reactor, (3) the mineralization of 2,4,6-TCP (2,4,6-TCP loading rate = 10-506 micromol/l per day), its intermediates of degradation and the other substrates [glucose + acetate + propionate (ratio 1:1:1) = COD loading rate = 500 mg COD/l per day] could be obtained under air-limited conditions if the culture parameters were strictly controlled [airflow = 36-48 vvd (volume of air/volume of liquid in the reactor per day), pH value at around 7.5]. Finally, the gel did not retain its structure during the whole culture (263 days) in the air-limited reactor, but the anaerobic and aerobic communities retained their activities and worked together for the mineralization.     

Sorbose mediated enhancement of cellulase biosynthesis inTrichoderma reesei

Addition of L-sorbose, a non-metabolizable non-inducing ketohexose, toTrichoderma reesei cultures growing on cellobiose or Avicel-cellulose lead to increased cellulase activities. Addition of sorbose resulted in a 6-fold increase in cellodextrins (cellotriose, cellotetraose, cellopentaose) concentration on day 3 in cellobiose cultures and 1.3-fold increase in cellodextrins concentrations on day 4 in Avicel cellulose cultures. This increase in intracellular cellodextrins concentration matched closely with the increase in endoglucanase activity at these time points. Treatment of the cell-free extracts with cellulase preparation led to disappearance of the cellodextrins and increase of glucose. These observations suggested a more direct involvement of cellodextrins in cellulase induction process. The cellulases produced in sorbose-supplemented cellobiose medium hydrolyzed microcrystalline cellulose as effectively as the ones produced on Avicel cellulose medium.     

High-Rate two-phase process for the anaerobic degradation of cellulose, employing rumen microorganisms for an efficient acidogenesis

A novel two-stage anaerobic process for the microbial conversion of cellulose into biogas has been developed. In the first phase, a mixed population of rumen bacteria and ciliates was used in the hydrolysis and fermentation of cellulose. The volatile fatty acids (VFA) produced in this acidogenic reactor were subsequently converted into biogas in a UASB-type methanogenic reactor.A stepwise increase of the loading rate from 11.9 to 25.8 g volatile solids/L reactor volume/day (g VS/L/day) did not affect the degradation efficiency in the acidogenic reactor, whereas the methanogenic reactor appeared to be overloaded at the highest loading rate. Cellulose digestion was almost complete at all loading rates applied. The two-stage anaerobic process was also tested with a closed fluid circuit. In this instance total methane production was 0.438 L CH(4)g VS added, which is equivalent to 98% of the theoretical value. The application of rumen microorganisms in combination with a high-rate methane reactor is proposed as a means of efficient anaerobic degradation of cellulosic residues to methane. Because this newly developed two-phase system is based on processes and microorganisms from the ruminant, it will be referred to as "Rumen Derived Anaerobic Digestion" (RUDAD-) process.     

Degradation of phenanthrene,fluorene and fluoranthene by pure bacterial cultures

Summary Bacterial mixed cultures able to degrade the polycyclic aromatic hydrocarbons (PAH) phenanthrene, fluorene and fluoranthene, were obtained from soil using conventional enrichment techniques. From these mixed cultures three pure strains were isolated:Pseudomonas paucimobilis degrading phenanthrene;P. vesicularis degrading fluorene andAlcaligenes denitrificans degrading fluoranthene. The maximum rates of PAH degradation ranged from 1.0 mg phenanthrene/ml per day to 0.3 mg fluoranthene/ml per day at doubling times of 12 h to 35 h for growth on PAH as sole carbon source. The protein yield during PAH degradation was about 0.25 mg/mg C for all strains. Maximum PAH oxidation rates and optimum specific bacterial growth were obtained near pH 7.0 and 30°C. After growth entered the stationary phase, no dead end-products of PAH degradation could be detected in the culture fluid.     

Hydrolytic activities of anaerobic fungi from wild blue bull (Boselaphus tragocamelus)

The anaerobic fungi play an active role in the plant fibre degradation by producing a wide array of potential hydrolytic enzymes in the rumen. In present study, 12 anaerobic fungal strains were isolated from the faecal samples of wild blue bull, and identified as species of Piromyces, Anaeromyces, Orpinomyces and Neocallimastix based on their morphological characteristics. Isolate WNG-12 (Piromyces sp.), showed maximum filter paper cellulase (23 mIU ml(-1)) and xylanase (127 mIU ml(-1)) activity, while WNG-5 (Piromyces sp.) showed maximum carboxymethyl cellulase activity (231 mIU ml(-1)). Based on the results obtained, it can be stated that Piromyces sp. WNG-12 may be a promising isolate in utilizing fibre rich diets in the rumen as evidenced by the production of hydrolytic enzymes in vitro.     

Rumen enzyme profile and fermentation characteristics in sheep as affected by treatment with sodium lauryl sulfate as defaunating agent and presence of ciliate protozoa

The efficiency of sodium lauryl sulfate as a defaunating agent and effect of rumen protozoa on nutrient utilization, fermentation characteristics and enzyme profile were evaluated in adult sheep maintained on a mixed ration containing 65:35% Pala (Ziziphus numularia) leaf: concentrate. Twenty-one adult Malpura sheep divided into three equal groups (DF, RF and F) were either defaunated by oral administration of sodium lauryl sulfate at the rate of 8 g/100 kg body weight (DF), or defaunated and again refaunated (RF), or maintained faunated (F). Daily dry matter intake was similar in defaunated, refaunated and faunated sheep. However, digestibility of cell wall and cell wall contents (NDF, ADF and cellulose) were lower (P < 0.01) in defaunated than refaunated and faunated sheep. Irrespective of the presence or absence of rumen protozoa, daily intake of DCP and DE were similar in the three experimental groups. Even with similar DM, DCP and DE intake, N-retention, blood glucose level, ruminal concentration of total VFA and total-N were higher (P < 0.01), while rumen pH and NH₃-N concentration were lower (P < 0.01) in defaunated sheep. Ruminal activity of amylase, xylanase, protease and urease enzymes were not influenced by presence or absence of ciliate protozoa. However, carboxymethyl cellulase enzyme activity was lower (P < 0.01) in the rumen of defaunated sheep. The total and differential counts of rumen protozoa were similar in refaunated and faunated sheep indicating lack of residual toxic effect of sodium lauryl sulfate. It is concluded that absence of ciliate protozoa increased ruminal TVFA, total-N with lower NH₃-N concentration and fibre digestibility in sheep. Moreover, sodium lauryl sulfate was fully effective for complete removal of rumen ciliate protozoa and successfully defaunated the sheep.     

Isolation and characterization of mesophilic cellulose-degrading bacteria from flower stalks-vegetable waste co-composting system

Fifteen mesophilic bacteria with high C(x) cellulase activities were isolated and purified from a mixed-culture enriched from a flower stalks-vegetable waste co-composting system. A CMCase test showed that the enzyme activity of these isolates ranged from 7.9 to 28.0 U ml(-1). Although filter paper degrading capability was low in single culture, significant synergetic cellulose degradation were detected in four groups of mixed cultures, their degradation rates were 23.5%, 26.3%, 19.4% and 24.5%, respectively. Study of morphological and physiological characters of five predominant isolates which possess high CMCase and had positive effect on synergetic cellulose degradation in mixed culture system showed that two of them were closely related to Bacillus pasteurii and Bacillus cereus, whereas the rest belong to the genus Halobacillus, Aeromicrobium and Brevibacterium, respectively.     

Effect of rumen ciliates on the digestive utilization of various carbohydrate-rich diets and on the end-products formed in the rumen. II. Utilization of inulin, saccharose and lactose

Three diets rich in inulin, saccharose and lactose, respectively, were given to 10 rumen-fistulated sheep. Two animals were defaunated, two were inoculated with either Polyplastron multivesibulatum or Entodinium sp., and two others were inoculated with both. The latter two were bred in conventional conditions. All animals ingested the same amounts of carbohydrates in the three diets (21-22 g/kg P0.75/day). Dietary nitrogen content was similar (table 1). The ciliate population was improved with the inulin diet (fig. 1; table 2). With a mixed population, the Entodinium improved with the inulin diet (fig 1; table 2). With a mixed population, the Entodinium sp. genus was always predominant. Holotrich protozoa (mainly Isotricha) in the rumen of the conventional sheep represented 15 to 30% of the total ciliate biomass, indicating that they were able to metabolize these soluble sugars. We also observed that P. multivesiculatum can ferment cellulose and all the soluble carbohydrates proposed in these diets. However, Entodinium sp. development occurred mainly in the presence of the sugard produced during carbohydrate hydrolysis by other ciliates or bacteria. The highest organic matter digestibility, noted in faunated animals (table 3) was confirmed by the VFA concentration in the rumen (table 4). This could be explained either by an activation of bacterial metabolism due to predation or by the direct effect of ciliates on fermentations, or both. Modifications in the VFA composition varied with ciliate inoculation, showing that ciliate metabolism may vary with the nature of the energy in the diet or that the observed results depended on various opposite effects in which the intensity of each component was influenced by the diet. In general, the acetic acid molar proportion increased and propionic acid decreased when there was a considerable Entodinium sp. population. The effect on butyric acid was low with these diets. Higher ammonia and lactic acid concentrations were observed in the rumen of faunated than defaunated sheep, irrespective of the ciliate inoculum.     

Anaerobic digestion of cellulose fraction of domestic refuse by means of rumen microorganisms

The anaerobic digestion of a cellulose-enriched fraction of domestic refuse by means of rumen microorganisms in an "artificial rumen" digester was studied. Various combinations of solid and liquid retention times and loading rates were applied to establish optimum conditions for the acidogenic phase digestion of the refuse fraction. An optimal substrate conversion of about 72% was obtained at a loading rate of 23.4 g volatile solids (VS)/L d and a solids retention time of 90 h. Variation of dilution rate between 1.04 and 3.14 fermentor volume turnovers per day had no effect on degradation efficiency. At a loading rate of 23.4 g VS/L d a differential removal rate of solids and liquids appeared to be necessary to obtain an effective degradation of the refuse fraction.     

In vitro primary satellite cell growth and differentiation within litters of pigs

Postnatal muscle growth is dependent on satellite cell (SC) proliferation, differentiation and fusion to increase the DNA content of existing muscle fibres and thereby the capacity to synthesize protein. The purpose of the present study was to examine the ability of isolated SCs from low, medium and high weaning weight litter mates of pigs to proliferate and differentiate, and to affect protein synthesis and degradation after fusion into myotubes. At 6 weeks of age, SCs from the lowest weight (LW), medium weight (MW) and highest weight (HW) female pigs within eight litters were isolated. Thereby, eight cultures of SCs were established for each of the three weight groups within litter, representing three groups of SCs from pigs exhibiting differences in postnatal muscle growth performance. Proliferation was estimated as the number of viable cells at different time points after seeding. SC differentiation was evaluated by measuring the activity of the muscle-specific enzyme, creatine phosphokinase, and protein synthesis and degradation were measured by incorporation and release of 3H-tyrosine, respectively. A tendency towards a difference in proliferation between SC cultures was found (P = 0.09). This was evident as the number of viable cells at day 3 was lower in cultures from LW pigs than from HW (P < 0.05) and MW (P < 0.01) pigs. Differentiation was significantly different between cultures (P < 0.05). There was a significant difference between LW and MW cultures at 72 h (P < 0.05), and a tendency towards a difference between LW and HW cultures at 45 h (P = 0.07). Protein synthesis per μg protein or per μg DNA did not differ among SC cultures from LW, MW and HW pigs. Neither did protein degradation rate differ significantly among SC cultures from LW, MW and HW pigs. Overall, the results show that SCs from LW pigs seem to proliferate and differentiate at a slower rate than SCs from MW and HW pigs. The results found in this study show no difference in the ability of SCs to affect protein synthesis or degradation between SCs from litter mates exhibiting different growth rates in vivo.