Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-1) to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (less than 20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena

Derepressed mutant PR-22 was obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg⁻¹ and cellulolytic activity from 1.9 to 4 U mg⁻¹; this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20–32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l⁻¹ of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.     

Partial characterization of the extracellular carboxymethylcellulase activity produced by the rumen bacterium Bacteroides succinogenes

In cultures of Bacteroides succinogenes, in which cellulose was the source of carbohydrate, from 70 to 80% of the carboxymethylcellulase (CMCase) activity was present in the culture fluid. The crude extracellular enzyme readily hydrolyzed acid-swollen cellulose with the production of glucose and cellobiose. Of this extracellular CMCase, 50-62% was associated with sedimentable membrane fragments, 9-13% with nonsedimentable material with a molecular weight greater than 4 X 10(6), and 28-38% with molecules having a molecular weight of approximately 45 000. Polyacrylamide gel electrophoresis (PAGE), in the presence of sodium dodecyl sulfate, revealed that both the nonsedimentable and the sedimentable fraction had complex protein compositions. The nonsedimentable and sedimentable CMCase fractions, after treatment with Triton X-100, were subjected to PAGE in the presence of 0.2% (w/v) Triton X-100. The results indicated the presence of fast- and slow-migrating CMCases in the former, and of a slow-migrating CMCase in the latter. An apparently uncharged CMCase, which probably corresponded to the slow-migrating component by PAGE, was partially purified from the concentrated culture supernate by solubilization in Triton X-100 and chromatography on DEAE--Sepharose, CM--Sepharose, and Phenyl--Sepharose. The partially purified CMCase had a pH optimum of 5.6-6.6 and a temperature optimum of 50 degrees C.     

Cloning and sequencing of a Bacteroides ruminicola B(1)4 endoglucanase gene.

Bacteroides ruminicola B(1)4, a noncellulolytic rumen bacterium, produces an endoglucanase (carboxymethylcellulase [CMCase]) that is excreted into the culture supernatant. Cultures grown on glucose, fructose, maltose, mannose, and cellobiose had high specific activities of CMCase (greater than 3 mmol of reducing sugar per mg of protein per min), but its synthesis was repressed by sucrose. B. rumincola did not grow on either ball-milled or acid-swollen cellulose even though the CMCase could hydrolyze swollen cellulose. The CMCase gene was cloned into Escherichia coli, and its nucleotide sequence contained a single open reading frame coding for a protein of 40,481 daltons. The enzyme was overproduced in E. coli under the control of the tac promoter and purified to homogeneity. The N-terminal sequence, amino acid composition, and molecular weight of the purified enzyme were similar to the values predicted from the open reading frame of the DNA sequence. However, the CMCase present in B. ruminicola was found to have a monomer molecular weight of 88,000 by Western immunoblotting. This discrepancy appeared to have resulted from our having cloned only part of the CMCase gene into E. coli. The amino acid sequence of the CMCase showed homology to sequences of beta-glucanases from Ruminococcus albus and Clostridium thermocellum.     

Monensin has no effect on growth and metabolism ofMegasphaera elsdenii

A rumen strain ofMegasphaera elsdenii was grown on glucose and lactate in monensin-free and monensin-supplemented medium (10 mg/L). Monensin had no effect on growth rate, growth yields, metabolic pattern and composition of cells. Growth yields of dry matter and protein were higher in cultures supplied with glucose than in cultures supplied with lactate. The bacterium compensated the lower gain of energy from fermentation of lactate by rapid utilization of this substrate. Cells grown on glucose contained more saccharide and less protein than lactate-grown cells.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Regulation of cellulases and xylanases from a derepressed mutant of Cellulomonas flavigena growing on sugar-cane bagasse in continuous culture

When the wild type Cellulomonas flavigena was grown on glycerol, xylose or cellobiose, it produced basal levels of carboxymethyl-cellulase (CMCase), filter-paperase (FPase) and xylanase activities. By comparison, a catabolic derepressed mutant strain of the same organism produced markedly higher levels of these enzymes when grown on the same carbon sources. Sugar-cane bagasse induced both the wild type and the mutant strain to produce three- to eight-time higher levels of FPase and xylanase than was observed with xylose or cellobiose. Continuous culture was used to determine the minimal cellobiose or glucose concentrations that repress the enzyme synthesis in both strains. 2.5 g l(-1) glucose repressed FPase and xylanases from wild type, while 1.6 times more glucose was needed to repress the same activities in the PN-120 strain. In the same way, twofold more cellobiose was needed to reduce by 75% the CMCase and xylanase activities in the mutant compared to the wild type. The FPase in the presence of 4 g l(-1) cellobiose did not change in the same strain. Therefore, its derepressed and feedback resistant characters of PN-120 mutant are evident. On the other hand, isoelectrofocused crude extracts of mutant and wild strains induced by sugar-cane bagasse, did not show differences in protein patterns, however, the Schiffs staining was more intense in the PN-120 than in the wild strain. These results point out that the mutational treatment did not apparently change the extracellular proteins from mutant PN-120 and this could affect their regulation sites, since derepressed and feed-back resistant enzymes may be produced.     

Production of alpha-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

alpha-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55 degrees C and 5.5, respectively, and the apparent K(m) for starch was 0.8 mg ml. The products of alpha-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of alpha-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial alpha-amylase. Activities of alpha-amylase up to 4.4 U ml (1 U represents 1 mumol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml), alpha-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed alpha-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of alpha-amylase (initial concentration, 2.5 mg ml), they were found to be less effective than starch, but maltose was almost as effective. The fungal alpha-amylase was found to be stable at 60 degrees C in the presence of low concentrations of starch (相似文献   

In situ identification of carboxymethyl cellulose-digesting bacteria in the rumen of cattle fed alfalfa or triticale

A method was developed and used to arrest and stain reducing sugars (glucose) produced by bacteria with cell-surface-associated carboxymethyl cellulase (CMCase) and endoglucanase activities (CMC bacteria) in the rumen of cows fed alfalfa or triticale. Precipitation of silver oxide on the surface of individual cells was observed using cellulolytic bacterial pure cultures with known CMCase activity and rumen mixed cultures. The CMC bacteria in the liquid and solid fractions of the rumen digesta were identified using fluorescence in situ hybridization (FISH) with currently available and newly designed oligonucleotide probes. The CMC bacteria contributed between 8.2% and 10.1% to the total bacterial cell numbers. Most of the CMC bacteria (75.2-78.5%) could be identified by FISH probing. The known cellulolytic populations Ruminococcus flavefaciens, R.?albus, and Fibrobacter succinogenes constituted 44.5-53.1% of the total. Other CMC bacteria identified hybridized with the probe Clo549 (11.2-23.0%) targeting members of an uncharacterized genus in Clostridia, the probe Inc852 (8.9-10.7%) targeting members of the family Incertae Sedis III and unclassified Clostridiales, and the probe But1243 (相似文献   

Description of two anaerobic fungal strains from the bovine rumen and influence of diet on the fungal population in vivo

Neocallimastix sp. NC71 and Piromyces sp. PC12 isolated from the calf remen grew optimally at 39 degrees C and pH 6.5-6.7, utilized a wide range of mono-, oligo- and polysaccharides and exhibited CMCase, Avicelase, cellobiase, amylase and xylanase activities. The end-products of wheat straw fermentation by both strains were acetate, formate, ethanol and lactate. The number of Neocallimastix sp. zoospores in the rumen of cows in the first 3 h after feeding with hay-silage-concentrate diets varied from 7 x 10(3) to 5.4 x 10(5) ml-1; the number of uniflagellate zoospores varied from 10(4) to 10(5) ml-1. Fungal zoosporgenesis and colonization of plant substrates in the rumen were induced by feed intake and were favoured by increased levels of crude fibre in the diet.     

Antibiotic activity of an isocyanide metabolite of Trichoderma hamatum against rumen bacteria

A metabolite of Trichoderma hamatum, 3-(3-isocyanocyclopent-2-enylidene)propionic acid, was tested for its effects on growth of and carbohydrate metabolism in 11 strains of functionally important rumen bacteria. To standardize the biological activity of this unstable metabolite, a rapid, aerobic disc diffusion assay was developed using Escherichia coli ATCC 11775. In an anaerobic broth dilution assay using a medium lacking rumen fluid and containing a soluble carbohydrate, the minimum inhibitory concentration of the metabolite which completely inhibited growth of the rumen bacteria for 18 h at 39 degrees C was generally less than 10 micrograms X mL-1; however, the minimum inhibitory concentrations for Megasphaera elsdenii B159 and Streptococcus bovis Pe(1)8 were 10-25 and 25-64 micrograms X mL-1, respectively. In general, the Gram-negative strains were more sensitive than the Gram positive. The minimum inhibitory concentration for Bacteroides ruminicola 23 grown with glucose was 1 micrograms X mL-1; for B. ruminicola GA33 (glucose), B. succinogenes S85 (cellobiose), and Succinivibrio dextrinosolvens 24 (maltose), it was 2 microgram X mL-1. When added to a cellulose-containing rumen fluid medium, 1-4 micrograms X mL-1 of the metabolite delayed cellulose hydrolysis by B. succinogenes S85, Ruminococcus albus 7, and R. flavefaciens FD1 for up to 4 days, and 6-7 micrograms X mL-1 prevented hydrolysis for at least 1 month. In the presence of the metabolite, the proportion of acetate produced from soluble carbohydrate by the majority of strains increased, but with some strains net production of acetate decreased relative to production of other acidic fermentation products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of various factors on ethanol yields from lignocellulosic biomass by Thermoanaerobacterium AK₁₇

The ethanol production capacity from sugars and lignocellulosic biomass hydrolysates (HL) by Thermoanaerobacterium strain AK(17) was studied in batch cultures. The strain converts various carbohydrates to, acetate, ethanol, hydrogen, and carbon dioxide. Ethanol yields on glucose and xylose were 1.5 and 1.1 mol/mol sugars, respectively. Increased initial glucose concentration inhibited glucose degradation and end product formation leveled off at 30 mM concentrations. Ethanol production from 5 g L(-1) of complex biomass HL (grass, hemp, wheat straw, newspaper, and cellulose) (Whatman paper) pretreated with acid (0.50% H(2) SO(4)), base (0.50% NaOH), and without acid/base (control) and the enzymes Celluclast and Novozyme 188 (0.1 mL g(-1) dw; 70 and 25 U g(-1) of Celluclast and Novozyme 188, respectively) was investigated. Highest ethanol yields (43.0 mM) were obtained on cellulose but lowest on hemp leafs (3.6 mM). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The influence of various factors (HL, enzyme, and acid/alkaline concentrations) on end-product formation from 5 g L(-1) of grass and cellulose was further studied to optimize ethanol production. Highest ethanol yields (5.5 and 8.6 mM ethanol g(-1) grass and cellulose, respectively) were obtained at very low HL concentrations (2.5 g L(-1)); with 0.25% acid/alkali (v/v) and 0.1 mL g(-1) enzyme concentrations. Inhibitory effects of furfural and hydroxymethylfurfural during glucose fermentation, revealed a total inhibition in end product formation from glucose at 4 and 6 g L(-1), respectively.     

Growth kinetics of Chinese hamster ovary cells in a compact loop bioreactor. 3. Selection and characterization of an anchorage-independent subline and medium improvement.

Four sublines of Chinese hamster ovary (CHO) cells were selected or cloned on a 10% fetal calf serum supplemented MEM-alpha medium. Three of them were monolayer cultures and could proliferate by 2000 times a week (mu = 1.1 d 1) in T-flasks. The other subline, S1, could grow in suspension even in static T-flask cultures. The stability in chromosome number of these cell lines was investigated. By evaluating the kinetic growth parameters, i.e. the specific rates of growth, glucose consumption and lactic acid production, and the yields of cells and lactic acid from glucose, the S1 cells were considered to be the most suitable subline for the bioreactor suspension culture. The S1 cells reached the greatest maximum of cell concentration among all cell lines tested because of their efficient glucose utilization. Observed nutrient limitations in the S1 cell culture was overcome by modification of the medium composition, that is addition of 10 mg l-1 hypoxanthine, 1 mg l-1 FeSO4.7H2O, and 0.1 mg l-1 sodium putrescine, elimination of glutamine, supplementation of 6 mM asparagine and double amount of isoleucine, leucine, methionine and vitamins other than ascorbic acid, cyanocobalamin and biotin, increase of NaHCO3 concentration from 26 to 40 mM, and finally decrease of NaCl concentration from 122 to 100 mM. With this modified medium, 7.2 X 10(6) ml-1 of the maximum cell concentration was observed in a glucose fed-batch culture, the cell concentration which was twice as much as in batch cultures with the original medium.     

Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.     

Quin's oval and other microbiota in the rumens of molasses-fed sheep

Two rumen-cannulated wether sheep were fed a diet containing 1 kg of a liquid-molasses mixture, 80 g of soybean oil meal, and 100 g of chopped wheat straw once a day. In 6 weeks and thereafter, the microbiota adapted such that Quin's oval, a very large bacterium, was present in huge numbers (11.3 X 10(10) and 1.3 X 10(10) ml-1 after 73 days). Direct microscopic counts were also done on small bacteria, moderate-sized Selenomonas spp., and small Entodinium spp., which were the only protozoa seen. After the necessary dilution of rumen contents to make the microbial cells visible, Quin's ovals were seen to be much smaller in sheep 1 than in sheep 2. Most-probable-number estimates indicated that Methanobrevibacter spp. were present at 10(7) ml-1, Methanosarcina spp. were present at 10(3) ml-1, and Eubacterium limosum-like bacteria were present at 10(5) to 10(6) ml-1. In the adapted sheep, the dry portion of the diet was rapidly consumed, but the molasses mixture was consumed over a 9- to 10-h period. Volatile fatty acids in the rumen were present in very low amounts just prior to feeding and were much higher during the consumption of the diet, with about a 1:1 molar ratio of propionate to acetate between 1 and 9 h after feeding. Data were obtained on hourly feed consumption, levels of volatile fatty acids, and pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quin's oval and other microbiota in the rumens of molasses-fed sheep.

Two rumen-cannulated wether sheep were fed a diet containing 1 kg of a liquid-molasses mixture, 80 g of soybean oil meal, and 100 g of chopped wheat straw once a day. In 6 weeks and thereafter, the microbiota adapted such that Quin's oval, a very large bacterium, was present in huge numbers (11.3 X 10(10) and 1.3 X 10(10) ml-1 after 73 days). Direct microscopic counts were also done on small bacteria, moderate-sized Selenomonas spp., and small Entodinium spp., which were the only protozoa seen. After the necessary dilution of rumen contents to make the microbial cells visible, Quin's ovals were seen to be much smaller in sheep 1 than in sheep 2. Most-probable-number estimates indicated that Methanobrevibacter spp. were present at 10(7) ml-1, Methanosarcina spp. were present at 10(3) ml-1, and Eubacterium limosum-like bacteria were present at 10(5) to 10(6) ml-1. In the adapted sheep, the dry portion of the diet was rapidly consumed, but the molasses mixture was consumed over a 9- to 10-h period. Volatile fatty acids in the rumen were present in very low amounts just prior to feeding and were much higher during the consumption of the diet, with about a 1:1 molar ratio of propionate to acetate between 1 and 9 h after feeding. Data were obtained on hourly feed consumption, levels of volatile fatty acids, and pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Morphometric evaluation of the specific growth rate of Aspergillus niger grown in agar plates at high glucose levels

Development of surface grown cultures of Aspergillus niger no. 10 was studied at two experimental levels: (a) following the time course of the biomass density (X [=] mg cm(-2)) and fitting the data by the logistic expression, which yielded a macroscopic specific growth rate expressed as mu(obs) = (dX/Xdt)[1-(X/X(max))](-1); and (b) measuring morphometric parameters like the specific elongation rate (k) of the germ tubes and their diameters (D(h)), the colony rate of radial extension (u(r)), and the mean length of distal hyphae (L(av)) to estimate the specific growth rate with the following proposed expression: mu(calc) = u(r)ln2[L(av)ln(L(av)/D(h))](-1). Increases in the initial glucose concentration (10, 40, 70, 120, 200, and 300 g L(-1)) caused reductions in the specific growth rates, the elongation kinetics of the germ tubes, and the hyphal diameter, nevertheless, u(r) and X(max) presented parabolic behavior, showing their maxima in the interval of 90 to 120 g L(-1) of glucose. The overall macroscopic effect of the tested concentrations of glucose on surface grown cultures of A. niger was to produce densely packed and slowly extending colonies, where changes in hyphal lengths and diameters were significant. There was good agreement between mu(obs) and mu(calc) values. Hence, this work validates a kinetic model based on morphometric data to estimate the specific growth rate of molds, obtained from dry weight data, using mold cultures grown in the same solid medium i.e., agar plates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 287-294, 1997.     

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent K_m for starch was 0.8 mg ml⁻¹. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml⁻¹ (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml⁻¹ in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml⁻¹), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml⁻¹), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.