Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 h. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given times was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration.     

Cellulolytic Activity of Botrytis cinerea in vitro and in vivo

Experiments were carried out to determine whether stepwise breakdown of native cellulose is carried out by B. cinereain vitro and in vivo. Protein fractions were obtained from ungerminated conidia, from culture filtrates 24, 48 and 96 h after inoculation with conidia, and from culture filtrates of 12 day-old cultures growing on cotton wool as the carbon source. In addition, petioles and fruits of tomato plants were inoculated and the protein fraction of the colonized tissues were tested. Using filter paper, carboxymethylcellulose and cellobiose as substrates, all fraction showed c₁, glucanase and cellobiase activity respectively.     

Cellulase production by Penicillium echinulatum on lactose

The inducer effect of lactose on cellulase activity in Penicillium echinulatum 9A02S1 was studied. Submerged cultivation was performed using different concentrations of lactose and cellulose, in which the pH, mycelial mass, soluble proteins, filter paper activity (FPA), and activity of β-glucosidases were measured. The cultures containing lactose only presented low FPAs (0.1 FPU/ml). The cultures with associated cellulose and lactose and those containing cellulose only presented similar enzymatic activities (1.5 FPU/ml), suggesting the possibility of up to 75% reduction in the cellulose concentration. In relation to the β-glucosidases, increasing the lactose/cellulose ratio results in a proportional increase of enzymatic activity. In the cultures using both inducers, there is a longer duration of the acid phase in relation to treatments using only cellulose or lactose, indicating diauxia and catabolic repression.     

Enhancement of anthocyanin production in callus cultures ofDaucus canota L. under the influence of fungal elicitors

Aqueous extracts of fungal biomass ofAspergillus niger, A. flavus, Penicillium notatum, Fusarium oxysporum and the filtrates of their culture media were analysed for elicitation capability to enhance anthocyanin production in callus cultures ofDaucus carota. The mycelial extract ofA. flavus at the 2.5% level gave maximum elicitation, which resulted in a two-fold increase in anthocyanin with maximal productivity of 23.7% on a dry weight basis. Whereas the media filtrates ofA. flavus induced a 1.25-fold increase in anthocyanin production with a yield of 20.6% on a dry weight basis,P. notatum andF. oxysporum were not as effective asA. flavus orA. niger. The contact time and the concentration required for maximal elicitation of anthocyanin differed with the type of elicitor used. Qualitative analysis of anthocyanins revealed the presence of cyanidin glycosides in control and elicited cultures.     

Proteomic characterization of lignocellulose-degrading enzymes secreted by Phanerochaete carnosa grown on spruce and microcrystalline cellulose

Proteins secreted by the white-rot, softwood-degrading fungus Phanerochaete carnosa during growth on cellulose and spruce were analyzed using tandem mass spectrometry and de novo sequencing. Homology-driven proteomics was applied to compare P. carnosa peptide sequences to proteins in Phanerochaete chrysosporium using MS BLAST and non-gapped alignment. In this way, 665 and 365 peptides from cellulose and spruce cultivations, respectively, were annotated. Predicted activities included endoglucanases from glycoside hydrolase (GH) families 5, 16, and 61, cellobiohydrolases from GH6 and GH7, GH3 β-glucosidases, xylanases from GH10 and GH11, GH2 β-mannosidases, and debranching hemicellulases from GH43 and CE15. Peptides corresponding to glyoxal oxidases, peroxidases, and glycopeptides that could participate in lignin degradation were also detected. Overall, predicted activities detected in extracellular filtrates of cellulose and spruce cultures were similar, suggesting that the adaptation of P. carnosa to growth on lignocellulose might result from fine tuning the expression of similar enzyme families.     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

Influence of 1‐methylcyclopropene (1‐MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture

Aims: Bacterial cellulose is an extracellular polysaccharide secreted by Acetobacter xylinum, which has become a novel material increasingly used in food and medical industries. However, its broad application is limited by its low yield and high cost. 1‐Methylcyclopropene (1‐MCP) is a potent inhibitor to either exogenous or endogenous ethylene during the biological senescence of plants, which has been broadly applied in commercial preservation of fruits and vegetables. The purpose of this study was to investigate the effects of 1‐MCP on both the growth of Acet.  xylinum and its cellulose production to demonstrate the potential enhancement of bacterial cellulose yield. Methods and Results: Three groups of samples were fermented under agitated culture with 125 rev min^?1 rotational speed. To the culture media, 0·14 mg of 1‐MCP contained in 100 mg dextrose powder was added on assigned days or on the first culture day only. Results from the measurement of bacterial cell concentration and bacterial cellulose yield at the end of a 12‐day culture demonstrated that cultures excluding 1‐MCP displayed a higher cell concentration and a lower cellulose production, while cultures containing 1‐MCP produced 15·6% more cellulose (1‐MCP added on day 1) and 25·4% (1‐MCP added on each assigned day) with less biomass. Conclusions: 1‐MCP was able to affect the growth of Acet. xylinum cells and resulted in increasing bacterial cellulose yield up to 25·4% over controls, which did not contain 1‐MCP. Significance and Impact of the Study: This was the first study to use the growth inhibitor of plants to investigate its effects on bacterial growth and production. It also demonstrated a significant enhancement of bacterial cellulose yield by the addition of 1‐MCP during the common agitated culture of Acet. xylinum.     

Rapid Tomato Seedling Assay for Virulent Isolates of Fusarium oxysporum F. sp. Radicis-lycopersici (FORL), the Tomato Crown and Root Rot Pathogen

A rapid tomato seedling assay was developed for determining the relative wilt capacity of isolates of Fusarium oxysporum f. sp. radicis-lyco-persici (FORL), a virulent strain of the tomato pathogen. The procedures for the assay require that 5-day-old cv. Bonny Best tomato seedlings be dipped in 30-day cell-free concentrated culture filtrates of FORL isolates, which were grown in Czapek-Dox medium with 2% Bacto-casamino acids (CDA). The seedlings in the culture filtrates were then incubated at 30 C under artificial light (1200 ftcandles) at 28% relative humidity in a wind stream of 100–150 m min. The relative pathogenicity of the isolates was determined by inoculating the roots of 18-day-old seedlings with cultures of FORL isolates. The pattern of cell-free filtrate wilt among the isolates was the same as that for the disease caused by cultures of the isolates. The seedlings treated with the filtrate from the most virulent isolate (Harrow HRS-182) wilted in 20 min. The filtrates from less virulent isolates took progressively longer. up to 90 min. to cause comparable wilt. Isolate HRS-082 was the first isolate also to induce disease in 10-day-old seedling assay. Both assays indicate three levels of wilt and disease capacities amongthe isolates examined. The utility of the assay in research and breeding for resistance is discussed.     

STIMULATORY PROPERTIES OF FILTRATE FROM THE GREEN ALGA HORMOTILA BLENNISTA. I. DESCRIPTION1,2

A laboratory phenomenon involving autostimulation of growth by filtrates of the green alga Hormotila blennista is described, and stimulation attributed primarily to the secretion of organic metabolites. Filtrates obtained from actively growing cultures 1 through 4 weeks old showed maximum growth rate stimulation values in excess of 100%. Stimulatory properties of filtrate were heat labile, were not closely controlled by the starting pH within the limits normally encountered in culture, and did not result from depletion of essential nutrients. Concomitant with growth rate stimulation, filtrates characteristically extended the lag phase of culture growth. H. blennista filtrate can support bacterial growth and selectively stimulate or inhibit 2 planktonic green algae. It was suggested that extracellular organic products secreted by H. blennista during active growth could be of survival value to the organism, and could also play regulatory roles among other microorganisms in nature.     

GROWTH AND EXCRETION IN PLANKTONIC ALGAE AND BACTERIA1

In short-term experiments using cultures of Chlorella pyrenoidosa, Anabaena flos-aquae, Asterionella formosa, and Navicula pelliculosa, both the proportion of photosynthetic products released from cells and the composition of these products altered, with age. In the first 3 species, percentage extracellular release values increased with increasing growth rates but the reverse trend was shown by Navicula. Fractionation of filtrates using Sephadex indicated that, in general, larger molecular weight compounds became predominant as cultures aged. Also a time-dependent shift in a similar direction occurred in cultures of all ages. In several lakes a predominance of large molecular weight compounds was apparent in filtrates even from short-term experiments. Filtrates of mixed cultures of planktonic bacteria growing on ¹⁴C glycolate were found to contain, large molecular weight organic compounds. It was demonstrated that in nonaxenic cultures of algae and in lake water, bacteria utilize low molecular weight extracellular metabolites of algal origin and larger molecular weight compounds are formed.     

Plant regeneration of NaCl-pretreated cells from long-term suspension culture of rice (Oryza sativa L.) in high saline conditions

Cells of a 2-year-old suspension culture of rice (Oryza sativa L.), grown under 1.5% NaCl stress for 3 months, gave rise to plants through embryogenesis in different saline conditions. The high regeneration potential (59.6%) on salt-free medium decreased rapidly with increasing concentration of salt in the regeneration medium. At 1.25% NaCl, healthy shoots were developed in 14.9% of the cultures. Under 1.5% salt stress, embryo formation and embryo germination (6.1%) was observed but further development into plants was inhibited. Cells not pretreated with salt produced plants at a low frequency (2.6–4.2%) both in salt-free and low saline condition (0.75–1% NaCl). Cells pretreated for 3 months with 0.75% salt did not give rise to plants on all tested media. Plants regenerated from the salt-stressed cultures were transferred to soil and grew to maturity in a greenhouse.Abbreviations BA 6-benzyladenine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Importance of Nutrient Competition and Allelopathic Effects in Suppression of the Green Alga Scenedesmus obliquus by the Macrophytes Chara, Elodea and Myriophyllum

Possible allelopathic effects of substances released from the macrophytes Chara globularis, Elodea canadensis, Myriophyllum spicatum on the common green alga Scenedesmus obliquus were tested in the laboratory with plastic plants and untreated medium as controls. A two-phase approach was used in which first the effects of physical presence of plants was studied (phase I) followed by the effects of plant culture filtrates (phase II). In the presence of plastic plants growth was reduced only marginally, but strong growth inhibition of Scenedesmus occurred in the physical presence of all macrophytes. In contrast, filtrates from Chara had no growth inhibitory effect on Scenedesmus. Myriophyllum filtrate reduced particle-based growth rate by 7% compared to filtration controls, while Elodea culture filtrate reduced volume-based growth by 12%, chlorophyll-based growth by 28% and particle-based growth by 15%. Photosystem II-efficiency of Scenedesmus was reduced in all three macrophyte treatments in phase I, but not in filtrates from macrophyte cultures (phase II). Thus, while enzyme activity or other physiological aspects may have been affected, the current study yielded no proof for allelopathically active compounds being directed at photosynthesis. Mean particle volume (MPV) of Scenedesmus was not influenced by macrophyte exudates and cultures remained dominated by unicells. The strong growth inhibitory effects found for Scenedesmus in the physical presence of macrophytes, but not in plastic controls, and no or weaker response in nutrient-enriched filtrates, suggest nutrient competition was a more powerful driving factor than allelochemicals. However, the experimental design does not exclude disappearance of allelochemicals during the filtration process.     

Dilute acid pretreatment of rye straw and bermudagrass for ethanol production

Ethanol production from lignocellulosic materials provides an alternative energy production system. Rye and bermudagrass that are used in hog farms for nutrient uptake from swine wastewater have the potential for fuel ethanol production because they have a relative high cellulose and hemicellulose content. Dilute sulfuric acid pretreatment of rye straw and bermudagrass before enzymatic hydrolysis of cellulose was investigated in this study. The biomass at a solid loading rate of 10% was pretreated at 121 degrees C with different sulfuric acid concentrations (0.6, 0.9, 1.2 and 1.5%, w/w) and residence times (30, 60, and 90 min). Total reducing sugars, arabinose, galactose, glucose, and xylose in the prehydrolyzate were analyzed. In addition, the solid residues were hydrolyzed by cellulases to investigate the enzymatic digestibility. With the increasing acid concentration and residence time, the amount of arabinose and galactose in the filtrates increased. The glucose concentration in the prehydrolyzate of rye straw was not significantly influenced by the sulfuric acid concentration and residence time, but it increased in the prehydrolyzate of bermudagrass with the increase of pretreatment severity. The xylose concentration in the filtrates increased with the increase of sulfuric acid concentration and residence time. Most of the arabinan, galactan and xylan in the biomass were hydrolyzed during the acid pretreatment. Cellulose remaining in the pretreated feedstock was highly digestible by cellulases from Trichoderma reesei.     

Antagonism between Gliodadium roseum,Trichoderma harzianum,or Trichothecium roseum and Phytophthora megasperma f. sp. glydnea

The antagonism between Gliodadium roseum, Trichoderma harzianum, or Trichothecium roseum and Phytophthora megasperam f. sp. glycinea (Pmg), cause of Phytophthora rot of soybeans (Glycine max), was studied. G. roseum, T. harzianum, and 17 isolates of T. roseum were grown separately on modified Czapek-Dox medium (MCD) in the dark for 25 days at 25 °C. Culture filtrates of T. roseum at 0.5% and 20.0% concentrations inhibited mycelial growth of Pmg at 3.1% and 90.4%; and those of G. roseum at –0.7% and 44.0%; and of T. harzianum at 0.7% and 46.0%, respectively. Culture filtrates of T. roseum at 0.5% concentration inhibited zoosporangenesis of Pmg at 98.0% and at 1.0% concentration prevented it. Culture filtrates of G. roseum and T. harzianum at 5% concentration significantly (P=0.05) inhibited zoosporangenesis of Pmg, but differences were not significant from that on MCD. Culture filtrates of 17 isolates of T. roseum inhibited mycelial growth and zoosporangenesis of Pmg at different percentages with different concentrations with those of isolate 9 showing the greatest inhibition of both. Mycelial growth of most of 16 races of Pmg was prevented at 10% concentration of the culture filtrates of isolate SS-2 of T. roseum, and all 16 races, except race 6, was prevented at 20% concentration. Zoosporangenesis of all races of Pmg was prevented at 2% the culture filtrate of SS-2. Culture filtrates of SS-2 inhibited zoosporangenesis of Pmg in soil.     

Cellulase production and ammonia metabolism in Trichoderma reesei on high levels of cellulose.

Trichoderma can be cultured in stirred-tank fermentors on high (8%) cellulose concentrations without increasing the salt concentration of the medium when NH4OH is used to control pH and as a nitrogen source. Approximately 90% of the ammonia consumed by the organism can be added as NH4OH. The advantage of using high concentrations of cellulose is that culture filtrates with greater cellulase activity are obtained. The advantage of a low salts medium is that unwanted solutes in the final enzyme preparation are reduced. The appearance of cellulase in the medium occurs later than net ammonia uptake so that only 20% of the final amount of cellulase has appeared when 80% of the maximum amount of ammonia has been consumed.     

In Vitro selection of groundnut cell lines fromCercosporidium personatum culture filtrates and regeneration of resistant plants through cell culture

Cell suspensions derived from immature leaves of the groundnut (Arachis hypogaea L.) were cultured in the presence and absence ofCercosporidium personatum pathotoxic culture filtrates. Cell viability and reactions of cell lines were determined after exposure to various concentrations (25–100%, v/v) of the filtrates. Cell lines have been selected for resistance to the toxin(s) produced byC. personatum. Selected cell lines were used for plant regeneration on regeneration media containingC. personatum culture filtrates. Plant regeneration frequency was found to be low in long-term cultures, whereas it was high in short-term cultures. The selfed progeny of the plants regenerated from the resistant cell lines showed resistance to the pathogen in the field. Six out of 82 plants exhibited enhanced resistance in the R₂ generation. The culture filtrate stimulated callus proliferation as well as plant regeneration at lower concentrations, a response that could prove to be very useful for obtaining disease resistant plants throughin vitro selection.     

Enzymatic hydrolysis of waste cellulose

Waste cellulose was a suitable carbon source for cellulose production by Trichoderma viride. The enzyme can be produced in submerged fermentation using newspaper as a growth substrate. A variety of pure and complex cellulosic materials were hydrolyzed by culture filtrates. Saccharification of 5% slurries after 48 hr ranged from 2–92%. The rate and extent of hydrolysis was controlled by degree of crystallinity, particle size, and presence of impurities. Newspaper was used to evaluate methods for the pretreatment of substrate. The best pretreatment was ball milling which gave good size reduction, maximum bulk density, and maximum susceptibility. Hammer milling, fluid energy milling, colloid milling, or alkali treatments were less satisfactory. Dissolving cellulose in cuprammonium, or carbon disulfide (Viscose) and then reprecipitating gave a susceptible, but low bulk density product. However the susceptibility was lost if the substrate was dried. Because of costs, low bulk density, necessity of keep ing the substrate wet, and generation of chemical waste streams dissolving cellulose to increase reactivity does not seem a practical approach. Cellulose fractions separated from municipal trash or agricultural residues such as milled fibres from bovine manure are promising substrates for conversion.     

Ethanol formation from cellulose by thermophilic bacteria

Summary A wild coculture of obligately thermophilic bacteria, including only a single cellulolytic species Clostridium, ferments 2% crystalline cellulose and produces 4.6–5.1 g·l^–1 of ethanol at 55°–60° C; that is, 0.96–1.1 moles of ethanol from 1 mole of glucose equivalent of cellulose degraded. However, the ethanol yield decreases with increasing cellulose concentration. Ethanolacetic acid ratio varies around 1 and cannot be influenced by substrate concentration. However, this ratio can be influenced by changing pH and temperature. For the ethanol production from cellulose, neutral and weekly alkaline media with a pH of 7.0–8.0 and a temperature of 55° C are optimal. Experiments in which the coculture was subjected to high ethanol concentrations showed that higher concentrations of added ethanol (up to 20 g·l^–1) suppress cellulose degradation by 50% and inhibit the actual production of ethanol.     

RETENTION OF DISSOLVED COMPOUNDS BY MEMBRANE FILTERS AS AN ERROR IN THE 14C METHOD OF PRIMARY PRODUCTION MEASUREMENT1

Membrane filters retain ¹⁴C bicarbonate, ¹⁴C glycollate, and other ¹⁴C labeled substances from filtrates of algal cultures and lake water. By refiltering different volumes of the filtrate from algal cultures and from lake water after incubation with ¹⁴C bicarbonate, it was shown that the labeled material retained was not proportional to volume but showed a saturation effect with increasing volume filtered. When the radioactivity retained by a filter is divided by the volume filtered, decreasing values are obtained with increased volume filtered. This radioactivity may represent a significant addition to the radioactivity in particulate material on the filters, resulting in a similar type of curve when different volumes of lake water or cultures are filtered. Values of radioactivity per milliliter were constant using Chlorella in Chu 10 medium. However, the curve could, be obtained by increasing pH and bicarbonate concentrations in the medium and on resuspending the algae in Lake Ontario (winter) filtrate. The values of cpm/ml retained from filtrates were low in Cryptomonas cultures and the curve was not obtained unless population density was reduced, thus increasing the relative contribution to the radioactivity on the filters. The curve was not always obtained in lake water. It was significant in 10 out of 14 experiments in Lake Ontario and in 2 out of 5 experiments in Grenadier Pond. Changes in lake water rather than in experimental techniques were probably responsible. On 2 occasions when values of cpm/ml were constant in Lake Ontario, addition of sodium bicarbonate without a pH change resulted, in a significant curve. Our experiments do not disprove the possibility of cell damage during Millipore filtration, however, it has been shown that ¹⁴C labeled substances retained from solutions can account for the entire range of decreasing values as a function of volume filtered.     

Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solaniAG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicilliumstrains with R. solani prepared in sterilized soil and when the Penicilliumstrains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicilliumspecies against R. solani is discussed.