Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

Chitosan production from hemicellulose hydrolysate of corn straw: impact of degradation products on Rhizopus oryzae growth and chitosan fermentation

Aims: To examine the potential use of hemicellulose hydrolysate (HH) for the production of chitosan by Rhizopus oryzae and investigate the influence of contents in HH on mycelia growth and chitosan synthesis. Methods and Results: Compared to xylose medium, HH enhanced mycelia growth, chitosan content and production of R. oryzae by 10·2, 64·5 and 82·1%, respectively. During sulfuric acid hydrolysis of corn straw, sugars (glucose, galactose, etc) and inhibitors (formic acid, acetic acid and furfural) were generated. Acetic acid (2·14 g l^?1) and formic acid (0·83 g l^?1) were stimulative, while furfural (0·55 g l^?1) was inhibitory. Inhibitors, at different concentrations, increased the mycelia growth and chitosan production by 24·5–37·8 and 60·1–207·1%. Conclusions: HH of corn straw is a good source for chitosan production. Inhibitors in HH, at proper concentrations, can enhance chitosan production greatly. Significance and Impact of the Study: This work for the first time reported chitosan production from HH. Chitosan production can be greatly enhanced by cheap chemicals such as inhibitors in HH.     

Production and conversion of chitosan with cultures of Mucor rouxii or Phycomyces blakesleeanus

Summary Highly deacetylated chitosan was accumulated in the mycelia ofMucor rouxii orPhycomyces blakesleeanus. These cultures also effected the deacetylation of the chitin ofAspergillus niger mycelium into chitosan. After 96 hours of incubation with these cultures the degree of acetylation of commercial crab shell chitosan was reduced from 25.0% to values between 4.3 and 8.6%. The potential exists for the production of chitosans with tailored physico-chemical properties from waste chitin.     

Improved production of viable cells of the salt-tolerant yeast Zygosaccharomyces rouxii by continuous culture

Summary A continuous culture system of the salt-tolerant yeast Zygosaccharomyces rouxii (soy yeast) was investigated in order to obtain high production efficiency of viable cells. The optimum pH and C/N ratio of the feed medium for cell production were about 5.0 and 16–20, respectively. About a fivefold increase in viable cell number and cell productivity (viable cell number per litre per hour) were obtained in glucose-limited culture at a dilution rate (D) of 0.06 h^–1 as compared with batch culture. However, the fermentative activity of the cells from glucose-limited culture was significantly lower than those from batch and dissolved-oxygen (DO)-limited cultures, and the former cells showed lower specific activity of glycolytic enzymes. On the other hand, at the boundary conditions between glucose and DO limitation almost the same cell productivity and higher fermentative activity of the cell were obtained as compared with glucose-limited conditions. The cultivation continued for about 60 days without any problems even if the D was altered. It was found that the continuous cultivation method was suitable for industrial production of viable cells of soy yeasts. Offprint requests to: T. Hamada     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

Continuous production of acetone and butanol with shear-activated Clostridium acetobutylicum

Summary When using shear activation of Clostridium acetobutylicum by pumping the cells through capillaries, the cell growth, glucose consumption and product formation rates are considerably increased. Shear-activated continuous cell culture can be used as an inoculum with a welldefined fermentation activity for batch cultures. Different runs of such batch cultivation yield well-reproducible results which could not be obtained from inocula of other cultures or even of heat-shocked spores. The cells can attain a growth rate higher than 1.6 h^-1.The shear-activated continous culture growth is affected already at a butanol concentration lower than 1.6 g/l^-1.     

Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi

An investigation was undertaken to understand the mechanism(s) by which chitosan exerts its antifungal effects against the wood-degrading fungi Sphaeropsis sapinea and Trichoderma harzianum. Exposure to increasing concentrations of chitosan caused an increase in the amount of hydrogen peroxide accumulation in cultures of S. sapinea, which was accompanied by a decrease in superoxide formation. The same effect was not observed in T. harzianum. Potassium ion leakage was an early event for both test fungi, leakage being more pronounced for S. sapinea than T. harzianum for the first 5 min, particularly at higher concentrations of chitosan treatment. Fluorescence microscopy provided evidence that the effect of chitosan on fungal hyphae was mediated through alterations in the plasma membrane properties. Chitosan also severely affected fungal morphology. Increasing concentrations of chitosan induced excessive branching, vacuolation, and a reduction in hyphal diameter. Transmission electron microscopy, which showed more severe ultrastructural changes in S. sapinea hyphae from chitosan treatment as compared to T. harzianum, provided valuable complementary information. The data suggest that the plasma membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected.     

The chitosan yield of zygomycetes at their optimum harvesting time

Fungi are a promising alternative source of chitosan. Fungi can be manipulated to give chitosan of more consistent and desired physico-chemical properties compared to chitosan obtained from crustacean sources. Chitosan was extracted from the mycelia of Rhizopus oryzae USDB 0602 at various phases of growth. The growth phase which produced the most extractable chitosan was determined to be the late exponential phase. In contrast to previous work on the screening of chitosan from fungal sources, mycelia of the fungi used in this study were harvested at their late exponential growth phase instead of at a fixed incubation time. The amount of extractable chitosan varied widely among the fungal strains. Gongronella butleri USDB 0201 was found to produce the highest amount of extractable chitosan per ml of substrate, followed by Cunninghamella echinulata and Gongronella butleri USDB 0428. However, in terms of yield of chitosan per unit mycelia mass, C. echinulata was the best strain among all fungi in the experiment. Therefore, besides G. butleri USDB 0201, C. echinulata can also be considered to be used in the commercial production of chitosan.     

Production and isolation of chitosan from Mucor rouxii.

A method for the lab-scale production and isolation of chitosan (polyglucosamine) from hyphal walls of Mucor rouxii was developed. Hyphal wall yields were generally 16 to 22% on a dry cell weight basis, of which 35 to 40% was glucosamine. Chitosan was readily extracted from purified, mycelial walls with acetic, formic, and hydrochloric acids; the last named was the most efficient. The yield of chitosan isolated ranged from 4 to 8% of the dry weight of the cell wall material.     

Improved algal oil production from Botryococcus braunii by feeding nitrate and phosphate in an airlift bioreactor

To improve biomass and microalgal oil production of Botryococcus braunii, fed‐batch culture was investigated in an airlift photobioreactor. The optimal feeding time of the fed‐batch culture was after 15 days of cultivation, where 1.82 g/L of the microalgal biomass was obtained in the batch culture. Nitrate nutrient was the restrictive factor for the fed‐batch cultivation while phosphate nutrient with high concentration did not affect the microalgal growth. The optimal mole ratio of nitrate to phosphate was 34.7:1, where nitrate concentration reached the initial level and phosphate concentration was one quarter of its initial level. With one feeding, the biomass of B. braunii reached 2.56 g/L after 18 days. Two feedings in 2‐day interval enhanced the biomass production up to 2.87 g/L after 19 days of cultivation. The hydrocarbon content in dry biomass of B. braunii kept at high level of 64.3% w/w. Compared with the batch culture, biomass production and hydrocarbon productivity of B. braunii were greatly improved by the strategic fed‐batch cultivation.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

Production of fungal chitosan by solid substrate fermentation followed by enzymatic extraction

An improved method is described for the production of chitosan from mycelia of the fungus Gongronella butleri, grown by solid substrate fermentation on sweet potato. The chitosan was extracted subsequently by 11 M NaOH at 45 °C, and 0.35 M acetic acid at 95 °C. The resulting extract was clarified using a heat-stable, commercial -amylase. The yield (4–6 g/100 g mycelia) and relative number average molecular weight (44–54 kDa) of the chitosan increased with increasing duration of fungal growth up to the sixth day.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Effect of chitosan elicitation and media components on the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture

To increase the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture, the effects of elicitation on the colorant production were investigated. Chitosan was the best biotic elicitor among nine plant derived and microbial derived polysaccharides. When elicited with 25 mg/L chitosan, the total production was increased approximately two times in a seven-day culture as compared to that in the unelicited cells. Anthraquinone production was increased in proportion to the contact period up to day 3. Maximum anthraquinone colorants were obtained with 3-day treatment of chitosan. During chitosan elicitation, the total production was increased 1.3 times in MS medium containing galactose as compared to that containing sucrose. The degree of deacetylation in chitosan and the use of growth regulator or addition of precursor did not affect the production of anthraquinone colorants. When madder cells were elicited at optimum condition, anthraquinone concentration and specific anthraquinone content increased 1.3 times (0.69 g/L) and 2.2 times (0.32 g/g DCW), respectively.     

Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus

Summary Continuous and batch cultures of Lactobacillus helveticus operated under different conditions were studied with respect to the limitation of growth and lactic acid production by increasing undissociated lactic acid and hydrogen ion concentrations, respectively. In a single-stage continuous culture without pH control a final pH of 3.8 and 65 mm undissociated lactic acid was obtained. In two-stage continuous cultures provided with different growth media and run at different pH values, 65–70 mm free acid was obtained in the second stage. Further batch-culture experiments showed growth limitation at 60–70 mm lactic acid. After growth ceased, production of lactate continued until a lactic acid concentration of about 100 mm was reached; obviously an uncoupling of growth and acid production had occurred. Examining the effect of different concentrations of either lactic acid or hydrochloric acid, added to growing batch cultures of L. helveticus, it was shown that the undissociated lactic acid concentration was responsible for growth limitation and lactic acid production in this organism, whereas the pH value had only an indirect effect.     

EVect of Chitosan on Growth and Toxin Production by Alternaria alternata f. sp. lycopersici

The antifungal activity of chitosan, a biopolymer of beta-1-4 glucosamine, against Alternaria alternata f. sp. lycopersici , causal agent of black mold of tomato, was investigated. Chitosan was incorporated into potato-dextrose broth at concentrations of 100-6400 mug ml - 1, and the growth and toxin production by the fungus were assessed after 15 days of incubation. At the higher concentrations, chitosan significantly aVected both fungal growth and toxin production. However, at lower concentrations toxin production was aVected more than growth. The fungus sporulated excessively in the presence of chitosan, but the spores were less viable. Chitosan also induced aggregation, abnormal shape, excessive branching and hyphal contortion of fungal cells, and leakage of proteins. The virulence of the toxin in culture filtrates of the fungus grown on diVerent concentrations of chitosan was assessed by administering toxin on tomato disks. The phospholipid content, electrolyte leakage and activities of xylanase and pectin methylesterase were measured in the tomato tissue administered with culture filtrates containing fungal toxin. Decreased trends in the tendency to cause electrolyte leakage, phospholipid degradation and activation of xylanase and pectin methylesterase in the tomato tissue were observed with increasing concentrations of chitosan. The results showed that toxin produced in the presence of chitosan was less eVective in causing degradation of tomato tissue compared with the control. Thus, chitosan is a potential antifungal agent which can interfere with the pathogenic factors of the fungus.     

A new method for the quantification of chitin and chitosan in edible mushrooms

Along with β-glucans, chitin is the dominant component of the fungal cell wall. Chitosan, the deacetylated form of chitin, has found quite a number of biomedical and biotechnological applications recently. Mushroom chitin could be an important source for chitosan production. A direct determination of chitin and chitosan in mushrooms is of expedient interest. In this paper, a new method for the quantification of chitin and chitosan is described. This method is based on the specific reaction between polyiodide anions and chitosan and on measuring the optical density of the insoluble polyiodide–chitosan complex. After deacetylation, chitin can also be quantified. The specificity of the reaction is used to quantify the polymers in the presence of complex matrices. With this new spot assay, the chitin content of mycelia and fruiting bodies from several basidiomycetes and an ascomycete were analysed. The presented method could also be used for the determination in other samples as well. The chitin content of the analysed species varies between 0.4 and 9.8 g chitin per 100 g of dry mass. Chitosan could not be detected in our mushroom samples, indicating that the glucosamine units are mostly acetylated.     

Cultural characteristics and extraction of the fungal pigment phleichrome from the phytopathogenic fungus<Emphasis Type="Italic">Cladosporium phlei</Emphasis>

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 10³ spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively. There was an equal contribution of the reported research by the first two authors.