Physicochemical properties and bioactivity of fungal chitin and chitosan

Chitinous material was extracted from mycelia of Aspergillus niger and Mucor rouxii grown in yeast peptone dextrose broth for 15 and 21 days, respectively. The extracted material was characterized for purity, degree of acetylation, and crystallinity and tested for antibacterial and eliciting properties. The maximum glucosamine level determined in the mycelium of A. niger was 11.10% dw and in the mycelium of M. rouxii was 20.13% dw. On the basis of the stepwise extraction of freeze-dried mycelia, it appeared that M. rouxii mycelia contained both chitin and chitosan, whereas A. niger contained only chitin. The yields of crude chitin from A. niger and M. rouxii were 24.01 and 13.25%, respectively, and the yield of chitosan from M. rouxii was 12.49%. Significant amounts (7.42-39.81%) of glucan were associated with chitinous compounds from both species and could not be eliminated by the extraction method used. The degrees of acetylation were determined to be 76.53 and 50.07% for chitin from A. niger and M. rouxii, respectively, and 19.5% for M. rouxii chitosan. The crystallinity of fungal chitin and chitosan was estimated to be less intense than in corresponding materials from shrimp shells. The extracted chitin and chitosan in a concentration of 0.1% reduced Salmonella Typhimurium DT104 2576 counts by 0.5-1.5 logs during a 4 day incubation in tryptic soy broth at 25 degrees C. Furthermore, all tested chitinous materials from fungal sources significantly reduced lesions caused by Botrytis cinerea and Penicillium expansum in harvested apples.     

Determination of glucosamine and N-acetyl glucosamine in fungal cell walls

A new method was developed to determine glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) in materials containing chitin and chitosan, such as fungal cell walls. It is based on two steps of hydrolysis with (i) concentrated sulfuric acid at low temperature and (ii) dilute sulfuric acid at high temperature, followed by one-step degradation with nitrous acid. In this process, chitin and chitosan are converted into anhydromannose and acetic acid. Anhydromannose represents the sum of GlcN and GlcNAc, whereas acetic acid is a marker for GlcNAc only. The method showed recovery of 90.1% of chitin and 85.7-92.4% of chitosan from commercial preparations. Furthermore, alkali insoluble material (AIM) from biomass of three strains of zygomycetes, Rhizopus oryzae, Mucor indicus, and Rhizomucor pusillus, was analyzed by this method. The glucosamine contents of AIM from R. oryzae and M. indicus were almost constant (41.7 +/- 2.2% and 42.0 +/- 1.7%, respectively), while in R. pusillus, it decreased from 40.0 to 30.0% during cultivation from 1 to 6 days. The GlcNAc content of AIM from R. oryzae and R. pusillus increased from 24.9 to 31.0% and from 36.3 to 50.8%, respectively, in 6 days, while it remained almost constant during the cultivation of M. indicus (23.5 +/- 0.8%).     

Sonication-assisted extraction of chitin from shells of fresh water prawns (Macrobrachium rosenbergii)

The effect of sonication during chitin extraction from freshwater prawn shells on yield, purity, and crystallinity of chitin was investigated. Dry prawn shells were suspended for 4 h in 0.25 M HCl at 40 degrees C while they were sonicated for 0, 1, and 4 h. Demineralized shells were lyophilized, resuspended in 0.25 M NaOH, and sonicated again for 0, 1, and 4 h for protein removal. The yield of chitin decreased from 8.28 to 5.02% for nonsonicated and sonicated samples, respectively, which was attributed to losses of depolymerized materials in the wash water. The application of ultrasound enhanced the removal of proteins. In nontreated shells, the amount of protein was 44.01% and was reduced to 12.55, 10.59, and 7.45% after 0, 1, and 4 h of sonication treatments. The glucosamine content slightly decreased with sonication probably because of losses due to depolymerization. The crystallinity indices of chitins decreased as the time of sonication increased. The degree of acetylation of chitins was unaffected by sonication, but the degree of acetylation of chitosans produced from sonicated chitin decreased from 70.0 to 68.7 and 61.4% for 1 and 4 h sonicated samples, respectively.     

Oxygenation of bisphenol A to quinones by polyphenol oxidase in vegetables

To understand conversion of bisphenol A and its related compounds under some chemical and biological environments, oxidation of these compounds was performed. Bisphenol A was oxidized to monoquinone and bisquinone derivatives by Fremy's salt, a radical oxidant; but salcomine and alkali did not catalyze the oxidation by molecular oxygen. Bisphenol A, bisphenol B, and 3,4'-(1-methylethylidene)bisphenol were converted to their monoquinone derivatives in the presence of oxygen and polyphenol oxidase from mushroom at 25 degrees C at pH 6.5. Among crude enzyme solutions of fruits and vegetables, potato, mushroom, eggplant, edible burdock, and yacon showed remarkable oxidative activity on bisphenol A. The highest activity was observed in potato, and the main product obtained by the enzymatic oxygenation was the monoquinone derivative of bisphenol A, accompanied by a small amount of the bisquinone derivative. The oxidation reactions found here will be useful for developing techniques for elimination of phenolic endocrine disrupters from the environment.     

Preparation of glucosamine from exoskeleton of shrimp and predicting production yield by response surface methodology

Chitin was prepared from Persian Gulf shrimp (Metapenaeus monoceros), and then, the obtained chitin was hydrolyzed by hydrochloric acid solutions. The production yield of glucosamine hydrochloride from chitin was optimized, and the effect of three factors (acid concentration, acid to chitin ratio, and reaction time) was investigated. A Box-Behnken design by Minitab software created 12 reactions with different conditions. Each reaction was performed in two replicates. Response surface methodology was used for predicting the glucosamine preparation. The optimum conditions for glucosamine hydrochloride preparation were 30 and 37% hydrochloric acid, 9:1 (v/w) acid solution to solid ratio, and 4 h of reaction time. Time ratio and time acid concentrations were the effective factors on the yield.     

Sonication-assisted extraction of chitin from North Atlantic shrimps (Pandalus borealis)

The influence of sonication during extraction of chitin from North Atlantic shrimp (NAS) shells (Pandalus borealis) on chitin yield, purity, and crystallinity was investigated. Shells were peeled, washed, lyophilized, ground, and suspended for 4 h in 0.25 M HCl (1:40) at 40 degrees C followed by ultrasonication at 41 W/cm(2) for 0, 1, and 4 h, respectively. Demineralized shells were lyophilized, resuspended in 0.25 M NaOH (1:40), and ultrasonicated at 41 W/cm(2) for 0, 1, and 4 h to remove proteins. The yield and mineral and protein contents were determined after each processing step. The purity of extracted chitin was determined from the total amount of glucosamine. The crystallinity index and size of crystals were calculated from wide-angle X-ray scattering measurements. Scanning electron microscope images were recorded to evaluate morphological changes in samples. The yield of chitin from NAS decreased from 16.5 to 11.4% for 0 and 1 h sonicated samples, respectively, which was attributed to increased concentrations of depolymerized materials in the wash water. Sonication did not enhance the removal of minerals. The application of ultrasound enhanced the removal of proteins from 39.8 to 10.6, 8.3, and 7.3% after 0, 1, and 4 h of sonication treatments. The crystallinity index of chitin decreased from 87.6 to 79.1 and 78.5% after 1 and 4 h of sonication, yielding chitosans with crystallinity indices of 76.7, 79.5, and 74.8% after deacetylation, respectively. Fourier transform infrared spectroscopy scans indicated that the degree of acetylation of chitins was unaffected by sonication. Comparison of the extraction results of NAS with that from freshwater prawns indicated that more impurities were left in NAS chitin, suggesting that composition and structural arrangement of chitin in shells influence the efficiency of ultrasound-assisted extraction.     

A modified chemical procedure for rapid determination of glucosamine and its application for estimation of mold growth in peanut kernels and koji.

One-step hydrolysis of chitin to release glucosamine for quantitation was achieved by combining a chitin-containing sample (10-200 mg of sample size) in a test tube with 1 mL of 10 M HCl followed by vacuum treatment for 10 min, incubation at 28 degrees C for 30 min, replenishment with 3 mL of deionized water, nitrogen flushing, screw capping, and heat treatment at 140 degrees C for 60 min. A phosphate buffer solution (pH 12.5, 0.2 M) was effective in pH stabilization and enhancing colorimetric determination of glucosamine content. When the modified procedure was applied to analyze glucosamine content in the mycelia of various molds, glucosamine content varied mainly depending on mold species. In estimations of mold growth of the uninoculated peanut kernels incubated under a humidified condition for 5 weeks, cooked rice and soybean inoculated with conidia of Aspergillus oryzae for koji preparation, logarithms of the internal mold populations and glucosamine contents both increased with increases of incubation time. The modified procedure provided a rapid and reliable estimation of mold growth in various substrates.     

Effects of ozone treatment on postharvest strawberry quality

The effect of ozone treatment on the postharvest quality of strawberry was evaluated. Strawberry fruits (Fragaria x ananassa Duch. cv. Camarosa) were stored at 2 degrees C in an atmosphere containing ozone (0.35 ppm). After 3 days at 2 degrees C, fruits were moved to 20 degrees C to mimic retail conditions (shelf life). The changes in several quality parameters such as fungal decay, color, sugar and acids distribution, and aroma were evaluated during the strawberries' shelf life. Ozone treatment was ineffective in preventing fungal decay in strawberries after 4 days at 20 degrees C. Significant differences in sugars and ascorbic acid content were found in ozone-treated strawberries. At the end of cold storage, the vitamin C content of ozonated strawberries was 3 times that of control fruits. A detrimental effect of ozone treatment on strawberry aroma was observed, with a 40% reduced emission of volatile esters in ozonated fruits.     

Migration of bisphenol A (BPA) from epoxy can coatings to jalapeño peppers and an acid food simulant

Effects of heat processing, storage time, and temperature on migration of bisphenol A (BPA) from an epoxy type can coating to an acid food simulant and jalape?o peppers were determined. Commercial jalape?o pepper cans (8 oz, dimensions 211 x 300) were stored at 25 degrees C for 40, 70, and 160 days. A solution of 3% acetic acid was canned in 211 x 300 cans from the same batch used for jalape?o peppers. Heat processing was applied to two-thirds of the cans, and the remaining cans were not heat processed. Cans were stored at 25 and 35 degrees C for 0, 40, 70, and 160 days. Results showed that there is a minimal effect of heat treatment. An effect of storage time on migration of BPA during the first 40 days at 25 degrees C was observed. An increase on migration of BPA was observed with storage time at 35 degrees C. The highest level of migration was 15.33 microg/kg of BPA at 160 days at 35 degrees C. A correction factor of approximately 0.4 was calculated for migration under simulating conditions of storage compared to the real ones. The highest level of BPA found in jalape?o peppers cans, surveyed from three supermarkets, was 5.59 +/- 2.43 microg/kg. Migration of BPA, performed according to the European and Mercosur conditions, was 65.45 +/- 5.29 microg/kg. All the migration values found in this study were below those legislation limits (3 mg/kg).     

Kinetics and products of the degradation of chitosan by hydrogen peroxide

Low concentrations of hydrogen peroxide induced random degradation of partially deacetylated chitin and chitosan. Average molecular weight decreased in accordance with first-order kinetics. The degradation rate was much faster than that of the ultrasonic degradation, and it was comparable to that of the enzymatic hydrolysis of chitosan. Chain-end scissions occurred after chitosan was degraded severely and produced significant amounts of oligosaccharides at temperatures > or =80 degrees C. Universal calibration moderated the change in molecular weight more closely than that calculated by the usual calibration using pullulan standards. Trace amounts of transition metal ions and the amino groups in chitosan were critical to the breakdown of the beta-1,4 glycosidic linkages. HPLC results of glucosamine and chito-oligosaccharides could be characterized by correlating the logarithmic values of retention time with the degrees of polymerization. The formation of glucosamine and chito-oligosaccharides depended on the concentration of H(2)O(2), temperature, and the physicochemical property of chitin/chitosan.     

Autolysis of lentinan, an antitumor polysaccharide, during storage of Lentinus edodes, shiitake mushroom

The lentinan contents in the Lentinus edodes fruit body during storage were examined by ELISA method using anti-lentinan antibodies. The lentinan content (12.8 mg.g(-)(1) dw) before storage decreased to 3.7 mg.g(-)(1) dw over 7 days at 20 degrees C. However, it only slightly decreased at 1 degrees C and only decreased to 9.3 mg. g(-)(1) dw at 5 degrees C. Glucanase activity, which seems to be associated with lentinan degradation, increased more during storage of L. edodes at 20 degrees C than it did at lower temperatures. In addition, only glucose was detected as a degraded product from lentinan by the glucanase. This suggested that this enzyme would fit the profile of an exo-type glucanase. Also, polyphenol oxidase activity, known as an index of freshness reduction in the mushroom, increased approximately 2.7-fold (to 61.5 units.mg(-)(1)) over 7 days during storage at 20 degrees C. However, its activity changed little during storage at lower temperatures. These results indicate that the reduction during storage of the quality of L. edodes as a functional food is accompanied by the decrease of lentinan, and by browning, and that exo-glucanase plays an important role in the decrease of lentinan content.     

Effect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles

Recent research suggests that blueberries are rich in total polyphenols and total anthocyanins. Phenolic compounds are highly unstable and may be lost during processing, particularly when heat treatment is involved. There is no systematic study available providing information on the fate of phenolic compounds during storage and how that affects their biological activity. We provide a systematic evaluation of the changes observed in total polyphenols (TPP), total anthocyanins (TACY), Trolox equivalent antioxidant capacity (TEAC), phenolic acids, and individual anthocyanins of blueberry extract stored in glass bottles and the ability of blueberry extract to inhibit cell proliferation. The extract was stored at different temperatures (-20 +/- 1, 6 +/- 1, 23 +/- 1, and 35 +/- 1 degrees C). Two cultivars, Tifblue and Powderblue, were chosen for the study. The recoveries of TPP, TACY, and TEAC in blueberry extract after pressing and heating were approximately 25, approximately 29, and approximately 69%, respectively, for both cultivars. The recovery of gallic acid, catechin, and quercetin was approximately 25%. Ferulic acid was not detected in the final extract in both Tifblue and Powderblue cultivars. The recovery of peonidin, malvidin, and cyanidin glycosides was approximately 20% in the final extract in both cultivars. Losses due to storage were less when compared with initial losses due to processing. At -20 degrees C, no statistically significant loss of TPP, TACY, and TEAC was observed up to 30 days (P < 0.05). At 6 degrees C storage, there was a significant loss observed from 15 to 30 days. Similar results were obtained at 23 and 35 degrees C (P < 0.05). There was retention of more than 40% of ellagic and quercetin after 60 days at 35 +/- 1 degrees C. Anthocyanins were not detected after 60 days of storage at 35 +/- 1 degrees C. Significant retention (P < 0.05) was obtained for malvidin (42.8 and 25.8%) and peonidin (74.0 and 79.5%) after 60 days of storage at 23 +/- 1 degrees C in glass bottles for Tifblue and Powderblue, respectively, when compared with other individual anthocyanins. A linear relationship was observed between TEAC values and total polyphenols or total anthocyanins. A cell viability assay was performed using HT-29 cancer cell lines and anthocyanins extracted from 30, 60, and 90 days of stored extract at 6 +/- 1 and 23 +/- 1 degrees C. A significant cell proliferation inhibition percentage was observed in 30 days, although this was reduced significantly after 30-90 days. These results suggest that heating and storage conditions significantly affect the phenolic compounds and their biological activities. Frozen and low temperature storage are suggested for blueberry extract in order to retain the bioactive components.     

Relationships between microbial indices in roots and silt loam soils forming a gradient in soil organic matter

Perennial rye grass (Lolium perenne) was grown in a greenhouse pot experiment on seven soils to answer the question whether the microbial colonisation of roots is related to existing differences in soil microbial indices. The soils were similar in texture, but differed considerably in soil organic matter, microbial biomass, and microbial community structure. Ergosterol and fungal glucosamine were significantly interrelated in the root material. This ergosterol was also significantly correlated with the average ergosterol content of bulk and rhizosphere soil. In addition, the sum of fungal C and bacterial C in the root material revealed a significant linear relationship with microbial biomass C in soil. The colonisation of roots with microorganisms increased apparently with an increase in soil microbial biomass. In the root material, microbial tissue consisted of 77% fungi and 23% bacteria. In soil, the fungal dominance was slightly, but significantly lower, with 70% fungi and 30% bacteria. Fungal glucosamine in the root material was significantly correlated with that in soil (r=0.65). This indicates a close relationship between the composition of dead microbial remains in soil and the living fraction in soil and root material for unknown reasons.     

Quality changes and nutrient retention in fresh-cut versus whole fruits during storage

The influences of processing and storage on the quality indices and nutritional content of fresh-cut fruits were evaluated in comparison to whole fruits stored for the same duration but prepared on the day of sampling. Fresh-cut pineapples, mangoes, cantaloupes, watermelons, strawberries, and kiwifruits and whole fruits were stored for up to 9 days in air at 5 degrees C. The postcutting life based on visual appearance was shorter than 6 days for fresh-cut kiwifruit and shorter than 9 days for fresh-cut pineapple, cantaloupe, and strawberry. On the other hand, fresh-cut watermelon and mango pieces were still marketable after 9 days at 5 degrees C. Losses in vitamin C after 6 days at 5 degrees C were < or = 5% in mango, strawberry, and watermelon pieces, 10% in pineapple pieces, 12% in kiwifruit slices, and 25% in cantaloupe cubes. No losses in carotenoids were found in kiwifruit slices and watermelon cubes, whereas losses in pineapples were the highest at 25% followed by 10-15% in cantaloupe, mango, and strawberry pieces after 6 days at 5 degrees C. No significant losses in total phenolics were found in any of the fresh-cut fruit products tested after 6 days at 5 degrees C. Light exposure promoted browning in pineapple pieces and decreased vitamin C content in kiwifruit slices. Total carotenoids contents decreased in cantaloupe cubes and kiwifruit slices, but increased in mango and watermelon cubes in response to light exposure during storage at 5 degrees C for up to 9 days. There was no effect of exposure to light on the content of phenolics. In general, fresh-cut fruits visually spoil before any significant nutrient loss occurs.     

The effect of post-harvest and packaging treatments on glucoraphanin concentration in broccoli (Brassica oleracea var. italica)

The effects of post-harvest and packaging treatments on glucoraphanin (4-methylsulfinylbutyl glucosinolate), the glucosinolate precursor of anticancer isothiocyanate sulforaphane [4-methylsulfinylbutyl isothiocyanate], were examined in broccoli (Brassica oleracea var. italica) during storage times. The results showed that at 20 degrees C, 55% loss of glucoraphanin concentration occurred in broccoli stored in open boxes during the first 3 days of the treatment and 56% loss was found in broccoli stored in plastic bags by day 7. Under both air and controlled atmosphere (CA) storage, glucoraphanin concentration appeared to fluctuate slightly during 25 days of storage and the concentrations under CA was significantly higher than those stored under air treatment. In modified atmosphere packaging (MAP) treatments, glucoraphanin concentration in air control packaging decreased significantly whereas there were no significant changes in glucoraphanin concentration in MAP with no holes at 4 degrees C and two microholes at 20 degrees C for up to 10 days. Decreases in glucoraphanin concentration occurred when the broccoli heads deteriorated. In the present study, the best method for preserving glucoraphanin concentration in broccoli heads after harvest was storage of broccoli in MAP and refrigeration at 4 degrees C. This condition maintained the glucoraphanin concentration for at least 10 days and also maintained the visual quality of the broccoli heads.     

Prevention of hydrolytic rancidity in rice bran during storage.

The effect of microwave heating, packaging, and storage temperature on the production of free fatty acids (FFA) in rice bran was examined. Freshly milled raw rice bran was adjusted to 21% moisture content and heated in a microwave oven at 850 W for 3 min. Raw and microwave-heated rice bran were packed in zipper-top bags or vacuum-sealed bags and stored at 4-5 or 25 degrees C for 16 weeks. FFA content of bran was measured at 4-week intervals. Total FFA increased rapidly over the 16-week period from the initial value of 2.5% in raw bran stored at 25 degrees C to 54.9% in vacuum bags and 48.1% in zipper-top bags. However, total FFA of raw bran stored at 4-5 degrees C increased at a slower rate from an initial value of 2. 5 to 25.4% in vacuum bags and 19.5% in zipper-top bags. After 16 weeks of storage, total FFA of microwave-heated bran stored at 25 degrees C increased from 2.8 to 6.9 and 5.2%, respectively, for samples stored in vacuum bags and zipper-top bags. Total FFA of microwave-heated samples stored at 4-5 degrees C did not change significantly with storage time. Results showed that hydrolytic rancidity of rice bran can be prevented by microwave heating and that the recommended storage condition for microwaved rice bran is 4-5 degrees C in zipper-top bags.     

Microbial colonisation of roots as a function of plant species

Fifteen plants species were grown in the greenhouse on the same soil and sampled at flowering to obtain rhizosphere soil and root material. In both fractions, the data on fungal and bacterial tissue obtained by amino sugar analysis were compared with the total microbial biomass based on fumigation-extraction and ergosterol data. The available literature on glucosamine concentrations in fungi and on muramic acid concentrations in bacteria was reviewed to prove the possibility of generating conversion values for general use in root material. All microbial properties analysed revealed strong species-specific differences in microbial colonisation of plant roots. The root material contained considerable amounts of microbial biomass C and biomass N, reaching mean levels of 10.9 and 1.4 mg g⁻¹ dry weight, respectively. However, the majority of CHCl₃ labile C and N, i.e. 89 and 55% was root derived. The average amount of ergosterol was 13 μg g⁻¹ dry weight and varied between 0.0 for Phacelia roots and 45.5 μg g⁻¹ dry weight for Vicia roots. The ergosterol content in root material of mycorrhizal and non-mycorrhizal plant species did not differ significantly. Fungal glucosamine was converted to fungal C by multiplication by 9 giving a range of 7.1-25.9 mg g⁻¹ dry weight in the root material. Fungal C and ergosterol were significantly correlated. Bacterial C was calculated by multiplying muramic acid by 45 giving a range from 1.7 to 21.6 mg g⁻¹ dry weight in the root material. In the root material of the 15 plant species, the ratio of fungal C-to-bacterial C ranged from 1.0 in mycorrhizal Trifolium roots to 9.5 in non-mycorrhizal Lupinus roots and it was on average 3.1. These figures mean that the microbial tissue in the root material consists on average of 76% fungal C and 24% bacterial C. The differences in microbial colonisation of the roots were reflected by differences in microbial indices found in the rhizosphere soil, most strongly for microbial biomass C and ergosterol, but to some extent also for glucosamine and muramic acid.     

A colorimetric method for estimating vesicular-arbuscular mycorrhizal infection in roots

A colorimetric method measuring the conversion of fungal chitin to glucosamine has been used to estimate the intensity of vesicular-arbuscular mycorrhizal infection in roots. The technique has been used successfully with four plant genera and with four different endophytes.     

Storage temperature effects on blood orange fruit quality.

Orange fruits of two blood varieties (Tarocco and Moro) were stored at 8 degrees C and 22 degrees C for 85 and 106 days, respectively, and analyzed periodically for standard quality parameters (total soluble solids, total acidity, ascorbic acid, juice yield, and rind color) and sensory influencing parameters (anthocyanins, and total and free hydroxycinnamic acids). A decrease in total acidity (TA) and juice yield during storage was observed for both cultivars; total soluble solids (TSS) increased only in the Tarocco oranges stored at 8 degrees C. The increase in TSS observed for Tarocco and the simultaneous decrease in TA in both varieties resulted in a higher maturity index (TSS/TA) for the two cultivars. No loss of vitamin C was noted in Tarocco orange at either temperature, whereas a sharp reduction in vitamin C occurred in the first 50 days of storage for Moro. A significant increase in anthocyanin content was observed in Tarocco and Moro stored at 8 degrees C. Overlong storage induces extensive hydrolysis of hydroxycinnamic derivatives to free acids in Moro orange and these, in turn, could develop the malodorous vinylphenols.     

Food additive lactic acid production by immobilized cells of Lactobacillus brevis on delignified cellulosic material

Improvements in yield and productivity in lactic acid fermentation by Lactobaccilus brevis cells immobilized on delignified cellulosic (DC) material are reported. The system proved to be more efficient in comparison with the work reported by other workers. Yields of 80 and 100% conversion using glucose were obtained at 30 degrees C in 1 day of fermentation time. Lactic acid fermentation using whey as substrate was obtained at 30 degrees C in 1-1.5 days, resulting in 70% yield, whereas the remaining lactose in whey was converted to alcohol byproduct, leading to a 90% lactose exploitation and 100% conversion. Cell immobilization of L. brevis on DC material was proved by its reuses in repeated batch fermentations and through electron microscopy. A series of 10 repeated batch fermentations without any loss in cell activity showed a tendency for high operational stability. The presence of DC material resulted in a drastic drop of the fermentation time from 48 to 13 h.