Culture method to study fungal growth in solid fermentation

Summary A new culture method is described to study the growth of Aspergillus niger on cassava meal in the solid state. This method uses preparations of the cooked starchy substrate as a homogeneous granulated product containing spores, salts and water. An incubation device aerates the mass with humidified air at a controlled temperature. Homogeneous development of mycelia, without sporulation, occurred in the substrate mass. From physiological studies, optimal conditions for A. niger growth on cassava in the solid state were 50–55% moisture, 35°C, a nitrogen source comprising 60% ammonium and 40% urea (on a nitrogen basis) and 2×10⁷ spores/g of substrate. Growth kinetics were established and changes in pH, protein, carbohydrate and water content were determined during the incubation. Growth rate and yield were quite similar to those described in the literature for A. niger cultivated in liquid media under optimal conditions.     

Enzyme hydrolysis of cassava peels: treatment by amylolytic and cellulolytic enzymes

Cassava peels provide a cheap non-food biomass waste that can be hydrolyzed to simple sugars as a useful feedstock. Unlike most crop wastes, they have high starch content as well as lignocellulose. In this study, an enzymatic treatment of cassava peels by various concentrations of amylase and glucoamylase is considered. Steam explosion pre-treatments reduced rate and yield of hydrolysis. Milled peels suspended at 10% w/v yielded a maximum reducing sugar of 0.41?g (as glucose) per gram of peels. HPLC analysis showed that levels of soluble oligosaccharides remained low throughout. A pretreatment with amylase at 95?°C slightly increased rates although final yield was the same. Additional treatment with cellulolytic enzymes increases the total hydrolysis yield to 0.61?g (as glucose) per gram of peels representing 91% of the carbohydrate in cassava peels.     

Isolation of a microbial consortium from activated sludge for the biological treatment of food waste

The bacterial community in the activated sludge of a local wastewater treatment plant was studied in an effort to understand and exploit the metabolic versatility of microorganisms for the efficient biological treatment of food waste. Microorganisms capable of and efficient in degrading domestic food waste were screened based on their ability to produce areas of clearing on selective media containing protein, fat, cellulose and starch. Nine microbial species belonging to the genera Flavobacterium, Pseudomonas, Micrococcus, Aeromonas, Xanthomonas, Vibrio and Sphingomonas were found to degrade all components of food waste. These bacteria were added to domestic wastewater and shown to cause a 60% reduction in the biochemical oxygen demand (BOD) level of wastewater compared to a control in which no microorganisms were added. The ability of the microbial consortium to degrade domestic wastewater as evidenced by the decrease in BOD levels suggests its potential for use in the biological treatment of food waste.     

The separation of solids from the liquefied mash of cassava tuber and continuous saccharification by immobilized glucoamylase

Summary The solid material in liquefied mash of cassava tuber was very efficiently separated by a mixture of Trichoderma cellulase and Aspergillus niger pectinase. The solid content of the residue after the treatment and centrifugation decreased from 29.5% to 7.0%. The transparent digested solution from cassava tuber after centrifugation was continuously saccharified by glucoamylase immobilized in a gel which was prepared using polyvinylpyrrolidone and -ray irradiation. The addition of 50 ppm of sulfite ion completely prevented microbial contamination during the 18 days of operation. The final DE (dextrose equivalent), glucose content and disaccharide content in the hydrolyzate were 98, 94.4 and 3.3%, respectively.     

Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation

A study was taken up to evaluate the role of some fermentation parameters like inoculum concentration, temperature, incubation period and agitation time on ethanol production from kinnow waste and banana peels by simultaneous saccharification and fermentation using cellulase and co-culture of Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC 1077. Steam pretreated kinnow waste and banana peels were used as substrate for ethanol production in the ratio 4:6 (kinnow waste: banana peels). Temperature of 30°C, inoculum size of S. cerevisiae G 6% and (v/v) Pachysolen tannophilus MTCC 1077 4% (v/v), incubation period of 48 h and agitation for the first 24 h were found to be best for ethanol production using the combination of two wastes. The pretreated steam exploded biomass after enzymatic saccharification containing 63 gL⁻¹ reducing sugars was fermented with both hexose and pentose fermenting yeast strains under optimized conditions resulting in ethanol production, yield and fermentation efficiency of 26.84 gL⁻¹, 0.426 gg ⁻¹ and 83.52 % respectively. This study could establish the effective utilization of kinnow waste and banana peels for bioethanol production using optimized fermentation parameters.     

Production of ethanol from raw cassava starch by a nonconventional fermentation method

Raw cassava root starch was transformed into ethanol in a one-step process of fermentation, in which are combined the conventional processes of liquefaction, saccharification, and fermentation to alcohol. Aspergillus awamori NRRL 3112 and Aspergillus niger were cultivated on wheat bran and used as Koji enzymes. Commercial A. niger amyloglucosidase was also used in this experiment. A raw cassava root homogenate–enzymes–yeast mixture fermented optimally at pH 3.5 and 30°C, for five days and produced ethanol. Alcohol yields from raw cassava roots were between 82.3 and 99.6%. Fungal Koji enzymes effectively decreased the viscosity of cassava root fermentation mashes during incubation. Commercial A. niger amyloglucosidase decreased the viscosity slightly. Reduction of viscosity of fermentation mashes was 40, 84, and 93% by commercial amyloglucosidase, A. awamori, and A. niger enzymes, respectively. The reduction of viscosity of fermentation mashes is probably due to the hydrolysis of pentosans by Koji enzymes.     

Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Screening of cassava (Manihot esculenta Crantz) accessions against Phytophthora palmivora induced tuber rot of cassava

Cassava (Manihot esculenta Crantz), is an important tropical tuber crop with global importance and plays a significant role in the food, nutritional and livelihood security of around 500 million people. In India, the low productivity of cassava attributes to the soil borne disease, particularly tuber rot caused by Phytophthora palmivora (Butl.) which is destructive and the attack is spreading in alarming rate in all the cassava growing regions causing heavy yield loss of more than 50%. Introduction of disease resistant varieties may alleviate the problem to a certain extent. This paper describes the screening procedures and findings on the disease resistant variety of cassava accession against tuber rot. Variety Sree Padmanabha imparted high resistance against tuber rot, while Sree Sahya was moderately resistant and all other accessions studied were found to be susceptible in in vitro and in field trials. In screening studies, a reproducible positive correlation was obtained between attached tubers in live plant with detached tubers which showed that detached tuber part can be used for the prediction of resistance in attached live plants of cassava for cultivar resistance. The procedure described here could be used as a simple, rapid and efficient method for screening of cassava accessions against tuber rot of cassava.     

Extraction of Zinc from Industrial Waste by a Penicillium sp

Zinc was extracted from a filter residue of a copper works (58.6% zinc) by a Penicillium sp. isolated from a metal-containing location. By isotachophoresis citric acid was identified as the leaching agent. Citrate was only formed when the leaching substrate was present. This production of citrate was different in several ways from that achieved by Aspergillus niger: glucose was utilized before fructose; the initial concentration of zinc was 50 to 500 times higher than usual in citrate fermentations with A. niger; citrate production stopped when 80 to 90% of the zinc was leached, although sufficient sugar for further synthesis was still present; and in synthetic media citrate production by A. niger needs an acidic environment (pH 2), while the formation of citric acid by Penicillium sp. occurred in a pH range of 7 to 4. Tests with different concentrations of waste material (0.5, 2.5, and 5%) showed that the highest yield of solubilized zinc occurred with a 2.5% substrate (93% zinc extracted after 13 days).     

Improving the quality of agro-wastes by solid-state fermentation: enhanced antioxidant activities and nutritional qualities

Solid substrate fermentations of some agro-wastes, namely cocoa pod husk (CPH), cassava peel (CP), and palm kernel cake (PKC) were carried out for the production of fructosyltransferase (FTase) by a newly isolated fungal strain Rhizopus stolonifer LAU 07. The fermented substrate were studied for improved nutritional quality by determining the crude protein, crude fibre, ash and lipid contents, and antioxidant activities. The cyanide content of cassava peels was also determined. Some levels of value-addition occured as a result of the fermentation. The protein contents of the substrates increased by 33.3, 55.4, and 94.8%, while the crude fibre contents decreased by 44.5, 8.6, and 7.2% in PKC, CP, and CPH, respectively. The cyanide content of cassava peel was reduced by 90.6%. Generally, fermentation of the substrates by R. stolonifer LAU 07 increased the antioxidant activity in a DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. The IC₅₀ (mg/ml) values of the methanolic extracts (fermented/unfermented) were obtained as 7.0/14.9, 4.4/10.6, and 5.5/14.7 mg/ml for PKC, CP, and CPH, respectively. Results herein reported showed that the nutritional qualities and antioxidant activities of all the investigated solid substrates were enhanced by fungal fermentation. Thus, scope exists for microbial upgrading of these low-quality agro-wastes and development of healthy animal feed supplements.     

Production of macerating enzymes of mandarin orange peel by fungal cultures

Summary Thirty-nine fungal cultures belonging to the genera of Aspergillus, Podospora, Sordaria, Cbaetomium, Iodophanus, Scleotinia, Coniella, Pellicularia and others, were examined for the production of enzymes which macerate the mandarin orange peel using a wheat bran as substrate. An isolated strain of Aspergillus niger was an excellent producer of macerating enzymes compared to other organisms tested. The peel of the mandarin orange could be macerated by the crude enzymes produced by isolated A. niger. The maceration of 1 g of peel/24 h yielded 0.57 g of reducing sugars. Expressed differently, 83% of solid peel materials were released from the peel into the water/24 h under the following conditions: a peel concentration of 8 g peel/l, a crude enzyme concentration of 1 g protein/l, a temperature of 40°C, a pH of 5, a 24 h incubation time and 120 rpm reciprocal shaking. The test of the macerating activity of commercially available hydrolases on the orange peel showed that the two samples of pectinase originating from A. niger had about the same activity as isolated A. niger whereas the two samples of cellulase originating from Trichoderma viride had remarkably lower activities than A. niger.     

Role of associative bacterial interaction in the induction of tuber rot incidence in cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz), one of the leading staples in the world, has considerable scope for integration into emerging markets through efficient and environmentally sound production of a diversified range of high quality, competitive products for food, feed and industry. One of the major reasons attributed to the low productivity of cassava in India is the incidence of tuber rot in cassava incited by Phytophthora palmivora. Usually, an array of microorganisms is involved in rotting of cassava tubers in the field and in postharvest conditions. Unfortunately, the disease management practices focus mainly on fungal pathogens. In this article, we tried to investigate the role of associative bacterial pathogens in induction of tuber rot of cassava, the potential of the bacteria with P. palmivora in causing disease incidence, spread and rotting. The bacteria and P. palmivora were originally isolated from tuber rot infected cassava from the field having a disease incidence percentage of above 40%, checked for pathogenicity and were proven for Koch postulate. The detached tubers of disease resistant (cv. Sree Pathmanabha) and susceptible cultivars (cv. M₄) of cassava were used for the study. The effect of bacterial association was studied in Cassava agar plate assay, in detached tubers and in in planta experiments in in vitro glass house conditions. The bacterial isolates were identified as Gram-negative coco bacilli of class Pseudomonas, Erwinia amylovora and Achromobacter denitrificans.     

Detoxification of cassava by Aspergillus niger B-1

 The effects of fermentation of cassava by Aspergillus niger B-1 β-glucosidase on its cyanide and protein content, and the optimal conditions for this enzyme’s activity, were examined. This fermentation process reduced the cyanide content of cassava by 95% to 2 mg/kg, and increased its total protein content by 50%, thereby improving its nutritional value. A significant decrease in cyanogenic glycosides was detected after 3 days of fermentation. The optimal pH for A. nigerβ-glucosidase activity on the cyanogenic glycoside linamarin was determined to be 3, the optimal temperature 55 °C, and its K _m 0.3 mM. The findings presented here will facilitate the development of an improved method for detoxification of cassava and for enhancement of its nutritional value. Received: 17 August 1995/Received revision: 27 October 1995/Accepted: 30 October 1995     

Root-surface mycoflora of cassava (Manihot esculenta) and post harvest rot of the tubers

The root-surface mycoflora of cassava were isolated from roots washed in serial changes of sterile distilled water and plated out on potato-dextrose agar. A small group of fungi which included Aspergillus niger, Botryodiplodia theobromae, Fusarium solani, Penicillium javanicum, Penicillium sp., and Trichoderma sp. were found to be consistently associated with the root surface. While the isolates, B. theobromae and F. solani were found to be aggressively pathogenic on freshly harvested cassava tubers causing extensive rot, A. niger was only mildly so. The root-surface mycoflora, therefore, includes fungi which have been reported as the most important in postharvest deterioration of the tubers. The removal of the rhizoplane microflora by surface-sterilization using calcium hypochlorite or Clorox and subsequent incubation in loosely tied polyethylene bags extended the storage life of the tubers considerably.     

不同来源溶藻菌的分离、鉴定及溶藻效果比较

为了探究从何种类型的自然生境中更易分离得到溶藻微生物,采用高氏1号培养基分别从水库底泥、湖泊底泥、农田土壤、林地土壤等四种来源共36份样品中分离了7 600株菌,并最终从中筛选得到了5株溶铜绿微囊藻(Microcystis aeruginosa)的溶藻菌,其中4株为假单胞菌(Pseudomonas sp.),1株为黄杆菌(Flavobacterium sp.),5株菌溶藻效率的变化范围为62%~95%。结果表明,当采用高氏1号培养基作为分离培养基时,湖泊底泥和水库底泥中的成功筛选概率最高,农田土壤次之,而林地土壤中则难以筛选得到,假单胞菌是较容易筛选得到的溶藻菌。     

The Potential of Bacterial Isolates for Emulsification with a Range of Hydrocarbons

A study was undertaken to investigate the distribution of biosurfactant producing and crude oil degrading bacteria in the oil contaminated environment. This research revealed that hydrocarbon contaminated sites are the potent sources for oil degraders. Among 32 oil degrading bacteria isolated from ten different oil contaminated sites of gasoline and diesel fuel stations, 80% exhibited biosurfactant production. The quantity and emulsification activity of the biosurfactants varied. Pseudomonas sp. DS10‐129 produced a maximum of 7.5 ± 0.4 g/l of biosurfactant with a corresponding reduction in surface tension from 68 mN/m to 29.4 ± 0.7 mN/m at 84 h incubation. The isolates Micrococcus sp. GS2‐22, Bacillus sp. DS6‐86, Corynebacterium sp. GS5‐66, Flavobacterium sp. DS5‐73, Pseudomonas sp. DS10‐129, Pseudomonas sp. DS9‐119 and Acinetobacter sp. DS5‐74 emulsified xylene, benzene, n‐hexane, Bombay High crude oil, kerosene, gasoline, diesel fuel and olive oil. The first five of the above isolates had the highest emulsification activity and crude oil degradation ability and were selected for the preparation of a mixed bacterial consortium, which was also an efficient biosurfactant producing oil emulsifying and degrading culture. During this study, biosurfactant production and emulsification activity were detected in Moraxella sp., Flavobacterium sp. and in a mixed bacterial consortium, which have not been reported before.     

Phenol degradation by microorganisms adsorbed on activated carbon

Summary The phenol degradation by Candida sp. and Pseudomonas sp. immobilized on activated carbon was investigated. Thanks to its great adsorptive surface, activated carbon is suited as supporting material for microorganisms and also provides a high adsorption capacity for phenol.The immobilization by adsorption avoids any unphysiological treatment of the microorganisms. One gram activated carbon adsorbed in 10 h about 4×10⁹ Pseudomonas cells and 3×10⁸ Candida cells. While the free cells did not tolerate more than 1.5 g/l phenol, the adsorbed microorganisms survived at temporary high phenol concentrations up to 15 g/l, and they degraded about 90% of the adsorbed phenol.The activated carbon operated like a depot: the adsorbed phenol diffused out of the carbon and could be metabolized by the microorganisms. The results give an explanation of the stimulating effect of activated carbon in the treatment of waste waters observed until now.     

In vitro standardisation of resistance screening methods in cassava against tuber rot disease

Cassava tuber rot caused by Phytophthora palmivora in growing regions of Tamil Nadu and Kerala, is causing yield loss up to 80%. In the present study, resistance reactions of 10 cassava cultivars were analysed on leaf, stem and tuberous roots by artificial inoculation method in search of a suitable in vitro resistant screening method. Leaf and tuber analysis showed positive correlation (0.883) but the stem-based results showed negative correlation with leaf and tuber analysis. The analysis exhibited the susceptibility of the cassava cultivars against P. palmivora. Leaf analysis was superior in discriminating even small variations in resistance reactions than tuber analysis. The cultivar Sree Padmanabha showed higher resistance than other cultivars and the level of resistance in a cultivar is heritable which could be helpful in breeding programme. Based on the results it can be concluded that leaves of cassava could be used for screening resistance in the host and also in analysing the virulence of the isolate. This is the first report on screening the resistance in cassava cultivars against root rot caused by P. palmivora.     

The Monosaccharide Composition of Cell Wall Material in Cassava Tuber (Manihot utilissima)

The monosaccharide composition of cell wall material (CWM) in the cassava tuber and the contents of the other constituents were determined for more advanced industrial utilization. Starch, 80% ethanol–soluble sugar, uronic acid, lignin, ash, and CWM contents in the cassava tuber 86.1, 2.4, 3.4, 0.5, 0.9, and 4.5%, respectively. Rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose contents in CWM were 1.9, 1.2, 2.6, 4.2, 2.0, 12.8, and 52.7%, respectively. Then, the degradation pattern of CWM by enzymatic and sequential acid hydrolysis was studied. Aspergillus niger cellulase preparation was the most effective, and 57.1 % of CWM was degraded by the enzyme preparation. On the other hand, about 50% of the hemicellulose part was extracted from CWM by hot water only.     

Production of cis,cis-muconate from benzoate and 2-fluoro-cis,cis-muconate from 3-fluorobenzoate by 3-chlorobenzoate degrading bacteria

Summary 3-Chlorobenzoate grown cells of Pseudomonas sp. strain B13 or Alcaligenes sp. strain A7-2 converted 3-fluorobenzoate to 2-fluoro-cis,cis-muconate with 87% yield. The latter strain produced 1.6 g/l. The type II muconate cycloisomerases of neither strain exhibit acitivity for 2-fluoro-cis,cis-muconate. Succinate grown cells of Pseudomonas sp. strain B13 converted benzoate to cis,cis-muconate (91% yield; 7.4 g/l). Enzyme tests confirmed that no muconate cycloisomerising enzyme was induced within 24 h.