Solid substrate fermentation of leached beet pulp withTrichoderma aureoviride

Optimum production of cellulases on leached beet pulp by native and mutant strains ofTrichoderma aureoviride in column-type, solid-substrate fermenters, at controlled temperature and aeration rate, was with a mutant strain, that produced 11 and 29 filter paper units of activity/g solids on raw and acid pre-treated leached beet pulp, respectively. The former value was 40% higher than in the native strain and 70% higher than in the reference strain. protein enrichment of residual solid was significant in all cases, with values up to 40% of total dry weight. Results are suitable for scale-up to pilot level.     

Growth of Chlorella pyrenoidosa in wastewater from cassava ethanol fermentation

In a cycle tubular photobioreactor, Chlorella pyrenoidosa was cultured in undiluted wastewater from ethanol fermentation using cassava powder as raw material. The results showed that the optimum cultivation conditions were initial pH of 6.0, temperature at 27°C, continuous illumination at 3,000 lux, and cycle speed of 110 ml min⁻¹. Under these optimum conditions, after the logarithmic phase of batch cultivation with wastewater of pH 6.0, the reactor could be continuously operated with natural pH wastewater (3.8) as feed solution. By a dilution ratio of 0.17 day⁻¹, it could be operated stably for over 30 days in continuous cultivation. pH, removal rate of chemical oxygen demand, and biomass (cell dry weight) concentration ranged from 6.22 to 6.47, 72.21 to 76.32% and 3.55 to 3.73 g l⁻¹, respectively. After treatment, the wastewater could be used again in the process of ethanol fermentation.     

Lignin degradation during softwood decaying by the ascomyceteChrysonilia sitophila

SoftwoodPinus radiata was degraded by the ascomyceteChrysonilia sitophila during 3 months. The total weight loss of the wood was 20% and the carbohydrate and lignin losses were 18% and 25%, respectively. Decayed wood was extracted with solvents of increasing polarity. Methanol and dioxane yielded extracts containing representative low molecular weight degraded lignins. The overall structure of the degraded lignins, as shown by U.V./visible, I.R.,¹H and¹³C NMR spectroscopy, GPC, functional group and elemental analyses, was compared with the structure of milled wood lignin extracted from undecayedP. radiata. The compilation of the data allows us to suggest oxidative C-C and -O-aryl cleavages for the mechanism of lignin degradation by this ascomycete. New saturated carbons on the side chain of the degraded lignins were detected. Based on these data a reductive ability of this microorganism was also suggested.     

Characterization ofgari andfu-fu preparation procedures in Nigeria

Processes for the production ofgari, East Nigerianfu-fu and West Nigerianfu-fu are described. Changes in pH value, moisture content, microflora and sugar content of cassava duringgari andfu-fu preparation are reported. Mannitol accumulated during thegari fermentation but not in either of thefu-fu fermentations. During each stage ofgari andfu-fu production, lactic acid bacteria predominated. Homofermentative organisms occurred most frequently in the early stages of each process and heterofermenters in the latter ones. Of the 179 microorganisms that were isolated and characterized fromgari andfu-fu, 52% were able to hydrolyse linamarin and 14% starch.     

Cellulase production by mixed fungi in solid-substrate fermentation of bagasse

Ammonia-treated bagasse with 80%(w/w) moisture content was subjected to mixed-culture solid-substrate fermentation (SSF) with Trichoderma reesei LM-UC4 and Aspergillus phoenicis QM 329, in flask or pot fermenters, for cellulase production. Significantly higher activities of all the enzymes of the cellulase complex were achieved in 4 days of mixed-culture SSF than in single-culture (T. reesei) SSF. The highest filter-paper-cellulase and -glucosidase activities seen in mixed-culture SSF were 18.7 and 38.6 IU/g dry wt, respectively, representing approx. 3- and 6-fold increases over the activities attained in single-culture SSF. The mixed-culture SSF process also converted about 46% of the cellulose and hemicellulose to reducing sugars and enriched the product with 13% fungal protein. The biomass productivity, 0.29 gl^-1.h, and enzyme productivity, 28.0 IU I^-1.h, were about twice as high in the mixed-culture than in the single-culture.R. Dueñas is with the Departamento de Biologia, Universidad Nacional San Antonio Abad, Cusco, Peru. R. Tengerdy is with the Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA. M. Gutierrez-Correa is with the Laboratorio de Micologia y Biotecnologia, Universidad Nacional Agraria La Molina, Apdo. Postal 456, Lima 1. Peru;     

Novel lignocellulolytic ability of Candida utilis during solid-substrate cultivation on apple pomace

Lignocellulolytic enzymes from conventional and non-conventional yeasts are not commonly studied, and they have never been described for Candida utilis species. After solid-substrate cultivation of C. utilis (CCT 3469) on apple pomace, degradation of cellulose, pectin and lignin fragments was observed. Production of the main lignocellulolytic enzymes by C. utilis was investigated and high activity for pectinase (239 U ml^–1) as well as a significant manganese-dependent peroxidase (19.1 U ml^–1) activity was found. Low cellulase (3.0 U ml^–1) and xylanase (1.2 U ml^–1) activities were also observed suggesting that C. utilis may have lignocellulose degradation ability.     

灵芝真菌液体发酵及其产物应用的研究进展

灵芝作为一种白腐真菌,同时也是珍稀的食药用真菌,富含多种生物活性成分。液体发酵技术生产周期短、效率高、产量大、品质稳定,是开发利用灵芝资源的重要途径。近年来,灵芝属真菌菌丝体液体发酵技术的开发与应用取得了较大进展。本文对灵芝属真菌液体发酵产物的主要活性成分及其药用效果、液体发酵工艺优化和发酵产物的应用进行综述,并对本领域的未来进行展望。     

Optimization of single-cell-protein production from cassava starch using Schwanniomyces castellii

Schwanniomyces castellii B5285 grew faster and produced greater biomass and higher protein yield than either S. alluvius ATCC 26074 or S. alluvius 81Y when these amylolytic yeasts were grown with 2% (w/v) cassava starch as sole C source. With 0.5% (w/v) glutamate as N source, S. castellii reached 7.12 g cell dry mass/l, with a protein yield of 6.4 g/100 g starch. The optimal agitation speed, aeration rate and pH for growth of this yeast in a fermenter were 400 rev/min, 1.67 vol./vol.min. and 5.0, respectively. Tween 80 at 0.1% increased cell dry mass to 8.90 g/l, cell yield to 44 g/100 g starch and protein yield to 7.4 g/100 g starch.The authors are with the Department of industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90110, Thailand     

A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen

Shoot tips of in vitro-grown plantlets of cassava (Manihot esculenta Crantz), representing a wide range of germplasm, were cryopreserved as follows: pre-cultured for 3 days, cryoprotected and dehydrated for 1 h, then frozen in liquid nitrogen using a six-step protocol. After 3 h in liquid nitrogen, the shoot tips were removed, rapidly warmed, and recultured sequentially in three recovery media. After 2 weeks, the regeneration of frozen shoot tips was completed. Genotypes with a low response were identified. Their response was attributed to the effects of pre and post-freezing steps. Refining the methodology led to a consistent 50–70% plant recovery.Abbreviations DMSO Dimethylsulfoxide - MS Murashige and Skoog medium (1962) - LN liquid nitrogen     

Low-temperature scanning electron microscopy of hyphal colonisation of Chenopodium quinoa during fermentation with Rhizopus oligosporus

In a study of tempe made by fermenting quinoa (Chenopodium quinoa Willd.) with Rhizopus oligosporus Saito, the interaction between the substrate and the fungus varied according to the raw material used. Low-temperature scanning electron microscopy confirmed that hyphal infiltration in quinoa was normally limited by the seed coat, but a massive penetration was found in fragments of quinoa where the testa was broken. This finding has implications for the texture and quality of tempe fermentation of grains and pulses.     

Origin of enzymes involved in detoxification and root softening during cassava retting

The origin of root softening during cassava retting was investigated in a natural retting and in a sterile fermentation. Softening only occurred in the natural retting. Although high activities of endogenous pectin methyl esterase were found in cassava extracts from both fermentations, the depolymerizing enzymes polygalacturonase, active at low pH, and pectate lyase were only found in the non-sterile retting. No cellulase or xylanase activity was observed. The role of pectinases in the softening of cassava roots was confirmedin vitro using commericial enzymes. Root softening is therefore due to the combined action of endogenous pectin methyl esterase and exogenous bacterial depolymerizing enzymes. Detoxification occurred in both fermentations, confirming that the linamarase responsible for the destruction of cassava cyanide glycosides was mainly endogenous, even though microbial -glucosidases may help in the detoxication.F. Ampe is and A. Brauman was with the Laboratoire de Microbiologie et de Biotechnologie, ORSTOM, BP 181-Brazzaville, Congo. A. Brauman is now with the Laboratoire d'Ecophysiologie des Invertébrés, Université Paris XII, Avenue du Général De Gaulle, 94 010 Créteil, France     

Implication of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in the production of pectinolytic enzymes during cocoa fermentation

The role of bacilli in cocoa fermentation is not well known. Their potential of production of pectinolytic enzymes during this process was evaluated. Bacillus growth was monitored and pectinolytic strains were screened for their use of pectin as sole carbon source. Effects of cocoa fermentation parameters susceptible to influence on enzyme production were analysed. Among 98 strains isolated, 90 were positive for pectin degradation and 80% of them presented detectable pectinolytic activities in submerged fermentation. Forty-eight strains produced polygalacturonase (PG), 47 yielded pectin lyase (PL) and 23 strains produced both enzymes. Bacilli growth was not significantly affected during fermentation. PL production was favoured by galactose, lactose, glucose as sugars, and arginine, glutamine, cysteine and ammonium sulphate as nitrogen compounds. Pectin at low concentration (0.05%) and iron stimulated PL production. It was strongly repressed by galacturonic acid (1%), and negatively affected by nitrogen starvation, zinc and temperatures above 45°C. PL yield was very weak below pH 4.0 and in anaerobic conditions. PG production was weakened by sucrose and cation depletion. It was increased slightly by cysteine, ammonium nitrate and nitrogen starvation and significantly above 40°C. PG synthesis was not affected by acidic pH (3.0–6.0) or oxygen availability. As fermentation products, lactate and acetate lowered the production of both enzymes while ethanol had no effect. The high proportion of pectinolytic producers among the strains studied and analysis of factors influencing pectinolytic enzymes production, suggest that Bacillus sp. is liable to produce at least one enzyme during cocoa fermentation.     

Pyruvate fermentation in light-grown cells ofRhodospirillum rubrum during adaptation to anaerobic dark conditions

Pyruvate fermentation inRhodospirillum rubrum (strains F₁, S₁, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F₁ and S₁ metabolized the produced formate further to H₂ and CO₂. A slight production of CO₂ from pyruvate, without additional H₂-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl Bacteriochlorophyll - CoA Coenzyme A - DSM Deutsche Sammlung von Mikroorganismen (Göttingen) - OD optical density - PHBA poly--hydroxybutyric acid - R Rhodospirillum     

Bacillus fermentation of soybeans

Kinema fermentations of Indian and Canadian No. 1 soybeans by Bacillus sp. DK-W1 and by mixed cultures of Bacillus sp. DK-W1 and Enterococcus faecium DK-C1 were essentially identical. The viable cell count of Bacillus increased from an initial 10⁵ to 10¹⁰ c.f.u./g wet wt after 48 h incubation at 37°C. The pH of the fermentation dropped from an initial 6.9 to about 6.4 after 8 h and then rose to 8.6 after 32 h, with a coincident increase in proteolytic activity and ammonia concentration. The fermentations containing E. faecium and Bacillus exhibited a greater initial pH decline and a slightly retarded subseqent increase in pH compared with fermentations with Bacillus only. The presence of E. faecium had no detectable effects on growth of the Bacillus, proteolytic activity, ammonia production or the final pH of the fermentations.P.K. Sarkar was and P.E. Cook and J.D. Owens are with the Food Microbial Interactions Laboratory, Department of Food Science and Technology, University of Reading, Reading RG6 2AP, UK; P.K. Sarkar is now with the Microbiology Laboratory, Centre for Life Sciences, University of North Bengal, Siliguri 734430, India.     

A geminivirus-induced gene silencing system for gene function validation in cassava

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.     

Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris

The Pichia pastoris expression system is widely used for the production of recombinant proteins. A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P. pastoris based on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described. An experimental procedure to allow precise calibration of the system and to reduce methanol sensor's interferences (>95% reduction) are also presented and discussed. Accuracy and measurement error were estimated about 0.05 g x l(-1) and 6%, respectively. The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.     

Potentials and limitations of mycorrhizal inoculation illustrated by experiments with field-grown cassava

Summary Field inoculation trials with cassava (Manihot esculenta Crantz) were conducted in Quilichao (typic Dystropept soil) and Carimagua (Haplustox soil). In Quilichao, with a large and effective native VA-mycorrhizal (VAM) population, inoculation withGlomus manihotis did not increase cassava yields significantly, neither when different sources and levels of inoculum material were used, nor with different cassava cultivars, or after stabilizing soil temperature through mulching. Field inoculation did result in a decrease of the coefficient of variation with respect to yield. The high dependency of cassava on an effective VAM association was indicated by a marked decrease in yield after eradication of native VAM by soil sterilization. In Carimagua, with a lower native VAM population, mycorrhizal inoculation withG. manihotis increased yields significantly at intermediate levels of 100 kg/ha of applied P, using either inoculum of cassava orPanicum maximum roots or inoculum of a soil-root mixture of maize or tropical kudzu. Higher or lower levels of P decreased the effect of inoculation on yield. There were no significant differences among P sources, ranging from highly soluble triple superphosphate to low solubility rock phosphates. Inoculation with different VAM isolates had a variable effect on cassava yields, and showed that there may be an interaction between P fertilizer level and isolate efficiency. It is concluded that there may be a potential to increase yields or decrease the fertilizer P requirements of cassava through field inoculation with effective VAM isolates, in the vast areas of acid infertile Oxisols and Ultisols with low native VAM fungal populations, represented by Carimagua.     

Effects of cassava varietal mixtures on the whiteflies Aleurotrachelus socialis and Trialeurodes variabilis in Colombia

The effects of cassava varietal mixtures on the population dynamics of the whiteflies Aleurotrachelus socialis Bondar and Trialeurodes variabilis (Quaintance) were investigated under outbreak conditions in Colombia. A regional variety of cassava (MCOL 2257) was grown in pure stand and mixed with a variety, CMC 40, believed to have partial resistance to T. variabilis. The dominant species in the systems, A. socialis, showed no varietal preference and population levels on the regional cultivar were similar between treatments. Of the two cultivars, CMC 40 was less preferred by T. variabilis and its presence in mixed variety plots lowered populations on the regional cassava. Yield losses, primarily due to A. socialis, were equal for regional cassava mixed with CMC 40 and in pure stand. Nevertheless, the use of CMC 40 in varietal mixtures provides a potential strategy for lowering herbivore load and increasing yields in zones with heavy T. variabilis attack.

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Résumé L'influence du mélange de différentes variétés de manioc sur A. socialis Bondar et T. variabilis Quaintance a été examinée dans des conditions de pullulation en Colombie. Une variété régionale (MCOL 2257) a été cultivée seule ou mélangée avec une variété (CMC 40), considérée comme ayant une résistance partielle à T. variabilis. A. socialis, espèce dominante dans la biocénose, n'a pas montré de préférence variétale et son niveau de population sur MCOL 2557 était le même quelles que soient les conditions de culture. T. variabilis a moins préféré CMC 40, dont la présence en culture mixte a réduit le niveau des populations sur manioc local. Les dégâts sur CMC 40, dus d'abord à A. socialis, ont été les mêmes, qu'il ait été seul ou mélangé à MCOL 2257. Les dégats sur MCOL 2257, dus avant tout à A. socialis ont été les mêmes en culture pure ou en mélange avec CMC 40. Quoi qu'il en soit, la culture de CMC 40 en association variétale constitue une méthode qui abaisse potentiellement le fardeau des herbivores en augmentant les récoltes dans les zones à forte contamination par T. variabilis.