Session 2 biological research

Inedible plant material, generated in a Controlled Ecological Life Support System (CELSS), should be recycled preferably by bioregenerative methods that utilize enzymes or micro-organisms. This material consists of hemicellulose, cellulose, and lignin with the lignin fraction representing a recalcitrant component that is not readily treated by enzymatic methods. Consequently, the white-rot fungus,Pleurotus ostreatus, is attractive since it effectively degrades lignin and produces edible mushrooms. This work describes an unstructured model for the growth ofP. ostreatus in a solid-state fermentation system using lignocellulosic plant materials fromBrassica napus (rapeseed) as a substrate at three different particle sizes. A logistic function model based on area was found to fit the surface growth of the mycelium on the solid substrate with respect to time, whereas a model based on diameter, alone, did not fit the data as well. The difference between the two measures of growth was also evident for mycelial growth in a bioreactor designed to facilitate a slow flowrate of air through the 1.5 cm thick mat of lignocellulosic biomass particles. The result is consistent with the concept of competition of the mycelium for the substrate that surrounds it, rather than just substrate that is immediately available to single cells. This approach provides a quantitative measure ofP. ostreatus growth on lignocellulosic biomass in a solid-state fermentation system. The experimental data show that the best growth is obtained for the largest particles (1 cm) of the lignocellulosic substrate.     

The use of steamed hemicellulose as substrate in microbial conversions

The suitability of acid- and enzymatically hydrolyzed birch hemicellulose as biotechnical raw material was studied usingCluconobacter oxydans, Fusarium oxysporum, andCandida utilis for production of xylonic acid, ethanol, and SCP, respectively. The fermentabilities of both hydrolyzates were rather similar and inhibition was evident in all cases at xylose concentrations of 257–30 g/L and higher. Potential identified fermentation inhibitors were the lignin-derived compounds sinapyl alcohol, coniferyl alcohol, vanillin, and syringaldehyde and the sugar degradation products furfural and 5-hydroxymethyl furfural.     

Induction and Purification by Three-Phase Partitioning of Aryl Alcohol Oxidase (AAO) from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Aryl alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin degradation by white rot fungi. Screening of lignolytic and AAO activity from twenty different fungal species were carried out. Among them, seven species showed lignolytic activity and three of them (Pleurotus ostreatus, Pleurotus eous, and Pleurotus platypus) were found to be AAO positive. Maximal AAO activity was observed in batch cultures of P. ostreatus and was found to be induced by aromatic amino acids and aryl alcohols up to a level of 289 U/l. Purification of AAO was carried out by three-phase partitioning (TPP). The 67 kDa enzyme was purified up to 10.19-fold by TPP with an overall recovery of 10.95%. Optimum pH and temperature for P. ostreatus AAO activity was found to be around 6 and 40 °C, respectively. From the LB plot, K _m value of AAO for oxidizing veratryl alcohol was determined to be 0.6 mM. Results of the study indicate that P. ostreatus is the best producers of AAO, and they could be employed as promising fungal species for biotechnological applications.     

Hybrid process for the conversion of lignocellulosic materials

Because of the recalcitrant nature of lignocellulosic materials, it is important to pretreat the biomass in order to obtain a suitable material for the bioconversion. In this study, two different types of pretreatments were performed. The first experiment used a 2-gal Parr reactor operated at 140, 150,160, and 170‡C with sulfuric acid concentrations varying from 0.5 to 2%. A second pretreatment was performed with a two-stage low-temperature process. The first-stage pretreatment was performed at 100 or 120‡C with sulfuric acid concentrations of 0.5, 2, and 5% followed by a secondstage pretreatment at 120‡C with 2% acid concentration. The best residues for enzymatic hydrolysis and simultaneous saccharification and fermentations (SSF) came from the higher temperature pretreatment with the Parr reactor. However, a large portion of the xylose fraction was degraded to furfural and glucose was degraded to HMF. On the contrary, the two-stage low temperature pretreatment resulted in a very low percentage of xylose degradation, and no glucose degradation. The residues from this two-stage pretreatment performed satisfactorily toward the production of ethanol by SSFs. This study discusses the results obtained from these experiments.     

Secretome analysis of Pleurotus eryngii reveals enzymatic composition for ramie stalk degradation

Pleurotus eryngii (P. eryngii) can secrete large amount of hydrolytic and oxidative enzymes to degrade lignocellulosic biomass. In spite of several researches on the individual lignolytic enzymes, a direct deconstruction of lignocellulose by enzyme mixture is not yet possible. Identifying more high‐performance enzymes or enzyme complexes will lead to efficient in vitro lignocelluloses degradation. In this report, secretomic analysis was used to search for the new or interesting enzymes for lignocellulose degradation. Besides, the utilization ability of P. eryngii to ramie stalk substrate was evaluated from the degradation of cellulose, hemicellulose, and lignin in medium and six extracellular enzymes activities during different growth stages were discussed. The results showed that a high biological efficiency of 71% was obtained; cellulose, hemicelluloses, and lignin decomposition rates of P. eryngii were 29.2, 26.0, and 51.2%, respectively. Enzyme activity showed that carboxymethyl cellulase, xylanase, laccase, and peroxidase activity peaks appeared at the primordial initiation stage. In addition, we profiled a global view of the secretome of P. eryngii cultivated in ramie stalk media to understand the mechanism behind lignocellulosic biomass hydrolysis. Eighty‐seven nonredundant proteins were identified and a diverse group of enzymes, including cellulases, hemicellulases, pectinase, ligninase, protease, peptidases, and phosphatase implicated in lignocellulose degradation were found. In conclusion, the information in this report will be helpful to better understand the lignocelluloses degradation mechanisms of P. eryngii.     

Biodegradation of Fluoranthene by Basidiomycetes Fungal Isolate <Emphasis Type="Italic">Pleurotus Ostreatus</Emphasis> HP-1

The biodegradation of fluoranthene, a high molecular weight polycyclic aromatic hydrocarbon (PAH), was investigated in submerged culture using the wood decaying fungus isolated from forest locality in Gujarat, India. The basidiomycete fungal isolate was found to have an ability to grow on sabaroud dextrose agar containing 50 mgl⁻¹ of each naphthalene, anthracene, acenaphthene, benzo (a) anthracene, pyrene, flouranthene, carbazole, and biphenyl. The involvement of extracellular fungal peroxidases such as manganese peroxidase (MnP) and laccase (Phenol oxidase) in the degradation of fluoranthene was studied. On the eighth day of incubation 54.09% of 70 mg l⁻¹ fluoranthene was removed. There after no PAHs removal was observed till the 20th day of the incubation period. The isolate was identified as Pleurotus ostreatus by 18S rRNA, 5.8S rRNA, and partial 28S rRNA gene sequencing. To the best of our knowledge this is the first time Pleurotus ostreatus have been reported to degrade such a high concentration of fluoranthene within much lower time period of incubation. Depletion in the residual fluoranthene in the culture medium was determined by HPLC. Attempts were made to identify the degradation product in the culture medium with the help of FT-IR, NMR, and HPTLC analysis. In the present study positive correlation between fluoranthene degradation and the ligninolytic enzyme (MnP and laccase) production is observed, thus this isolate can play an effective role for bioremediation of PAHs contaminated sites.     

Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics

The cellulose reactivity of two lignocellulosic feedstocks, switchgrass and poplar, was evaluated under straight saccharification (SS) and simultaneous saccharification and fermentation (SSF) conditions following dilute sulfuric acid pretreatments designed for optimum xylose yields. The optimum pretreatment conditions, within the constraints of the experimental system (Parr batch reactor), were 1.2% acid, 180°C, and 0.5 min for switchgrass and 1% acid, 180°C, and 0.56 min for poplar. The cellulase enzyme preparation was from Trichoderma reesei and fermentations were done with Saccharomyces cerevisiae. Time courses for SS were monitored as the sum of glucose and cellobiose; those for SSF as the sum of glucose, cellobiose, and ethanol. Percentage conversions under SS conditions were 79.1% and 91.4% for the pretreated poplar and switchgrass feedstocks, respectively. Analogous values under SSF conditions were 73.0% and 90.3% for pretreated poplar and switchgrass, respectively.     

Screening of Oleaginous Yeast Strains Tolerant to Lignocellulose Degradation Compounds

High cost of triacylglycerol lipid feedstock is the major barrier for commercial production of biodiesel. The fermentation of oleaginous yeasts for lipid production using lignocellulose biomass provides a practical option with high economic competitiveness. In this paper, the typical oleaginous yeast strains were screened under the pressure of lignocellulose degradation compounds for selection of the optimal strains tolerant to lignocellulose. The inhibitory effect of lignocellulose degradation products on the oleaginous yeast fermentation was carefully investigated. Preliminary screening was carried out in the minimum nutritious medium without adding any expensive complex ingredients then was carried out in the lignocellulosic hydrolysate pretreated by dilute sulfuric acid. Seven typical lignocellulose degradation products formed in various pretreatment and hydrolysis processing were selected as the model inhibitors, including three organic acids, two furan compounds, and two phenol derivatives. The inhibition of the degradation compounds on the cell growth and lipid productivity of the selected oleaginous yeasts were examined. Acetic acid, formic acid, furfural, and vanillin were found to be the strong inhibitors for the fermentation of oleaginous yeasts, while levulinic acid, 5-hydroxymethylfurfural, and hydroxybenzaldehyde were relatively weak inhibitors. Trichosporon cutaneum 2.1374 was found to be the most adopted strain to the lignocellulose degradation compounds.     

Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid in a two-zone fermentor-extractor system

Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid was investigated using a two-zone bioreactor. The system is composed of an immobilized cell reactor, a separate column reactor containing the lignocellulosic substrate and a hollow-fiber membrane. It is operated by recirculating the cell free enzyme (cellulase) solution from the immobilized cell reactor to the column reactor through the membrane. The enzyme and microbial reactions thus occur at separate locations, yet simultaneously. This design provides flexibility in reactor operation as it allows easy separation of the solid substrate from the microorganism, in situ removal of the product and, if desired, different temperatures in the two reactor sections. This reactor system was tested using pretreated switchgrass as the substrate. It was operated under a fed-batch mode with continuous removal of lactic acid by solvent extraction. The overall lactic acid yield obtainable from this bioreactor system is 77% of the theoretical.     

Mn2+ alters peroxidase profiles and lignin degradation by the white-rot fungus Pleurotus ostreatus under different nutritional and growth conditions

The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn²⁺ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn²⁺ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of ¹⁴C-lignin mineralization was measured in the Mn²⁺-amended BSM, as determined during the course of 47 d of fermentation. Mn²⁺ amendment of the PM increased mineralization rate to that obtained in the Mn²⁺-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn²⁺ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn²⁺ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.     

Thermophilic Bacillus coagulans Requires Less Cellulases for Simultaneous Saccharification and Fermentation of Cellulose to Products than Mesophilic Microbial Biocatalysts

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g^?1 cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g^?1 cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Ethanol Production from Wet-Exploded Wheat Straw Hydrolysate by Thermophilic Anaerobic Bacterium Thermoanaerobacter BG1L1 in a Continuous Immobilized Reactor

Thermophilic ethanol fermentation of wet-exploded wheat straw hydrolysate was investigated in a continuous immobilized reactor system. The experiments were carried out in a lab-scale fluidized bed reactor (FBR) at 70°C. Undetoxified wheat straw hydrolysate was used (3–12% dry matter), corresponding to sugar mixtures of glucose and xylose ranging from 12 to 41 g/l. The organism, thermophilic anaerobic bacterium Thermoanaerobacter BG1L1, exhibited significant resistance to high levels of acetic acid (up to 10 g/l) and other metabolic inhibitors present in the hydrolysate. Although the hydrolysate was not detoxified, ethanol yield in a range of 0.39–0.42 g/g was obtained. Overall, sugar efficiency to ethanol was 68–76%. The reactor was operated continuously for approximately 143 days, and no contamination was seen without the use of any agent for preventing bacterial infections. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol. The work reported here also demonstrates that the use of FBR configuration might be a viable approach for thermophilic anaerobic ethanol fermentation.     

Fungal Pretreatment by Phanerochaete chrysosporium for Enhancement of Biogas Production from Corn Stover Silage

Corn stover silage (CSS) was pretreated by Phanerochaete chrysosporium in solid-state fermentation (SSF), to enhance methane production via subsequent anaerobic digestion (AD). Effects of washing of corn stover silage (WCSS) on the lignocellulosic biodegradability in the fungal pretreatment step and on methane production in the AD step were investigated with comparison to the CSS. It was found that P. chrysosporium had the degradation of cellulose, hemicellulose, and lignin of CSS up to 19.9, 32.4, and 22.6 %, respectively. Consequently, CSS pretreated by 25 days achieved the highest methane yield of 265.1 mL/g volatile solid (VS), which was 23.0 % higher than the untreated CSS. However, the degradation of cellulose, hemicellulose, and lignin in WCSS after 30 days of SSF increased to 45.9, 48.4, and 39.0 %, respectively. Surface morphology and Fourier-transform infrared spectroscopy analyses also demonstrated that the WCSS improved degradation of cell wall components during SSF. Correspondingly, the pretreatment of WCSS improved methane production by 19.6 to 32.6 %, as compared with untreated CSS. Hence, washing and reducing organic acids (such as lactic acid, acetic acid, propionic acid, and butyric acid) present in CSS has been proven to further improve biodegradability in SSF and methane production in the AD step.     

Influence of lignocellulose-derived aromatic compounds on oxygen-limited growth and ethanolic fermentation by Saccharomyces cerevisiae

Phenolic compounds released and generated during hydrolysis inhibit fermentation of lignocellulose hydrolysates to ethanol by Saccharomyces cerevisiae. A wide variety of aromatic compounds form from lignin, which is partially degraded during acid hydrolysis of the lignocellulosic raw material. Aromatic compounds may also form as a result of sugar degradation and dare present in wood as extractives. The influence of hydroxy-methoxy-benzaldehydes, diphenols/quinones, and phenylpropane derivatives on S. cerevisiae cell growth and ethanol formation was assayed using a defined medium and oxygen-limited conditions. The inhibition effected by the hydroxy-methoxy-benzaldehydes was highly dependent on the positions of the substituents. A major difference in inhibition by the oxidized and reduced form of a diphenol/quinone was observed, the oxidized form being the more inhibitory. The phenylpropane derivatives were examined with respect to difference in toxicity depending on the oxidation-reduction state of the γ-carbon, the presence and position of unsaturated bonds in the aliphatic side chain, and the number and identity of hydroxyl and methoxyl substituents. Transformations of aromatic compounds occuring during the fermentation included aldehyde reduction, quinone reduction, and double bond saturation. Aromatic alcohols were detected as products of reductions of the corresponding aldehydes, namely hydroxy-methoxy-benzaldehydes and coniferyl aldehyde. High molecular mass compounds and the corresponding diphenol were detected as products of quinone reduction. Together with coniferyl alcohol, dihydroconiferyl alcohol was identified as a major transformation products of conifery aldehyde.

     

Mechanism and potential applications of bio-ligninolytic systems in a CELSS

A large amount of inedible plant material, generated as a result of plant growth in a Controlled Ecological Life Support System (CELSS), should be pretreated and converted into forms that can be recycled on earth as well as in space. The main portion of the inedible biomass is lignocellulosic material. Enzymatic hydrolysis of this cellulose would provide sugars for many other uses by recycling carbon, hydrogen, oxygen, and nitrogen through formation of carbon dioxide, heat, and sugars, which are potential foodstuffs. To obtain monosaccharides from cellulose, the protective effect of lignin should be removed. White-rot fungi degrade lignin more extensively and rapidly than other microorganisms.Pleurotus ostreatus degrades lignin effectively, and produces edible and flavorful mushrooms that increase the quality and nutritional value of the diet. This mushroom is also capable of metabolizing hemicellulose, thereby providing a food use of this pentose containing polysaccharide. This study presents the current knowledge of physiology and biochemistry of primary and secondary metabolisms of basidiomycetes, and degradation mechanism of lignin. A better understanding of the ligninolytic activity of white-rot fungi will impact the CELSS Program by providing insights on how edible fungi might be used to recycle the inedible portions of the crops.     

Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation

This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Enzyme Production by Solid Substrate Fermentation of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Trametes versicolor</Emphasis> on Tomato Pomace

A process of solid state fermentation (SSF) on tomato pomace was developed with the white-rot fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support. Operative parameters (humidity, water activity, and size of substrate particles) guaranteeing a good colonization of tomato pomace by both fungi were defined and conditions for production at high titers of the industrially relevant enzymes laccase, xylanase and protease were identified. Significant laccase activity levels (up to 36 U g⁻¹ dry matter) were achieved without any optimization of culture conditions, neither by nutrient addition nor by O₂ enrichment. Furthermore, protease activity levels up to 34,000 U g⁻¹ dry matter were achieved, being higher than those reported for the fungi typically considered as the best protease producers such as Aspergillus strains. Moreover, as one of the most significant results of this study, analysis of P. ostreatus tomato SSF samples by zymogram revealed two bands with laccase activity which had not been detected so far.     

Biological pretreatment of lignocellulose for enzymatic hydrolysis of cellulose

Pretreatment of lignocellulosic materials is considered as the rate-limiting step in an economically feasible process for enzymatic hydrolysis of cellulose. Biological delignification techniques have not been developed as intensively as physical and chemical methods. However, white-rot fungi are effective degraders of lignin, and some of them even preferentially remove lignin from wood compared with carbohydrates, and therefore might be suitable for biological pretreatment of lignocellulose. White-rot fungi were cultivated on wheat straw and the residue was hydrolyzed withTrichoderma reesei cellulase. Of nineteen fungi examined,Pleurotus ostreatus, Pleurotus sp. 535,Pycnoporus cinnabarinus 115,Ischnoderma benzoinum 108,Phanerochaete sordida 37,Phlebia radiata 79, and two unidentified fungi were found suitable for pretreatment of straw: the yields of reducing sugars and glucose based on original straw were markedly better compared with uninoculated straw, and these fungi also gave better results thanPolyporus versicolor, a nonselective reference fungus (Cowling, 1961). In the best cases the efficiency of the biological pretreatment was comparable with that of alkali treatment (2% NaOH, 0.4 g NaOH/g straw, 10 min at 115‡C), but the fungal treatment resulted in a higher proportion of glucose in the hydrolyzates. Combined fungal and (strong) alkali treatment did not give better results than alkali or fungal treatment alone. When culture flasks were periodically flushed with oxygen the treatment time could be reduced by about 1 wk with the two fungi,P. sordida 37 andP. cinnabarinus 115, tested. The effect of oxygen in pretreatment reflected the effect of oxygen in the degradation of¹⁴C-lignin of poplar wood to¹⁴CO₂ by these fungi (Hatakka and Uusi-Rauva, 1983). The economic feasibility of the biological pretreatment process is poor due to the long cultivation times needed. The best results were obtained with the longest treatment time studied, which was 5 wk. However, the rapid progress in the field of biological lignin degradation may help to accelerate the delignification process, and also find factors that favor lignin degradation, but suppress the utilization of carbohydrates.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.