Continuous wine making by delignified cellulosic materials supported biocatalyst

The continuous making of wine by a delignified cellulosic (DC) material-supported biocatalyst is reported. It was prepared by immobilizing the alcohol resistant strain AXAZ-1 on DC material. The product was found suitable for the continuous process in industrial applications. The operational stability was maintained for 2 mo with monitoring the ethanol concentration, wine, and alcohol productivities as well as the stability of °Be density at the outlet. Wine productivity was three to sixfold higher than obtained by natural fermentation, alcohol concentrations of the wine was in the range of 9.3-211.2% v/v and low volatile acidities of 0.15-20.36 g acetic acid/L were obtained. The effect of total acidity and flow rate of must were also examined. To demonstrate that the operational stability of the bioreactor is due to DC material that promotes the fermentation, and it takes place at even higher ethanol levels, an analogous system of kissiris supported biocatalyst was studied. Likewise, the tolerance in the alcohol concentration, as compared with free cells, were studied by their stability of the activity in the repeated batch fermentation of must.     

Low-temperature brewing by freeze-dried immobilized cells

We propose a novel biocatalyst in brewing. A cryotolerant strain of Saccharomyces cerevisiae was immobilized on delignified cellulosic material followed by freeze-drying of the immobilized cells without the use of any cryoprotectant. The freeze-dried immobilized biocatalyst was used in repeatedbatch fermentation of wort and showed reduced fermentation time and increased productivities as compared with free freeze-dried cells (FFDCs). It also demonstrated suitability for low-temperature brewing (5 and 0°C). The fermentation time in repeated-batch fermentations at 15°C was 1.5–2 d for a period of 13 mo, showing a high operational stability of the system. At 0°C the freeze-dried immobilized biocatalyst showed a 2- to 3.5-fold decrease in fermentation time in comparison with FFDCs. Polyphenol contents, bitterness, and diacetyl concentration were lower in beers produced by freezedried immobilized cells as compared with FFDCs. At 0°C polyphenols were 40% lower than at 15°C. Higher alcohols were reduced and ethyl acetate increased in comparison with FFDCs. Amyl alcohols at 0°C were lower than half of their content at 15°C, while ethyl acetate was 31 mg/L at 0°C and 18 mg/L at 15°C. These data justify the improved aroma and taste of beers produced by freeze-dried immobilized biocatalyst mainly at low temperatures.     

Fermentation Efficiency of Cells Immobilized on Delignified Brewers' Spent Grains after Low- and High-Temperature Thin Layer Thermal Drying

Low-cost dried yeasts immobilized on delignified brewers' spent grains for use in wine making and brewing were produced by simple thermal drying techniques. To optimize the thermal drying process, vacuum and air stream conditions were examined. Drying of thin layers of the biocatalysts was performed at low (30–38 °C) and high temperatures (40–70 °C). The fermentation efficiency of the thermally dried biocatalysts was acceptable, with immobilized cells showing a significantly higher thermotolerance compared with free cells. Immobilized cells dried at high temperatures presented slightly improved glucose fermentation efficiency compared with the low-temperature dried biocatalysts. Gas chromatography–mass spectrometry analysis of aroma volatiles of the fermented products revealed an increase of esters, lower higher alcohol formation, and significantly lower concentration of carbonylic compounds.     

Dry Red Wine Making Using Yeast Immobilized on Cork Pieces

The commercial Saccharomyces cerevisiae Uvaferme 299 wine yeast was immobilized on cork pieces to produce a biocatalyst for use in dry red wine making. The immobilized biocatalyst was suitable for clarified and non-clarified grape must fermentation at ambient and low temperatures (0–25 °C). Fermentation times were low and very low amounts of residual sugar were detected, showing suitability for dry wine production. The presence of suspended solids in the non-clarified must did not affect the activity of the immobilized cells. Complete fermentation of sugars at 0 °C was possible with immobilized Uvaferme 299, although not a cryotolerant strain, indicating a cryoprotective effect of cork. The presence of cork did not affect the composition of major volatiles with methanol and acetaldehyde kept at low levels. Reduction of amyl alcohols on total volatiles was also observed in wines fermented at low temperatures. Differences in the headspace aroma profile in wines produced by immobilized and free cells were found by GC–MS analysis, but no cork taint compounds were detected.     

High-temperature wine making using the thermotolerant yeast strain Kluyveromyces marxianus IMB3

Kluyveromyces marxianus IMB3 yeast cells were immobilized on delignified cellulosic material, apple, and quince separately. Both immobilized and free cells were used in high-temperature wine making, and their fermented grape must contained 3 to 4% alcohol. Semisweet wines were produced by the addition of potable alcohol to the fermented must. Preliminary sensory evaluation of the produced semisweet wines showed good flavor and aroma. The final product contained extremely low levels of higher and amyl alcohols while ethyl acetate was at levels usually present in wines. The ferment produced may be blended with other products to improve their quality.     

Volatiles Formation from Grape Must Fermentation Using a Cryophilic and Thermotolerant Yeast

Grape must fermentation performance and volatiles formation by simultaneously cryophilic and thermotolerant yeast (strain AXAZ-1), isolated from grapes in Greece, was evaluated in a wide temperature range (5?C40°C). Yeast strain was immobilized on brewer??s spent grains (BSG) and the formed biocatalyst was introduced into a Multi-Stage Fixed Bed Tower (MFBT) bioreactor. Almost complete sugar utilization from the aforementioned biocatalyst was observed in a wide temperature spectrum, ranging from 5?°C to 37?°C, while at 40?°C residual sugar was up to 29?g/l. Time to complete fermentation with the immobilized yeast ranged from 290?h at 5?°C and 120?h at 40?°C to 25?h at 33?°C. The daily ethanol productivity reached maximum (88.6?g/l) and minimum (5.6?g/l) levels at 33?°C and 5?°C, respectively. The aroma-related compounds?? profiles of immobilized cells at different fermentation temperatures were evaluated by using solid phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS). Must fermentation resulted in a high-quality fermentation product due to the low concentrations of higher and amyl alcohols at all temperatures tested. AXAZ-1 is a very promising strain for quality wine production, as it is capable of performing fermentations of high ethanol concentration and productivities in both low and high temperatures.     

Freeze-Dried Saccharomyces cerevisiae Cells Immobilized on Potato Pieces for Low-Temperature Winemaking

A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on potato pieces. This biocatalyst was subjected to freeze-drying, and the effect of several protective agents and storage at 5 °C, up to 9 months, on viability and fermentative activity of yeasts cells were studied. From several protective agents tested, sodium glutamate preserved the viability of immobilized yeast cells at high levels even after 9-month storage. The freeze-drying biocatalyst was used for repeated batch fermentations of grape must at low temperatures until 5 °C. The produced wines analyzed for volatile byproducts by GC and GC/MS and the results showed that the freeze-dried biocatalysts, with sodium glutamate as protectant, produced wines with higher formation of esters than free cells and having at least similar aromatic profile to those produced by wet biocatalysts.     

Ethanol production from pretreated olive tree wood and sunflower stalks by an SSF process

Olive tree wood and sunflower stalks are agricultural residues largely available at low cost in Mediterranean countries. As renewable lignocellulosic materials, their bioconversion may allow both obtaining a value-added product, for fuel ethanol, and facilitating their elimination. In this work, the ethanol production from olive tree wood and sunflower stalks by a simultaneous saccharification and fermentation (SSF) process is studied. As a pretreatment, steam explosion at different temperatures was applied. The water insoluble fractions of steam-pretreated sunflower stalks and steamed, delignified olive tree wood were used as substrates at 10% w/v concentration for an SSF process by a cellulolytic commercial complex and Saccharomyces cerevisiae. After 72-h fermentation, ethanol concentrations up to 30 g/L were obtained in delignified steam-pretreated olive tree wood at 230°C and 5 min. Sunflower stalks pretretated at 220°C and 5 min gave maximum ethanol concentrations of 21 g/L in SSF experiments.     

纤维素酶降解纤维素机理的研究

纤维素是自然界中含量最多的一类碳水化合物,同时它也是地球上数量最大的可再生资源。纤维素酶是一种高活性生物催化剂,在纤维素类资源的利用方面发挥重要的作用。本文综述了纤维素、纤维素酶的分子结构和纤维素酶对纤维素的降解机理,影响酶解的主要因素以及提高酶解效率的主要措施,并对纤维素酶研究存在的问题以及今后的发展作了进一步展望。     

纤维素乙醇产业化

由于能发挥缓解能源紧张、减少环境污染、促进农村发展等重要作用,利用年产量巨大的植物纤维资源,生产可再生性液体替代燃料乙醇的技术受到了巨大的关注,成为工业生物技术的研究热点。酶法生产纤维素乙醇面临多种困难:纤维素原料比重轻,收集运输不便;原料结构复杂,需要深度预处理;纤维素酶系的酶解效率有待提高;半纤维素中的木糖难以发酵转化为乙醇等。经过多年研究,新技术已经取得重大进展,开始接近实用化。紧迫的社会需求正在迫使国内外政府和企业界大量投资,开展纤维素乙醇的中试研究和试生产,力求在短时期内克服上述难点,尽快实现产业化。充分利用植物纤维资源中的多种组分,联合生产乙醇和部分高值产品的生物精练技术,是实现纤维素乙醇产业化的重要突破口和必然途径。玉米芯生物精练生产乙醇和木糖相关产品的技术正在进行产业化。本文综述了纤维素乙醇产业化的研究进展并做了展望。     

Lactic acid production from cellulosic material by synergetic hydrolysis and fermentation

The hydrolysis process on corncob residue was catalyzed synergetically by the cellulase from Trichoderma reesei and the immobilized cellobiase. The feedback inhibition to cellulase reaction caused by the accumulation of cellobiose was eliminated efficiently. The hydrolysis yield of corncob residue was 82.5%, and the percentage of glucose in the reducing sugar reached 88.2%. The glucose in the cellulosic hydrolysate could be converted into lactic acid effectively by the immobilized cells of Lactobacillus delbrueckii. When the enzymatic hydrolysis of cellulose and the fermentation of lactic acid were coupled together, no glucose was accumulated in the reaction system, and the feedback inhibition caused by glucose was also eliminated. Under the batch process of synergetic hydrolysis and lactic acid fermentation with 100 g/L of cellulosic substrate, the conversion efficiency of lactic acid from cellulose and the productivity of lactic acid reached 92.4% and 0.938 g/(L·h), respectively. By using a fed-batch technique, the total concentration of cellulosic substrate and lactic acid in the synergetic process increased to 200 and 107.5 g/L, respectively, whereas the dosage of cellulase reduced from 20 to 15 IU/g of substrate in the batch process. The results of the bioconversion of renewable cellulosic resources were significant.     

Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates

The Department of Energy’s Office of the Biomass Program has set goals of making ethanol cost competitive by 2012 and replacing 30% of 2004 transportation supply with biofuels by 2030. Both goals require improvements in conversions of cellulosic biomass to sugars as well as improvements in fermentation rates and yields. Current best pretreatment processes are reasonably efficient at making the cellulose/hemicellulose/lignin matrix amenable to enzymatic hydrolysis and fermentation, but they release a number of toxic compounds into the hydrolysate which inhibit the growth and ethanol productivity of fermentation organisms. Conditioning methods designed to reduce the toxicity of hydrolysates are effective, but add to process costs and tend to reduce sugar yields, thus adding significantly to the final cost of production. Reducing the cost of cellulosic ethanol production will likely require enhanced understanding of the source and mode of action of hydrolysate toxic compounds, the means by which some organisms resist the actions of these compounds, and the methodology and mechanisms for conditioning hydrolysate to reduce toxicity. This review will provide an update on the state of knowledge in these areas and can provide insights useful for the crafting of hypotheses for improvements in pretreatment, conditioning, and fermentation organisms.     

Organic Phase Synthesis of Ethyl Oleate Using Lipases Produced by Solid-state Fermentation

This paper reports a study of the enzymatic esterification of oleic acid and ethanol. The reaction was catalyzed by lipases produced by solid-state fermentation with Rhizopus sp. Olive oil and perlite were used as an inducer and inert support, respectively. Synthesis of ethyl oleate was carried out in a 10-mL batch reactor with magnetic stirring. The effects of substrate ratios, biocatalyst concentration, and temperature on the reaction rate and conversion efficiency were evaluated. The highest reaction rate (1.64 mmol/L min) was reached with an oleic acid/ethanol mol ratio of 1:5 (oleic acid 50 mM:ethanol 250 mM) and 1 g of biocatalyst. Conversions approaching 100% were obtained after 60 min of reaction at 45 degrees C with n-hexane as a solvent. The initial reaction rate increased proportionally with respect to biocatalyst concentration, which suggests that the reaction rate was not controlled by mass transfer. The biocatalyst retained more than 80% of its catalytic activity after 7 months of storage at 4 degrees C. The results demonstrate that the biocatalyst produced by Rhizopus sp. in solid-state fermentation can be successfully used for ethyl oleate synthesis over short reaction periods under conditions when ethanol is in excess.     

Pilot-Scale Fermentation of Aqueous-Ammonia-Soaked Switchgrass

Aqueous-ammonia-steeped switchgrass was subject to simultaneous saccharification and fermentation (SSF) in two pilot-scale bioreactors (50- and 350-L working volume). Switchgrass was pretreated by soaking in ammonium hydroxide (30%) with solid to liquid ratio of 5 L ammonium hydroxide per kilogram dry switchgrass for 5 days in 75-L steeping vessels without agitation at ambient temperatures (15 to 33 °C). SSF of the pretreated biomass was carried out using Saccharomyces cerevisiae (D₅A) at approximately 2% glucan and 77 filter paper units per gram cellulose enzyme loading (Spezyme CP). The 50-L fermentation was carried out aseptically, whereas the 350-L fermentation was semiaseptic. The percentage of maximum theoretical ethanol yields achieved was 73% in the 50-L reactor and 52–74% in the 350-L reactor due to the difference in asepsis. The 350-L fermentation was contaminated by acid-producing bacteria (lactic and acetic acid concentrations approaching 10 g/L), and this resulted in lower ethanol production. Despite this problem, the pilot-scale SSF of aqueous-ammonia-pretreated switchgrass has shown promising results similar to laboratory-scale experiments. This work demonstrates challenges in pilot-scale fermentations with material handling, aseptic conditions, and bacterial contamination for cellulosic fermentations to biofuels.     

Development of continuous fermentation using immobilized yeast cells for wine cooler process development

The continuous wine fermentation process, which employs a newly designed tapered column type bioreactor and immobilized yeast cells (Montrachet 522), was studied and its fermentation performance was compared with batch and suspended cell continuous wine fermentation systems. It was found that a stable continuous culture fermentation process could be maintained for a period of 2–3 mo when the new bioreactor system packed with immobilized yeast cells was employed. The new bioreactor containing immobilized yeast cells performed significantly better than the suspended cell culture system or batch culture. The effluent wine from the continuous fermentor system contained 7.1% (v/v) ethanol and 0.18% (w/v) residual sugar at 0.01 h^-1 dilution rate. The new continuous bioreactor system also gave 17–34 times higher maximum ethanol productivity compared to the conventional batch wine fermentation. At a low dilution rate, 0.01^-1, as high as 92% sugar to ethanol yield was achieved. Based on the results obtained from this study, the possibility of developing a continuous wine cooler fermentation process was demonstrated. A two-stage continuous wine fermentation system may be designed and operated. The grape juice can be fed into the first-stage that is operated at about 0.2 h^-1 dilution rate and the effluent from the first-stage is fed into the second-stage continuous fermentor operated at about 0.01 h^-1 dilution rate. By doing so, a wine cooler can be produced continuously and efficiently, by employing the newly designed tapered column type bioreactor charged with the immobilized yeast cells.     

Reinforced absorbent material: a cellulosic composite of TEMPO-oxidized MFC and CTMP fibres

This research aims to develop new materials based on renewable resources that can fulfill the functions necessary in the absorption core of a disposable diaper. Absorbent foam was recently produced from softwood kraft pulp by TEMPO oxidation, disintegration and freeze drying. In this study, the TEMPO-oxidized MFC was mixed with pulp fibres, thus forming a cellulosic composite, in an attempt to improve the mechanical stability of the freeze-dried absorbent material. The fibres were added in different amounts and the freeze-dried materials were evaluated for their absorption and retention properties. The results of this study suggest that the composite material has a better mechanical stability than the absorbent foam without fibres. It was shown that using spruce CTMP fibres in the composite resulted in better absorption and retention capacities than in a composite with softwood kraft pulp fibres. The higher stiffness of the CTMP fibres is a probable explanation for this difference. For the composite material with CTMP fibres, liquid porosimetry showed that pore size distribution was more or less retained when put under load. Furthermore, it was seen that the retention properties reached a maximum around 85 % CTMP fibres and 15 % TEMPO-oxidized MFC. In the centrifuge retention test, the retention of the TEMPO-oxidized MFC in the composite material reached about the same capacity as conventional superabsorbent polymers.     

Characteristics of black zones associated with delignified wood

Several white-rot fungi cause two micromorphologically distinct types of decay. White-rot fungi typically cause a simultaneous removal of all cell wall components in close proximity to fungal hyphae. This type of degradation results in erosion troughs and holes in the cell walls. In addition, a selective removal of lignin and hemicellulose can occur intermittently throughout the decayed wood. Selectively delignified wood can be characterized by the complete removal of middle lamellae, resulting in a defibration of cells and exposure of cellulosic macrofibrils within cell walls. A common observation of decay in the field is the association of dark zones with selectively delignified wood. Swollen hyphae containing pigmented materials were commonly associated with the dark zones in wood delignified byHeterobasidion annosum, Ganoderma applanatum, G. tsugae, Ischnoderma resinosum, Perenniporia medulla-panis, orDichomitus squalens. Dark zones examined using scanning electron microscopy in conjunction with energy dispersive X-ray analysis were found to contain high concentrations of manganese. Multielement analyses using inductively coupled plasma atomic emission spectrometry indicated approximately a 50-fold increase of Mn in black zones. Pigmented substances containing Mn were readily decolorized with acids. Since large concentrations of Mn were only found in selectively delignified wood, manganese may influence the selective degradation of lignin.     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

Isolation and Identification of Residual Chromophores in Cellulosic Materials

A general procedure was developed for the isolation of residual chromophores in or on cellulosic material, which were hitherto inaccessible to structure elucidation due to their extremely low content in the ppb concentration scale. It is applicable to cellulosic pulp, cellulosic fibers (viscose, Lyocell) and cellulose derivatives (acetate, carbonyl-labeled cellulose) as well. The chromophore identification comprises treatment of the cellulosic material with boron trifluoride – acetic acid complex (BF₃*2HOAc) containing sulfite, chromatographic separation of the resulting chromophore-containing mixture, and structure determination of the main constituents by NMR / MS and comparison to authentic samples. Both adsorbed and covalently bound aromatic and quinoid compounds are selectively released by the treatment. Covalent ester, ether and secondary alkyl links between chromophore and cellulose are broken. Two cellulosic example substrates have been analyzed for their chromophore content: Lyocell fibers and non-bleached viscose fibers, and up to eleven chromophores per sample have been identified.     

Monolithic macroporous albumin/chitosan cryogel structure: a new matrix for enzyme immobilization

A novel monolithic macroporous material was developed by cross-linking hen egg albumin (HEA) and chitosan with glutaraldehyde at subzero temperatures. A macroporous cryogel structure allowed efficient mass transport of solutes within the material. In one application, albumin was partially replaced with active enzymes (glucose oxidase and horseradish peroxidase) resulting in the production of macroporous biocatalyst preparations suitable for flow-injection analysis of glucose in the low millimolar range. In another application, the proteolytic enzymes savinase and esperase were coupled to the macroporous structure via free amino groups on the pore walls using glutaraldehyde as cross-linker/spacer agent. The low hydraulic resistance of the matrix allowed for the development of a generic, high-performance online protein digestion system utilizing the wall-bound proteases.