Zsolt Szengyel

Yields from Glucose, Xylose, and Paper Sludge Hydrolysate During Hydrogen Production by the Extreme Thermophile Caldicellulosiruptor saccharolyticus

This study addressed the utilization of an industrial waste stream, paper sludge, as a renewable cheap feedstock for the fermentative production of hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus. Hydrogen, acetate, and lactate were produced in medium in which paper sludge hydrolysate was added as the sole carbon and energy source and in control medium with the same concentration of analytical grade glucose and xylose. The hydrogen yield was dependent on lactate formation and varied between 50 and 94% of the theoretical maximum. The carbon balance in the medium with glucose and xylose was virtually 100%. The carbon balance was not complete in the paper sludge medium because the measurement of biomass was impaired owing to interfering components in the paper sludge hydrolysate. Nevertheless, >85% of the carbon could be accounted for in the products acetate and lactate. The maximal volumetric hydrogen production rate was 5 to 6 mmol/(L·h), which was lower than the production rate in media with glucose, xylose, or a combination of these sugars (9–11 mmol/[L·h]). The reduced hydrogen production rate suggests the presence of inhibiting components in paper sludge hydrolysate.

Simultaneous detoxification and enzyme production of hemicellulose hydrolysates obtained after steam pretreatment

In the process of making ethanol from lignocellulosic materials, compounds inhibitory to microorganisms are generated during steam pretreatment of the wood. Water-soluble inhibitors and pentoses are liberated and washed from the cellulose structure which is further enzymatically hydrolyzed. To make the process economically feasible, the pentoses have to be fermented to ethanol. A major drawback with the pentose-fermenting organisms that have been suggested for this purpose is that they do not tolerate an inhibitory environment and therefore, the pentose stream has to be detoxified prior to fermentation. An alternative use of the hemicellulose hydrolysate obtained after steam-pretreatment of willow is to use it for enzyme production by the cellulolytic fungus Trichoderma reesei. The sugars in the pentose fraction are almost completely utilized, and simultaneously the hemicellulose hydrolysate is detoxified and can be recirculated in the process to minimize the need for freshwater.

Design and operation of a bench-scale process development unit for the production of ethanol from lignocellulosics

A bench-scale unit for the development of an enzymatic process for the bioconversion of lignocellulosics to ethanol has been used to study the recycling of waste-water streams to minimize fresh-water requirements and reduce effluent streams. Willow, after impregnation with sulphur dioxide, was steam-pretreated, enzymatically hydrolysed, and the sugars produced were fermented using S. cerevisiae. The fermentation broth was distilled and the stillage was fractionated by evaporation into six separate condensate fractions and a residue. The overall yield of ethanol from willow was 65% of the theoretical yield based on total fermentable sugars. The inhibitory effect of the evaporation condensates was assessed by fermentation using S. cerevisiae. The non-volatile residue of the stillage was found to be inhibitory to fermentation. The ethanol yield decreased from 0.37 g/g in a pure sugar reference to 0.31 g/g in the residue and the average ethanol fermentation rate decreased fi om 6.3 g/(l h) to 2.7 g/(l h), respectively. The evaporation condensates, containing the volatile components, showed no negative effects on fermentation. The intermediate evaporation condensate fractions, fractions 4 and 5, had the lowest chemical oxygen demand (GOD), 1560 and 1120 mg/l, compared with 33 300 mg/l for the stillage. Therefore, these fractions can be released directly into the effluent without further treatment. Copyright (C) 1997 Elsevier Science Ltd. (Less)

Cellulase production by T. reesei

Steam-pretreated willow has been found to be a suitable substrate for ethanol production in Sweden. The production of Trichoderma reesei cellulases on steam-pretreated willow (SPW) of varying cellulose content has been studied. Batch fermentations were performed in shake flasks, and in 4- and 22-l fermenters. SPW was found to be a suitable carbon source for cellulase production. At an initial solids concentration corresponding to 10 g/l cellulose, the yield was 108 filter paper units (FPU)/g cellulose after 3 days in the 22-l fermenter. However, the yield was below 100 FPU/g cellulose in the shake flasks and 4-l fermenter. The solid residue of enzymatically hydrolysed SPW was found to be nearly as good a substrate as SPW, although the cellulose content was as low as 20% in this hydrolysis residue. Using delignified SPW, the yield of cellulases was low, indicating that lignin does not inhibit cellulase production. By applying a pressure of 1.7 bar in the fermenter, the initial fermentation rate was increased. The productivity was increased from 12.2 FPU/l/h to 16.7 FPU/l/h, due to the higher level of dissolved oxygen at the higher pressure.

Chemical pretreatments of corn stover for enhancing enzymatic digestibility

Corn stover, the most abundant agricultural residue in Hungary, is a potential raw material for the production of fuel ethanol as a result of its high content of carbohydrates, but a pretreatment is required for its efficient hydrolysis. In this article, we describe the results using various chemicals such as dilute H2SO4, HCl, and NaOH separately as well as consecutively under relative mild conditions (120°C, 1h). Pretreatment with 5% H2SO4 or 5% HCl solubilized 85% of the hemicellulose fraction, but the enzymatic conversion of pretreated materials increased only two times compared to the untreated corn stover. Applying acidic pretreatment following a 1-d soaking in base achieved enzymatic conversion that was nearly the theoretical maximum (95.7%). Pretreatment with 10% NaOH decreased the lignin fraction >95%, increased the enzymatic conversion more than four times, and gave a 79.4% enzymatic conversion. However, by increasing the reaction time, the enzymatic degradability could also be increased significantly, using a less concentrated base. When the time of pretreatment was increased three times (0.5% NaOH at 120°C), the amount of total released sugars was 47.9 g from 100 g (dry matter) of untreated corn stover.

Cellulase production of Trichoderma reesei Rut C 30 using steam-pretreated spruce - Hydrolytic potential of cellulases on different substrates

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hem icellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and logen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C30. However, the conversion of cellulose using these enzymes was higher than that obtained with logen or Celluclast cellulases using steam-pretreated spruce as substrate.

Effect of pH on cellulase production of Trichoderma reesei RUT C30

Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, β-glucosidase production was increased greatly.

Ethanol fermentation of various pretreated and hydrolyzed substrates at low initial pH.

Lignocellulosic materials represent an abundant feedstock for bioethanol production. Because of their complex structure pretreatment is necessary to make it accessible for enzymatic attack. Steam pretreatment with or without acid catalysts seems to be one of the most promising techniques, which has already been applied for large variety of lignocellulosics in order to improve enzymatic digestibility. During this process a range of toxic compounds (lignin and sugar degradation products) are formed which inhibit ethanol fermentation. In this study, the toxicity of hemicellulose hydrolysates obtained in the steam pretreatment of spruce, willow, and corn stover were investigated in ethanol fermentation tests using a yeast strain, which has been previously reported to have a resistance to inhibitory compounds generated during steam pretreatment. To overcome bacterial contamination, fermentations were carried out at low initial pH. The fermentability of hemicellulose hydrolysates of pretreated lignocellulosic substrates at low pH gave promising results with the economically profitable final 5 vol% ethanol concentration corresponding to 85% of theoretical. Adaptation experiments have shown that inhibitor tolerance of yeast strain can be improved by subsequent transfer of the yeast to inhibitory medium.

Effect of acetic acid and furfural on cellulase production of Trichoderma reesei RUT C30

Because of the high temperature applied in the steam pretreatment of lignocellulosic materials, different types of inhibiting degradation products of saccharides and lignin, such as acetic acid and furfural, are formed. The main objective of the present study was to examine the effect of acetic acid and furfural on the cellulase production of a filamentous fungus Trichoderma reesei RUT C30, which is known to be one of the best cellulase-producing strains. Mandels’s mineral medium, supplemented with steam-pretreated willow as the carbon source at a concentration corresponding to 10 g/L of carbohydrate, was used. Four different concentration levels of acetic acid (0–3.0 g/L) and furfural (0–1.2 g/L) were applied alone as well as in certain combinations. Two enzyme activities, cellulase and β-glucosidase, were measured. The highest cellulase activity obtained after a 7-d incubation was 1.55 FPU/mL with 1.0 g/L of acetic acid and 0.8 g/L of furfural added to the medium. This was 17% higher than that obtained without acetic acid and furfural. Furthermore, the results showed that acetic acid alone did not influence the cellulase activity even at the highest concentration. However, β-glucosidase activity was increased with increasing acetic acid concentration. Furfural proved to be an inhibiting agent causing a significant decrease in both cellulase and β-glucosidase production.

Hydrogen production from paper sludge hydrolysate

The main objective of this study was to develop a system for the production of “renewable” hydrogen. Paper sludge is a solid industrial waste yielding mainly cellulose, which can be used, after hydrolysis, as a feedstock in anaerobic fermentation by (hyper)thermophilic organisms, such as Thermotoga elfii and Caldicellulosiruptor saccharolyticus. Tests on different medium compositions showed that both bacteria were able to produce hydrogen from paper sludge hydrolysate, but the amount of produced hydrogen and the requirement for other components differed. Hydrogen production by T. elfii strongly depended on the presence of yeast extract and salts. By contrast, C. saccharolyticus was less dependent on medium components but seemed to be inhibited by a component present in the sludge hydrolysate. Utilization of xylose was preferred over glucose by C. saccharolyticus.