Band 2, Heft 1

Efficient starch saccharification is an essential step towards achieving improved ethanol yields by fermentation. Sorghum grains are important starch sources for bioconversion to ethanol. In the present study, disease degraded (spoilt) husked grains from Nigerian sorghum cultivars were obtained from field sites and subjected to bioprocessing to ethanol. The crude husked grains (comprising husks, spikelet, awn, rachis and pubescence materials) were hammer milled and each meal separately mashed with enzyme cocktails comprising amylase, glucanase and protease enzymes. The saccharified worts obtained were then fermented with the yeasts, Saccharomyces cerevisiae and Pichia stipitis (aka Scheffersomyces stipitis), without exogenous nutrient supplementation. Sugars liberated during mashing were determined and it was found that enzymatic hydrolysis of milled sorghum grains was effective in yielding favourable levels of fermentable sugars up to 70g sugar/100g substrate with one particular cultivar (KSV8). Ethanol and carbon dioxide production was measured from subsequent trial fermentations of the sorghum mash and it was found that S. cerevisiae produced ethanol levels equating to 420 L/t that compares very favourably with yields from wheat and barley. Our findings show that crude degraded sorghum grains represent favourable low-cost feedstocks for bioconversion to ethanol with reduced energy input and without additional costs for nutrient supplementation during fermentation. Consequently, our results suggest some economic benefits could be derived from spoilt or degraded sorghum grains.

Paulownia elongata is a short-rotation fast growing tree and is known for high biomass accumulation and carbon sequestration potential. Optimization of protocols for nucleic acid extraction, PCR, RT-PCR, and other molecular biology techniques are required for better understanding of cellulose synthesis and to assess the potential of Paulownia as a biofuel tree. The main objective of this work was to study a putative cellulose synthase amplicon expression under various environmental conditions and evaluate the potentials of Paulownia as a biofuel tree. Using cross-species PCR an amplicon representative of a putative cellulose synthase gene from Paulownia was identified. This 177-bp long DNA sequence was 46% similar with cellulose synthase genes from Arabidopsis as expected. Gene specific primers for this particular Paulownia cellulose synthase gene were designed and reverse transcription PCR was performed to confirm its transcription. We report an inexpensive cDNA dot-blot method to study expression of this gene under various environmental conditions. We observed that cold and, to a lesser extent, heat stress downregulated its expression. This information will help to understand cellulose deposition in plant cell wall under stressful conditions. To the best of our knowledge this is the first characterization of a cDNA sequence from Paulownia elongata.

Fuel ethanol is an environmentally friendly alternative liquid fuel to the widely used petroleum derived transportation liquid fuels. Since 2007, worldwide fuel ethanol production has increased. Currently ethanol is primarily produced from carbohydrates such as sucrose and starch by fermentation using the yeast Saccharomyces cerevisiae. In this work, new approaches for the selection of S. cerevisiae strains with increased ethanol production from hydrolyzed corn meal are described. An industrial production strain of Saccharomyces cerevisiae AS400 was subjected to positive selection of mutants resistant to toxic concentrations of oxythiamine, trehalose, 3-bromopyruvate, glyoxylic acid, and glucosamine. The selected mutants are characterized by 5-8% increase in ethanol yield (g g-1 of consumed glucose) as compared to the parental industrial ethanol-producing strain. A three-step selection approach that consisted of the use of glyoxylic acid, glucosamine and bromopyruvate resulted in a 12% increase in ethanol yield during fermentation on industrial media. These results indicate that the selected strains are promising candidates for industrial ethanol production.

Bioconversion of lignocellulosic materials into ethanol requires an intermediate pretreatment step for conditioning biomass. Sugar yields from wheat straw were previously improved by the addition of a mild alkali pretreatment step before bioconversion by the white-rot fungus Irpex lacteus. In this work, an alternative alkaline treatment, which significantly reduces water consumption, was implemented and optimized. Sugar recovery increased 117% with respect to the previously developed alkaline wash process at optimal process conditions (30°C, 30 minutes and 35.7% (w/w) of NaOH). In order to further reduce operational costs, a system for alkali recycling was implemented. This resulted in the treatment of 150% more wheat straw using the same amount of NaOH. Finally, enzymatic hydrolysis was optimized and resulted in a reduction of enzyme dose of 33%.

The search for new ethanol production technologies is due to this biofuel being a renewable and environmentally friendly option. Immobilized cell systems for ethanol production have been studied; however, the phenomenon involved in cell sorption on raw materials has been poorly explored. Therefore, this work evaluates P. stipitis immobilization on sugarcane bagasse pretreated with sulphuric acid, as well as ethanol production in batch culture. The results obtained showed that the Guggenheim-Anderson-de Boer (GAB) model explained the sorption phenomenon. The selected inoculum size for immobilization was the same as the monolayer sorption capability (1.17 gl-1). Using 1:100 g ml- 1 solid-liquid ratio, at 250 rpm, ethanol yield and productivity of 0.404 gg-1 glucose and 0.41 gl-1h-1 were obtained, respectively. The immobilized systems were stable for up to twenty-five repeated batches (36 h each). Ethanol production was increased from the first to the twenty-fifth batch (18.1 and 24.7 gl-1 ethanol). The use of complex media, such as molasses “B” or sugarcane hydrolyzates, caused an increase in process efficiency 2.4 and 1.8-fold respectively, compared with free cells systems. Biotechnological ethanol production from lignocellulosic hydrolyzates could be improved by the use of the immobilization cell sorption on pre-treated raw materials.

Olive stones are the main solid byproducts obtained from olive oil production and from table olives production. As a lignocellulosic material, the use of olive stones for ethanol and other chemicals production has been proposed, particularly under the biorefinery concept. As part of such a process, this work deals with the fractionation of the lignocellulosic material by dilute acid autoclave pretreatment at 2% sulfuric acid, 130°C, 60 min and 1:1 liquid to solid ratio. Moreover, the work addresses the fermentation of the liquors obtained after pretreatment. The released sugars are composed mainly by xylose and other hemicellulosic sugars. The fermentation performance of three xylose-fermenting microorganisms, e.g. two Escherichia coli species and Scheffersomyces stipitis, are compared. The study analyzes in a first step the microorganism behavior on synthetic liquors, with a similar composition to that of the real liquors. Finally, and taken into account the results from the previous steps, the real liquor obtained from olive stones pretreatment is fermented. Results show that E. coli MM160 is the best ethanol producer out of the three microorganisms studied. Globally, the pretreatment produced a liquor containing 140 g hemicellulosic sugars/l and requiring firstly dilution by 50% and a detoxification step by overliming. The fermentation of this liquor by E. coli MM160 results in a 25 g ethanol/l solution equivalent to 50 g ethanol/kg olive stone, in spite of 20 g acetic acid/l also present. These results confirm both olive stones and E. coli MM160 as promising feedstock and microorganism for ethanol production.

Development of biofuels such as lignocellulosic ethanol represents a sustainable alternative in the transport sector. Wheat straw is a promising feedstock for bioethanol production in Europe due to its large production and high carbohydrates content. In a process to produce cellulosic ethanol, previous to the enzymatic hydrolysis to obtain fermentable sugars and the subsequent fermentation, a pretreatment step to break down the recalcitrance of lignocellulose fiber is essential. In this work, a range of steam explosion pretreatment conditions were evaluated according to different parameters: sugars recovery, degradation products generation, and enzymatic hydrolysis yields. Moreover, the enzymatic hydrolysis process was also studied at high substrate loadings, since operating at high solids loading is crucial for large scale development of ethanol production. Pretreatment at 200°C - 10 min resulted in higher enzymatic hydrolysis yield (91.7%) and overall glucose yields (35.4 g glucose/100 g wheat straw) but also higher production of toxic compound. In turn, the characteristics of the pretreated wheat straw at lower severity (Log R0=3.65) correspond to 190°C and 10 min, with minimal sugars degradation and toxics formation indicated a great potential for maximizing total sugars production by using optimal enzyme combinations including accessory enzymes in the enzymatic hydrolysis step.

In this work, the ethanol dehydration production process is carried out using the Mathematical Modeling Pressure Adsorption Process. A new model is suggested, it has two equalization steps, and is compared with the Industrial Pressure Swing Process operating cycle. An analysis of the effects of introducing the pressure equalization step is performed on four main response variables: purity, production, recovery and energy consumption and it is compared with the current cycle configuration operating in the industry. We used Aspen Adsorption for the valuation and simulation of the cyclic PSA process. We analyzed and processed the simulation results in Statgraphics Centurion to obtain optimum operating conditions for the process. This evaluation shows that purity decreases slightly, whereas recovery and production increase. The most important thing is that the energy consumption is reduced. These results clearly show that by modifying the operating cycle schema, optimum operating conditions also change. The optimization of the new cycle was executed considering as variables bed pressure, adsorption time and purging flow. We found that a smaller column is more productive for the equalization cycle than that of a 14m bed, which is optimal in the industrial cycle with a consequent reduction in adsorbent material.

The high cost of cellulolytic enzymes used in the ethanol production process has led to a growing interest in situ production. The evaluation of the influence of several factors in the fungus Penicillium sp. cellulase production using pretreated sugarcane bagasse is very interesting. Penicillium sp. cellulase production by using filter paper as cellulosic substrate and the use of glucose, sucrose and lactose like co-substrates was assessed. In the experiments using filter paper as a cellulosic substrate, the highest FPase enzyme activity obtained was 280 FPU.L-1 using sucrose as co-substrate. Subsequently, the study of pretreated sugarcane bagasse was conducted using Plackett-Burman experimental design with analysis of 6 factors influencing the process. The highest FPase activity was 615.1 FPU.L-1. The factors influencing FPase and β- glucosidase activity were the use of molasses and the solid loading. The successful use of molasses as co-substrate opens perspectives for future researches.

Pre-treatment is a process that releases simple sugars from complex lignocellulosic biomass by using chemicals like acid and alkali which are one of the simplest and cost effective techniques. In this study, the conditions for sulphuric acid and sodium hydroxide pretreatment of de-oiled oil seed cake (DOSC) were optimized by response surface methodology (RSM). The levels of factors (DOSC concentration, agitation speed, sulphuric acid (H2SO4), sodium hydroxide (NaOH) concentration and reaction time) that affect release of reducing sugars by pre-treatment were obtained by one factor at a time (OFAT) approach of which only H2SO4 concentration, NaOH concentration and reaction time showed significance. The levels of factors were optimized by central composite design. Optimized conditions were found to be 11.65% (v/v) of H2SO4 concentration at 1.28h and, 4 N of NaOH at 3.7 h for acid and alkali hydrolysis respectively. Under optimized conditions, the release of reducing sugars was found to be 0.69 g/L (41.36 mg RRS/ g cellulose) and 0.40 g/L (23.98 mg RRS/ g cellulose) for acid hydrolysis and alkali hydrolysis of DOSC, respectively. Hence, RSM was found to be an efficient technique to optimize the hydrolysis process and ensure maximum release of reducing sugars.

Residues from olive oil industry such as Extracted Olive Pomace (EOP) are potential substrates for bioethanol production. In this work, enzymatic hydrolysis of EOP pretreated by dilute acid hydrolysis (DAH) was assessed, and the enzymatic hydrolysis and bioconversion were carried out both by separate hydrolysis and fermentation (SHF) and pre-saccharification followed by simultaneous saccharification and fermentation (PSSF). DAH led to a significant removal hemicellulose, but the subsequent enzymatic treatments showed that the resulting residue was still partially recalcitrant to cellulase hydrolysis. Size reduction and further treatment of EOP-DAH with an alkaline solution were also tested. Alkaline post-treatment allowed a decrease in lignin content, but had little effect on enzymatic saccharification comparing to size reduction. Hence fermentation study was performed with ground EOP-DAH. The PSSF process showed a relatively higher bioethanol fermentation yield (0.46 gg-1) when compared to the SHF process.

The economics for production of secondgeneration (2G) ethanol from sugarcane bagasse in large scale, competing with the cogeneration of electric energy, is still not consolidated. In this scenario, the key for feasibility may be the biorefinery concept, a multiproduct industry using biomass fractions to produce energy, chemicals and by-products. Xylooligosaccharides (XOS) are oligomers of xylose often used as additives in food, animal feeds, and drugs. The effect of NaOH pretreatment on the recovery of xylan for XOS production from sugarcane bagasse under different conditions, namely 121°C, 4-7% NaOH loading, was investigated. The best condition was 4% NaOH and 60 min of reaction, achieving 55% of xylan extraction, without monomer production. In order to produce XOS, soluble and immobilized xylanases were used to hydrolyze commercial birchwood xylan (as control) and the sugarcane bagasse xylan. The immobilized endoxylanase produced XOS with 37% of xylobiose and 20% of xylotriose (w/w). The small production of xylose clearly indicated the purity of the xylan extracted from sugarcane bagasse. The biocatalyst had more than 90% of its activity preserved after 5 reaction cycles. The results showed the suitability of sugarcane bagasse as a raw material for production of ethanol and of XOS using immobilized xylanase.

The addition of laccase enzymes reduces the amount of phenols present in lignocellulosic pretreated materials and increases their fermentability. However, laccase addition in combination with cellulases reduces hydrolysis yields. In this work, hybrid hydrolysis and fermentation (HHF) configuration allowed overcoming the negative effect of laccase treatment on enzymatic hydrolysis. Furthermore, the effects of different laccase dosages, length of detoxification time and inoculum size on ethanol production were evaluated. In the evaluated configurations, the different laccase dosages did not show any significant effect on enzymatic hydrolysis. The lowest laccase dosage (0.5 IU/g DW) removed ~70% of total phenols which was enough to reach the highest ethanol production yields (~10 g/L) using K. marxianus CECT 10875. Shorter detoxification times and larger inoculum sizes had a positive impact on both ethanol production and volumetric productivity. These optimal detoxification conditions enable the fermentation of inhibitory slurries by reducing the overall time and cost of the process.

The use of macroalgae (seaweed) as a potential source of biofuels has attracted considerable worldwide interest. Since brown algae, especially the giant kelp, grow very rapidly and contain considerable amounts of polysaccharides, coupled with low lignin content, they represent attractive candidates for bioconversion to ethanol through yeast fermentation processes. In the current study, powdered dried seaweeds (Ascophylum nodosum and Laminaria digitata) were pre-treated with dilute sulphuric acid and hydrolysed with commercially available enzymes to liberate fermentable sugars. Higher sugar concentrations were obtained from L. digitata compared with A. nodosum with glucose and rhamnose being the predominant sugars, respectively, liberated from these seaweeds. Fermentation of the resultant seaweed sugars was performed using two non-conventional yeast strains: Scheffersomyces (Pichia) stipitis and Kluyveromyces marxianus based on their abilities to utilise a wide range of sugars. Although the yields of ethanol were quite low (at around 6 g/L), macroalgal ethanol production was slightly higher using K. marxianus compared with S. stipitis. The results obtained demonstrate the feasibility of obtaining ethanol from brown algae using relatively straightforward bioprocess technology, together with non-conventional yeasts. Conversion efficiency of these non-conventional yeasts could be maximised by operating the fermentation process based on the physiological requirements of the yeasts.

Banana is second largest produced fruit of total world’s fruits. Cooking banana or plantains processing industry is generating enormous amount of waste in the form of unripe banana peel at one place, thus important to study waste management and utilization. Therefore, unripe banana peel was investigated for ethanol production. This study involved chemical characterization, optimization of acid hydrolysis, selection of yeast strain and optimization of fermentative production of ethanol from dried unripe banana peel powder (DUBPP). Ethanol concentration was determined using gas chromatography flame ionization detector (GC-FID). Characterization of DUBPP revealed notably amount of starch (41% w/w), cellulose (9.3% w/w) and protein (8.4% w/w). 49.2% w/w of reducing sugar was produced by acid hydrolysis of DUBPP at optimized conditions. Three yeast strains of Saccharomyces cerevisiae were screened for ethanol conversion efficiency, osmotolerance, ethanol tolerance, thermotolerance, fermentation ability at high temperature and sedimentation rate. Further, fermentation conditions were optimized for maximum ethanol production from acid hydrolysate of DUBPP. At optimized fermentation conditions, 35.5 g/l ethanol was produced using selected strain of Saccharomyces cerevisiae NCIM 3095. Hence, unripe banana peel waste can be good feedstock for ethanol production.