BIOCONVERSION OF LIGNO CELLULOSIC BIOMASS IN TO ETHANOL

Biorefinery concepts refer to the integrated production of food, fuel, power, commodity chemicals, and polymers from biomass, mimicking the structure of a fossil-based refinery. The use of lignocellulosic biomass as feedstock for biorefineries has been promoted as a long-term strategy to mitigate climate change and foster food security. One of the critical bottlenecks for the development of these systems is the recalcitrance of the feedstock. Costly and energy intensive physicochemical and/or enzymatic pretreatment are therefore needed to release simple sugars from the lignocellulose matrix. This and other challenges would penalise the conversion efficiency or the environmental impact. This thesis puts the focus on the techno-economic and environmental assessment of sustainability of biorefinery concepts. On the one hand, it proposes an alternative approach to the conventional economic performance indicators for biorefineries. The proposed economic evaluation method addresses the lack of a formal market for crop residues and other intermediate agro-industrial streams. The approach allows for the comparison of process variants in terms of the maximum willingness-to-pay of a process unit for the bioresource that enters the system. This is calculated as the global biorefinery revenues, estimated from the definition of competitive target prices, minus the processing costs. This thesis also investigated the inclusion of parameter and scenario uncertainty into the LCA of lignocellulosic biorefineries. Scale and state-of-technology issues behind modelling assumptions are not always explicitly presented and justified. In order to facilitate decision-making, a robust comparison among different biorefinery options must include an explicit estimation of the uncertainty associated to process design parameters. The sensitivity of the LCA indicators to methodological choices such as allocation of impacts among co-products, choice of system boundaries and uncertainty in the values of background flows to the system has been evaluated by using systematic process simulation and global sensitivity analysis techniques.The proposed methods are illustrated by two case studies based on process flowsheets adapted from the second generation (2G) ethanol model developed by the US National Renewable Energy Laboratory (NREL). Different alternatives for feedstock, biomass pretreatment and valorization of extracted sugars were considered. They were simulated with Aspen Plus® process simulator. Case study 1 was used to illustrate the techno-economic assessment method. It consists in a basic 2G ethanol facility, refining sugarcane bagasse. Two technological variants were considered: The first corresponded to the NREL benchmark design, using dilute acid pretreatment and producing ethanol (only fuel-OF). The second variant consisted of a low-severity liquid hot water (LHW) pretreatment producing ethanol from cellulose and a pentoses syrup (Feed and Fuel – FF). [...]

As a main stream of waste from the paper and pulp industry, paper sludge is regarded as a threat to environmental safety as most of it is disposed via landfill or incineration, which can cause severe soil, water or air pollution. However, alkaline paper sludge has the potential to be a catalyst in biomass pretreatment, and also contains a large amount of fiber that can be used as a feedstock for ethanol production. The dissolved lignin in alkaline pretreatment can be recovered as a good quality co-product such as lignin pellets. The ash in paper sludge can also be recovered to replace sand as a raw material in mortar production. However, the use of paper sludge for commercialized ethanol production is currently a challenge due to its low solid content and high pH. This paper therefore proposes a novel conceptual design for combining paper sludge and agricultural residues (e.g. rice straw in this study) to produce ethanol and other co-products. The net present value is used to assess the economic potential of two proposed process designs: (1) a current technology of turning C5 sugars into biogas; and (2) a prospective technology of co-fermenting both C5 and C6 sugars into ethanol. It is found that both approaches could be economically feasible, but at different scales of production. A sensitivity analysis is also conducted to investigate the effects of plant size, feedstock stream composition (the ratio between rice straw and paper sludge) and subsidies on the economic performance of the proposed projects.

Enhanced bio-ethanol production from Chlorella sp. biomass by hydrothermal pretreatment and enzymatic hydrolysis

The potential of 89 culturable cold-adapted isolates from uncontaminated habitats, including 61 bacterial and 28 yeast strains, to utilize representative fractions of petroleum hydrocarbons (n-alkanes, monoaromatic and polycyclic aromatic hydrocarbons) for growth and to produce various enzymes at 10 degrees C was investigated. The efficiency of bacterial and yeast strains was compared. The growth temperature range of the yeast strains was significantly smaller than that of the bacterial strains. Sixty percent of the yeasts but only 8% of the bacteria could be classified as true psychrophiles, showing no growth above 20 degrees C. A high percentage (89%) of the yeast strains showed lipase activity. More than one-third of the 61 bacterial strains produced amylase, beta-lactamase, beta-galactosidase or lipase; more than two-thirds were protease producers. Only 6% of the bacterial strains but 79% of the yeast strains utilized n-hexadecane for growth; 13% of the bacterial strains and 21-32% of the yeast strains utilized phenol, phenanthrene or anthracene for growth. Only four yeast strains but none of the bacterial strains could grow with all hydrocarbons tested. The biodegradation of phenol was investigated in fed-batch cultures at 10 degrees C. Three yeast strains degraded phenol concentrations as high as 10 mM (one strain) or 12.5 mM (two strains). Of eight bacterial strains, two strains degraded up to 10 mM phenol. The optimum temperature for phenol degradation was 20 degrees C for all eight bacterial strains and for two yeast strains. Biodegradation by five yeast strains was optimal at 10 degrees C and faster at 1 degrees C than at 20 degrees C. All phenol-degrading strains produced catechol 1,2 dioxygenase activity.

Paper mill sludge is a solid waste material generated from pulping and papermaking operations. Because of high glucan content and its well-dispersed structure, paper mill sludges are well suited for bioconversion into value-added products. It also has high ash content originated from inorganic additives used in papermaking, which causes hindrance to bioconversion. In this study, paper mill sludges from Kraft process were de-ashed by a centrifugal cleaner and successive treatment by sulfuric acid and sodium hydroxide, and used as a substrate for cellulase production. The treated sludge was the only carbon source for cellulase production, and predominantly inorganic nutrients were used as the nitrogen source for this bioprocess. The cellulase enzyme produced from the de-ashed sludge exhibited cellulase activity of 8 filter paper unit (FPU)/mL, close to that obtainable from pure cellulosic substrates. The yield of cellulase enzyme was 307 FPU/g glucan of de-ashed sludge. Specific activity was 8.0 FPU/mg protein. In activity tests conducted against the corn stover and alpha-cellulose, the xylanse activity was found to be higher than that of a commercial cellulase. Relatively high xylan content in the sludge appears to have induced high xylanase production. Simultaneous saccharification and fermentation (SSF) was performed using partially de-ashed sludge as the feedstock for ethanol production using Sacharomyces cerevisiae and the cellulase produced in-house from the sludge. With 6% (w/v) glucan feed, ethanol yield of 72% of theoretical maximum and 24.4 g/L ethanol concentration were achieved. These results were identical to those of the SSF using commercial cellulases.

Effect of pretreatment on the ethanol and fusel alcohol production during fermentation of sugarcane press-mud

Every year, millions of tonnes of municipal solid waste are generated in the city of Toronto from residential and non-residential sources. A large fraction of the municipal solid waste is composed of organic materials. This valuable resource has traditionally been disposed of in landfills, which in turn contributes to the pollution of the environment and the generation of green house gases. This places a great emphasis on the need for the design and implementation of more sustainable waste management practices and the adequate supportive infrastructures in order to achieve sustainability. The city of Toronto has been experiencing a huge challenge over the past few years regarding its waste problem, and having inadequate infrastructure for effective waste management practices. In the year 2000, the City of Toronto established a goal of 100% waste diversion by the year 2010 (Task Force, 2001). In the year 2005, the City of Toronto collected approximately 100,000 tonnes of source separated organic waste (SSO) from single-family households (Butts, 2005). SSO is an excellent source of fermentable carbohydrates including free sugars, starch, cellulose, hemicellulose and other degradable organic materials. However, the main obstacle is the release of some of its carbohydrates, such as cellulose and hemicellulose, from their bondage to lignin before conversion to fermentable sugars. Cellulose and hemicellulose in SSO are bonded to lignin and are not easily separated and fermented to ethanol. Therefore, for utilizing SSO as a feedstock for ethanol production, a deep understanding of the nature of lignocellulosic materials is essential in order to overcome the challenges in the biological conversion to ethanol. As an initial part of a multi-staged project, this thesis is to examine the potential of SSO for utilization as a feedstock for ethanol production. A set of experiments were conducted on SSO in order to determine the amenability of SSO to ethanol production The experimental results show a relatively high amount of carbohydrates in the SSO samples, indicating potential of SSO to be utilized as an ethanol production feedstock. Comparing result of the characteristics study with other cellulosic feedstocks, indicates that SSO has a reasonable amount of fermentable sugars and can be utilized for ethanol production instead of using other cellulosic feedstocks such as herbaceous energy crops. A technology for the biological conversion of SSO to ethanol was proposed based on the current techniques and the results from the characterization study on SSO. It is foreseen that the findings of this study will enhance the overall understanding of the nature of SSO and the possibility of using it for ethanol production, and provide technical data and information for the decision makers in the assessment of the potential of SSO for ethanol production.

The impact of yeast and lactic acid bacteria strains on the fruity aroma of red wines was investigated by sensory and analytical strategies. The ester composition of four different Bordeaux red wines was quantified by HS-SPME-GC/MS. These wines, made with selected yeast and bacteria strains, were investigated at the end of alcoholic fermentation and regularly until 12 months of aging, during 2011 and 2012 vintages. Sensory analyses of wines after 3 and 12 months of aging revealed significant differences with regard to yeast strains. Bacteria seemed to have only a slight impact on changes in aromatic profile. Ester levels were strongly influenced by yeast strain and very little affected by malolactic fermentation and aging. Differences and similarities between sensory data and ester profile are discussed. This study highlights the importance of yeast strains in red winemaking. Their sensory impact remains despite the other vinification steps after alcoholic fermentation.

Objective. To determine and evaluate the indicators of changes in the water regime of the most common varieties of potatoes (on the 60th and 85th day of germination) in the conditions of central Polissya of Ukraine, namely the coefficients of water recovery, water retention, drought resistance and water deficit. Methods. Field and laboratory-based, selection and statistical. Results. The coefficients of water retention, water recovery capacity and drought resistance and water deficit of plant leaves are determined. The highest water content of potato leaves was observed among the following varieties: Tyras, Nahoroda, Serpanok, Slovianka and Chervona Ruta. It is established that the mechanism of water recovery in potato varieties of different maturity groups is different, the highest percentage of this indicator in early-maturing varieties is observed on the 60th day from germination, in the mid ripening group on the 85th day from germination. High value of the integrated indicator of water recovery was demonstrated by the following varieties: Tyras, Vyhoda, Yavir, Lietana, Slovianka Volodarka, Bazaliia and Chervona Ruta. It was found that on the 60th day from germination among the following potato varieties: Chervona Ruta, Serpanok, Avanhard, Mezhyrichka, Nahoroda, Oleksandryt, Radomysl and Bazaliia, the drought resistance coefficient was ≤ 80%. It was found that only in the varieties Tyras and Slovianka, on the 85th day after germination, there is an increase in the numerical expression of the drought resistance coefficient, which indicates an increased degree of adaptation of plants to soil moisture deficiency. In the first and second record the indicator of water deficit of the most of the studied varieties of potatoes was within acceptable limits, indicating a slight negative effect of weather conditions on the physiological state of plants. A slightly increased indicator of water deficit was observed on the 60th day of germination among the following potato varieties: Tyras, Volodarka and Levada, but within two weeks it decreased to a safe level. Conclusions. We recommend growing potato varieties that have shown high values of integrated indicators of water regime in the area of central Polissya and use as a source material for breeding purposes in the creation of new drought-resistant genotypes.

Microalgal biomass is a sustainable and valuable source of lipids with omega-3 fatty acids. The efficient extraction of lipids from microalgae requires fast and alternative extraction methods, frequently combined with biomass pre-treatment by different procedures. In this work, Pressurized liquid extraction (PLE) was optimized and compared with traditional lipid extraction methods, Folch and Bligh and Dyer, and with a new Ultrasound Assisted Extraction (UAE) method for lipids from microalgae Isochrysis galbana. To further optimize PLE and UAE, enzymatic pre-treatment of microalga Isochrysis galbana was studied with commercial enzymes Viscozyme and Celluclast. No significant differences were found for lipid yields among different extraction techniques used. However, advanced extraction techniques with or without pre-treatment are a green, fast, and toxic solvent free alternative to traditional techniques. Lipid composition of Isochrysis was determined by HPLC-ELSD and included neutral and polar lipids, showing that each fraction comprised different contents in omega-3 polyunsaturated fatty acids (PUFA). The highest polar lipids content was achieved with UAE (50 °C and 15 min) and PLE (100 °C) techniques. Moreover, the highest omega-3 PUFA (33.2%), eicosapentaenoic acid (EPA) (3.3%) and docosahexaenoic acid (DHA) (12.0%) contents were achieved with the advanced technique UAE, showing the optimized method as a practical alternative to produce valuable lipids for food and nutraceutical applications.

Цель. Экспериментальное обоснование возможности применения препарата «Миелопид» в лечении экспериментальной критической ишемии конечностей.Материалы и методы. Опыты проводились на 130 белых крысах-самцах линии Wistar. Экспериментальные животные были разделены на пять групп: интактную, контрольную, первую, вторую и третью опытные. В интактную группу вошло 10 крыс, у которых оценен нормальный уровень микроциркуляции. В остальных группах было включено по 30 крыс-самцов в каждую, у которых моделировали критическую ишемию правой задней конечности путем иссечения бедренной и подколенной артерий. В контрольной группе лечение не проводилось. В первой группе экспериментальные животные получали препарат «Миелопид» по 50 мкг/кг в мышцы бедра через 3 часа после операции, затем через одни, двое, трое суток. Во второй группе крысам вводили препарат «Актовегин», а в третьей - «Солкосерил» интраперитонеально в дозе 50 мкг/кг через 3 часа после операции, затем ежедневно в течение пяти суток. Оценка уровня микроциркуляции в мышцах голени крыс выполнялась методом лазерной допплеровской флоуметрии (ЛДФ) на 10-е, 21-е и 28-е сутки эксперимента, на тех же сроках производили гистологическое исследование ишемизированных мышц, во время которого определяли динамику среднего диаметра артериол, капилляров, венул, мышечных волокон, уровня артерио-венулярного шунтирования, площади некроза и плотности капиллярной сети.Результаты. Уровень микроциркуляции в первой группе животных превосходил на 10-е сутки эксперимента аналогичный показатель во второй и третьей группах в 1,24, на 21-е сутки - в 1,18, а на 28-е сутки во второй группе в 1,42, а в третьей - в 1,71 раза. Уровень артерио-венулярного шунтирования у животных, пролеченных миелопидом, был ниже на 21-е сутки эксперимента на 8,1%, чем у животных, пролеченных актовегином и на 17,3% - пролеченных солкосерилом. На 10-е сутки площадь некроза мышечных волокон у животных, пролеченных миелопидом, была достоверно меньше на 13,2%, чем в группе животных, которым вводили актовегин и на 7,5% в третьей группе животных, лечившихся солкосерилом, на 21-е сутки соответственно на 10,8% и на 11,1%, на 28-е сутки - на 5% и на 6%. Плотность капиллярной сети на 10-е сутки в первой группе превосходила в 1,25 плотность во второй группе и в 1,34 в третьей, на 21-е сутки соответственно в 1,2 и 1,4, на 28-е сутки – в 1,46 и 1,47 раза.Заключение. Лечение экспериментальной критической ишемии нижних конечностей препаратом «Миелопид» патогенетически обосновано и эффективно.Ключевые слова: критическая ишемия, конечности, лечение, миелопид, актовегин, солкосерил.

Conversion of cellulosic biomass such as agricultural residues to biofuels offers major economic, environmental, and strategic benefits. Sorghum is one of the important grain crops in US. It represents a renewable resource that is currently grown on six to ten million acres in the U.S. However, at present, there is not enough scientific information and knowledge about the use of sorghum stover for biofuel production. The objective of this research was to evaluate and characterize sorghum biomass as a feedstock for ethanol production. Five types of sorghum biomass, including brown midrib (bmr) sorghum, forage sorghum, grain sorghum, photosynthesis sorghum, and sweet sorghum, were characterized and used for ethanol production. Pretreatment with dilute acid was used to increase fermentable sugars yield. The effect of sulfuric acid concentration, treatment temperature, and residence time on fermentable sugars yield were studied. AccelleraseTM 1000 was used to hydrolyze cellulose into glucose at 50 oC and pH 4.8 for 96 h.

Short-chain fructooligosaccharides (FOS) stimulate selectively the growth and activity of microorganisms in the colon providing positive health effects and well-being in humans and animals. The lack of accurate isolation methods, however, hampers the possibility of getting new potential fructo-oligosaccharides (FOS)-fermenting yeast or bacterial strains. A valuable screening procedure to visually detect bacterial and yeast strains able to ferment FOS in liquid or solid rich media supplemented with an innocuous pH indicator is described. Using this assay, 15 FOS-consuming strains isolated from different sources were successfully evaluated to prove the utility of the method. This screening procedure is a new and valuable tool in rapid large-scale detection of potential FOS fermenting-strains useful in food and health science. Key words:  FOS-fermenting microorganisms, prebiotics, probiotics, symbiotics.

BackgroundSimultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover, degradation products resulting from biomass pretreatment impair fermentation of sugars, especially xylose, and can synergize with high temperature stress. One approach to resolve both concerns is to utilize a strain background with innate tolerance to both elevated temperatures and degradation products.ResultsIn this study, we screened a panel of 108 wild and domesticated Saccharomyces cerevisiae strains isolated from a wide range of environmental niches. One wild strain was selected based on its growth tolerance to simultaneous elevated temperature and AFEX™ (Ammonia Fiber Expansion) degradation products. After engineering the strain with two copies of the Scheffersomyces stipitis xylose reductase, xylitol dehydrogenase and xylulokinase genes, we compared the ability of this engineered strain to the benchmark 424A(LNH-ST) strain in ethanol production and xylose fermentation in standard lab medium and AFEX pretreated corn stover (ACS) hydrolysates, as well as in SSCF of ACS at different temperatures. In SSCF of 9% (w/w) glucan loading ACS at 35°C, the engineered strain showed higher cell viabilities and produced a similar amount of ethanol (51.3 g/L) compared to the benchmark 424A(LNH-ST) strain.ConclusionThese results validate our approach in the selection of wild Saccharomyces cerevisiae strains with thermo-tolerance and degradation products tolerance properties for lignocellulosic biofuel production. The wild and domesticated yeast strains phenotyped in this work are publically available for others to use as genetic backgrounds for fermentation of their pretreated biomass at elevated temperatures.

Saccharification of starchy food waste through thermochemical and enzymatic pretreatment, towards enhanced bioethanol production via newly isolated non-conventional yeast strains