Unraveling the structure of sugarcane bagasse after soaking in concentrated aqueous ammonia (SCAA) and ethanol production by Scheffersomyces (Pichia) stipitis

Abstract Background Fuel ethanol production from sustainable and largely abundant agro-residues such as sugarcane bagasse (SB) provides long term, geopolitical and strategic benefits. Pretreatment of SB is an inevitable process for improved saccharification of cell wall carbohydrates. Recently, ammonium hydroxide-based pretreatment technologies have gained significance as an effective and economical pretreatment strategy. We hypothesized that soaking in concentrated aqueous ammonia-mediated thermochemical pretreatment (SCAA) would overcome the native recalcitrance of SB by enhancing cellulase accessibility of the embedded holocellulosic microfibrils. Results In this study, we designed an experiment considering response surface methodology (Taguchi method, L8 orthogonal array) to optimize sugar recovery from ammonia pretreated sugarcane bagasse (SB) by using the method of soaking in concentrated aqueous ammonia (SCAA-SB). Three independent variables: ammonia concentration, temperature and time, were selected at two levels with center point. The ammonia pretreated bagasse (SCAA-SB) was enzymatically hydrolysed by commercial enzymes (Celluclast 1.5 L and Novozym 188) using 15 FPU/g dry biomass and 17.5 Units of β-glucosidase/g dry biomass at 50°C, 150 rpm for 96 h. A maximum of 28.43 g/l reducing sugars corresponding to 0.57 g sugars/g pretreated bagasse was obtained from the SCAA-SB derived using a 20% v/v ammonia solution, at 70°C for 24 h after enzymatic hydrolysis. Among the tested parameters, pretreatment time showed the maximum influence (p value, 0.053282) while ammonia concentration showed the least influence (p value, 0.612552) on sugar recovery. The changes in the ultra-structure and crystallinity of native SCAA-SB and enzymatically hydrolysed SB were observed by scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The enzymatic hydrolysates and solid SCAA-SB were subjected to ethanol fermentation under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) by Scheffersomyces (Pichia) stipitis NRRL Y-7124 respectively. Higher ethanol production (10.31 g/l and yield, 0.387 g/g) was obtained through SSF than SHF (3.83 g/l and yield, 0.289 g/g). Conclusions SCAA treatment showed marked lignin removal from SB thus improving the accessibility of cellulases towards holocellulose substrate as evidenced by efficient sugar release. The ultrastructure of SB after SCAA and enzymatic hydrolysis of holocellulose provided insights of the degradation process at the molecular level.

Ultra-structural mapping of sugarcane bagasse after oxalic acid fiber expansion (OAFEX) and ethanol production by Candida shehatae and Saccharomyces cerevisiae

Background Diminishing supplies of fossil fuels and oil spills are rousing to explore the alternative sources of energy that can be produced from non-food/feed-based substrates. Due to its abundance, sugarcane bagasse (SB) could be a model substrate for the second-generation biofuel cellulosic ethanol. However, the efficient bioconversion of SB remains a challenge for the commercial production of cellulosic ethanol. We hypothesized that oxalic-acid-mediated thermochemical pretreatment (OAFEX) would overcome the native recalcitrance of SB by enhancing the cellulase amenability toward the embedded cellulosic microfibrils. Results OAFEX treatment revealed the solubilization of hemicellulose releasing sugars (12.56 g/l xylose and 1.85 g/l glucose), leaving cellulignin in an accessible form for enzymatic hydrolysis. The highest hydrolytic efficiency (66.51%) of cellulignin was achieved by enzymatic hydrolysis (Celluclast 1.5 L and Novozym 188). The ultrastructure characterization of SB using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform–near infrared spectroscopy (FT-NIR), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) revealed structural differences before and after OAFEX treatment with enzymatic hydrolysis. Furthermore, fermentation mediated by C. shehatae UFMG HM52.2 and S. cerevisiae 174 showed fuel ethanol production from detoxified acid (3.2 g/l, yield 0.353 g/g; 0.52 g/l, yield, 0.246 g/g) and enzymatic hydrolysates (4.83 g/l, yield, 0.28 g/g; 6.6 g/l, yield 0.46 g/g). Conclusions OAFEX treatment revealed marked hemicellulose degradation, improving the cellulases’ ability to access the cellulignin and release fermentable sugars from the pretreated substrate. The ultrastructure of SB after OAFEX and enzymatic hydrolysis of cellulignin established thorough insights at the molecular level.

Cold-Start Emissions testing of Snowmobiles Using Ethanol and Gasoline

Increasing prices for fuel with depletion and instability in foreign oil imports has driven the importance for using alternative and renewable fuels. The alternative fuels such as ethanol, methanol, butyl alcohol, and natural gas are of interest to be used to relieve some of the dependence on oil for transportation. The renewable fuel, ethanol which is made from the sugars of corn, has been used widely in fuel for vehicles in the United States because of its unique qualities. As with any renewable fuel, ethanol has many advantages but also has disadvantages. Cold startability of engines is one area of concern when using ethanol blended fuel. This research was focused on the cold startability of snowmobiles at ambient temperatures of 20 °F, 0 °F, and -20 °F. The tests were performed in a modified 48 foot refrigerated trailer which was retrofitted for the purpose of cold-start tests. Pure gasoline (E0) was used as a baseline test. A splash blended ethanol and gasoline mixture (E15, 15% ethanol and 85% gasoline by volume) was then tested and compared to the E0 fuel. Four different types of snowmobiles were used for the testing including a Yamaha FX Nytro RTX four-stroke, Ski-doo MX Z TNT 600 E-TEC direct injected two stroke, Polaris 800 Rush semi-direct injected two-stroke, and an Arctic Cat F570 carbureted two-stroke. All of the snowmobiles operate on open loop systems which means there was no compensation for the change in fuel properties. Emissions were sampled using a Sensors Inc. Semtech DS five gas emissions analyzer and engine data was recoded using AIM Racing Data Power EVO3 Pro and EVO4 systems. The recorded raw exhaust emissions included carbon monoxide (CO), carbon dioxide (CO2), total hydrocarbons (THC), and oxygen (O2). To help explain the trends in the emissions data, engine parameters were also recorded. The EVO equipment was installed on each vehicle to record the following parameters: engine speed, exhaust gas temperature, head temperature, coolant temperature, and test cell air temperature. At least three consistent tests to ensure repeatability were taken at each fuel and temperature combination so a total of 18 valid tests were taken on each snowmobile. The snowmobiles were run at operating temperature to clear any excess fuel in the engine crankcase before each cold-start test. The trends from switching from E0 to E15 were different for each snowmobile as they all employ different engine technologies. The Yamaha snowmobile (four-stroke EFI) achieved higher levels of CO2 with lower CO and THC emissions on E15. Engine speeds were fairly consistent between fuels but the average engine speeds were increased as the temperatures decreased. The average exhaust gas temperature increased from 1.3-1.8% for the E15 compared to E0 due to enleanment. For the Ski-doo snowmobile (direct injected two-stroke) only slight differences were noted when switching from E0 to E15. This could possibly be due to the lean of stoichiometric operation of the engine at idle. The CO2 emissions decreased slightly at 20 °F and 0 °F for E15 fuel with a small difference at -20 °F. Almost no change in CO or THC emissions was noted for all temperatures. The only significant difference in the engine data observed was the exhaust gas temperature which decreased with E15. The Polaris snowmobile (semi-direct injected two-stroke) had similar raw exhaust emissions for each of the two fuels. This was probably due to changing a resistor when using E15 which changed the fuel map for an ethanol mixture (E10 vs. E0). This snowmobile operates at a rich condition which caused the engine to emit higher values of CO than CO2 along with exceeding the THC analyzer range at idle. The engine parameters and emissions did not increase or decrease significantly with decreasing temperature. The average idle engine speed did increase as the ambient temperature decreased. The Arctic Cat snowmobile (carbureted two-stroke) was equipped with a choke lever to assist cold-starts. The choke was operated in the same manor for both fuels. Lower levels of CO emissions with E15 fuel were observed yet the THC emissions exceeded the analyzer range. The engine had a slightly lower speed with E15.

Revenue diversification opportunities from sugarcane

Sugarcane is a major global agricultural crop that produces significant quantities of sugar and biomass in tropical and sub-tropical regions. Over many centuries, the crop has been grown primarily for its high sugar content which traditionally contributes over 95% of the revenue derived from the crop. While the production of renewable electricity from bagasse and rum from molasses has a long history, in more recent decades significant advances have been made in the production of cogeneration products and fuel ethanol at large scale. Sugarcane biorefineries producing fuels, green chemicals, biopolymers and bio-products offer great potential for improving the profitability of sugarcane production. This paper will address the opportunities available for sugarcane biorefineries to contribute to future profitability and sustainability of the sugarcane industry.

Low carbon fuels and commodity chemicals from waste gases – systematic approach to understand energy metabolism in a model acetogen

Gas fermentation using acetogenic bacteria offers a promising route for the sustainable production of low carbon fuels and commodity chemicals from abundant, inexpensive C1 feedstocks including industrial waste gases, syngas, reformed methane or methanol. Clostridium autoethanogenum is a model gas fermenting acetogen that produces fuel ethanol and 2,3-butanediol, a precursor for nylon and rubber. Acetogens have already been used in large scale industrial fermentations, they are ubiquitous and known to play a prominent role in the global carbon cycle. Still, they are considered to live on the thermodynamic edge of life and potential energy constraints when growing on C1 gases pose a major challange for the commercial production of fuels and chemicals. We have developed a systematic platform to investigate acetogenic energy metabolism, exemplified here by experiments contrasting heterotrophic and autotrophic metabolism. The platform is built from complete omics technologies, augmented with genetic tools and complemented by a manually curated genome-scale mathematical model. Together the tools enable the design and development of new, energy efficient pathways and strains for the production of chemicals and advanced fuels via C1 gas fermentation. As a proof-of-platform, we investigated heterotrophic growth on fructose versus autotrophic growth on gas that demonstrate the role of the Rnf complex and Nfn complex in maintaining growth using the Wood–Ljungdahl pathway. Pyruvate carboxykinase was found to control the rate-limiting step of gluconeogenesis and a new specialized glyceraldehyde-3-phosphate dehydrogenase was identified that potentially enhances anabolic capacity by reducing the amount of ATP consumed by gluconeogenesis. The results have been confirmed by the construction of mutant strains.

Análise do mercado sucroalcooleiro e das elasticidades preço e renda da demanda por etanol hidratado

Este trabalho tem como objetivo estudar o mercado de etanol combustível e estimar as elasticidades preço e renda da demanda de etanol hidratado, no Brasil, no período de janeiro de 2003 a setembro de 2012. O método econométrico utilizado para analisar os dados e obter os resultados referentes à estimativa dos parâmetros das equações de demanda foi a metodologia de Johansen. Baseado na teoria da demanda incluiu-se, num primeiro momento, como variáveis explicativas: o preço do bem, o preço do bem substituto e a renda, num segundo momento incluiu-se também a frota de veículos, estimando assim o modelo VAR/VEC. Os resultados encontrados foram significativos e estão de acordo com a teoria econômica, nos levando a concluir que a demanda por etanol é bem elástica ao preço do etanol e ao preço da gasolina. Antes de desenvolvermos o modelo, analisamos o mercado de cana-de-açúcar, de etanol e da commodity concorrente ao etanol, o açúcar. Destacando a preocupação com o meio ambiente e a importância do etanol como energia renovável.

鲜甘薯快速乙醇发酵条件的初步研究

本文对不同菌种（酵母菌和运动发酵单胞菌）快速生产燃料乙醇的条件进行了研究，实现了鲜甘薯快速转化为燃料乙醇。全文分为两部分： 第一部分：酵母菌快速生产燃料乙醇的条件研究。通过单因素试验，酵母菌快速生产燃料乙醇的条件为：发酵方式采用边糖化边发酵（SSF），蒸煮温度为85 ℃，料水比2:1（初始糖浓度 210 g/kg），糖化酶用量0.75 AGU/g 鲜甘薯，接种量10%（v/w）。在最优条件下，经过24 h发酵，乙醇浓度可达97.44 g/kg, 发酵效率为92%，发酵强度为4.06 g/kg/h。由于采用了低温蒸煮和SSF，可以大大节约能耗，从而降低乙醇生产的成本。同时，利用摇瓶优化的条件，进行了10 L，100 L，500 L发酵罐的放大试验，由于发酵罐初期可以人为通氧，使菌体能迅速积累，发酵时间缩短2 h，发酵效率在90%以上。 第二部分：运动发酵单胞菌快速生产燃料乙醇条件研究。通过单因素试验和正交试验获得了发酵的最佳参数：初始pH值6.0-7.0，硫酸铵5.0 g/kg，糖化酶量1.6 AUG/kg淀粉，初始糖浓度200 g/kg，接种量12.5%（v/w）。经过21 h发酵，乙醇浓度为95.15 g/kg，发酵效率可达94%。同时对不灭菌发酵也进行了研究，发酵效率可达92%。为鲜甘薯运动发酵单胞菌燃料乙醇的工业化生产打下基础。 对发酵结束后的残糖进行了研究。通过薄层层析和葡萄氧化酶测定证明：无论是酵母菌还是运动发酵单胞菌发酵结束后的发酵液中都不含葡萄糖。经过HPLC进一步分析残糖说明：发酵液中已没有葡萄糖成分；经糖化酶水解后仍没有葡萄糖出现；但经酸水解后又出现了葡萄糖，说明结束后的残糖是一些低聚糖结构。有关残糖的结构需要进一步研究。可以通过开发高效的低聚糖水解酶来降低发酵液的残糖，提高原料的利用率。 A new technology for rapid production fuel ethanol from fresh sweet potato by different microorganisms (Saccharomyces cerevisiae and Zymomonas mobilis) was gained in this research. The paper involved two parts: Part 1: The study on fuel ethanol rapid production from fresh sweet potato by Saccharomyces cerevisiae. The following parameters of Saccharomyces cerevisiae was investigated by a series of experiments: fermentation models, cooking temperature, initial sugar concentration and glucoamylase dosage. The results showed that SSF (simultaneous saccharification and fermentation) not only reduced the fermentation time (from 30 to 24h) but also enhanced the ethanol concentration (from 73.56 to 95.96 g/kg). With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg which the fermentation yield could reach to 92% and ethanol productivity 4.06 g/kg/h from sweet potato enzymatic hydrolysis. Furthermore, the savings in energy by carrying out the cooking (85 ℃) and saccharification (30 ℃) step at low temperature had been realized. The results were also verified in 10 L, 100 L and 500 L fermentor. The fermentation yield was no less than 90%. The fermentation time of fermenter was shorter than Erlenmeyer flask. This may be that the aeration in the early fermentation period is available, which lead to the rapidly commutations of biomass. Part 2: The technology of ethanol rapid production with simultaneous saccharification and fermentation ( SSF ) by Zymomonas mobilis，using fresh sweet potato as raw material was studied. The effects of various factors on the yield of ethanol were investigated by the single factor and the orthogonal experiments. As a result, the optimal technical conditions were obtained from those experiments：initial pH value 6.0-7.0, nitride 5.0 g/kg，(NH4)2SO4, glucoamylase 1.6 AUG/kg starch, inoculums concentration 12.5% (v/w). The Zymomonas mobilis was able to produce ethanol 95.15 g/kg, with 94% of the theoretical yield, from fresh sweet potato after 24 h fermentation. The fermentation efficiency of non-sterilized was also reach to 92%. We also analyzed the final fermentation residual sugars of Saccharomyces cerevisiae and Zymomonas mobilis. When the residual sugars were analyzed by thin-layer chromatogram and glucose oxidase, there was no glucose. The analysis of reducing sugars by HPLC showed that there was no glucose existed in the fermentation liquor. However, the glucose appeared after being hydrolyzed by acid. It is indicated that the residual sugars in the final fermentation liquor were the configuration of oligosaccharide, which was linked by the special glycosidic bonds. It was feasible for reducing residual sugars to develope the enzyme that can degradation the oligosaccharide.

酿酒酵母和运动发酵单胞菌木薯乙醇发酵条件研究

本文结合我国燃料乙醇发展的方针政策，以酿酒酵母和运动发酵单胞菌为菌种研究其在非粮能源作物木薯中乙醇发酵的情况，为木薯原料更好地应用于生产中提供了理论依据。 酿酒酵母木薯高浓度乙醇发酵的研究。实验采用的木薯干淀粉含量约70-75%。以酿酒酵母为菌种进行高浓度乙醇发酵的工艺条件研究，最佳条件为：木薯干粉碎细度为35目，料水比1：2，α-淀粉酶用量0.09 KNU/g淀粉，蒸煮温度85 ℃，蒸煮时间15 min。采用30 ℃同步糖化发酵工艺，糖化酶用量为3.4 AGU/g淀粉，发酵时间30 h。在10 L发酵罐中，乙醇质量比达127.88 g/kg，发酵效率为88.28%，发酵强度4.263 g/kg/h，100 L中试研究中乙醇浓度为127.75 g/kg，发酵强度4.258 g/kg/h。利用高效液相色谱对发酵液中残糖进行了分析，证明葡萄糖、果糖等单糖已完全被菌体利用，剩余糖为二糖，三糖等不可发酵的低聚糖。 运动发酵单胞菌快速乙醇发酵的研究。对实验室保藏的8株运动发酵单胞菌进行比较，选择发酵速度最快的Zymomonas mobilis232B进行研究。该菌在纯葡萄糖中的最佳发酵条件为：葡萄糖浓度18%，起始pH 6-7，发酵温度30 ℃，发酵时间18 h，乙醇浓度88 g/kg。在以木薯为底物同步糖化快速乙醇发酵中，采用Full Factorial设计和最速上升实验确定了培养基成分中的2个显著性因子及其最适浓度：酵母粉4 g/kg，硫酸铵0.8 g/kg。在最适培养基条件下，对木薯料水比和糖化酶用量进行了优化，得到Z.mobilis232B木薯乙醇发酵最佳料水比1：3，糖化酶浓度4 AGU/g淀粉，乙醇发酵4.915 g/kg/h。利用高效液相色谱对发酵液中残糖进行了分析，剩余糖为二糖，三糖等，但成分较酵母发酵后复杂。 According to the fuel ethanol development plans and policies in our country, the ethanol production from cassava by Saccharomyces cerevisiae and Zymomonas mobilis was studied. It provided theoretical basis for ethanol fermentation by cassava in industry. Part 1 is the study of VHG (very high gravity) ethanol fermentation by Saccharomyces cerevisiae. The content of starch in cassava was 70-75%. Compared with the performances under different experimental conditions, the following optimal conditions for VHG fermentation were obtained: Granule size of dry cassava 35 mashes, hydromodulus of cassava to water at 1:2, α-amylase enzyme dosage 0.09 KNU/g starch, cooking temperature 85 ℃ for 15 min, using the SSF process (simultaneous saccharification and fermentation) and the amount of glucoamylase 3.4 AGU/g starch. Accordingly, the final ethanol concentration was up to 127.88 g/kg; the ethanol yield reached 88.28%, and ethanol productivity was 4.263 g/kg/h after 30 h. When the fermentation scale expanded to 100 L, the final ethanol concentration was 127.75 g/kg, and the ethanol productivity was 4.258 g/kg/h in 30 h. The residual sugar was analyzed by high performance liquid chromatography, and proved that there was no glucose and fructose. The residual reducing sugar was some unfermentable oligosaccharide Part 2 is the study of the rapid ethanol production by Zymomonas mobilis. Compare with other seven stains, Zymomonas mobilis 232B was selected for research. The optimum condition in glucose medium was as follow: glucose concentration 18%, initial pH 6-7, and fermentation temperature 30 ℃. The ethanol concentration was 88g/kg in 18 h. After that, rapid ethanol production from cassava in SSF by Zymomonas mobilis 232B was studied. Through a series of experiments aided by Full Factorial Design and steepest ascent search, the optimal concentration yeast extract and ammonium sulfate were determined: 4 g/kg and 0.8 g/kg, each. Under optimum medium conditions, the optimal hydromodulus of cassava to water and glucoamylase dosages were obtained: hydromodulus of cassava to water at 1:3 and glucoamylase dosages 4 AGU/g starch. The ethanol production reached 4.915 g/kg/h. The residual sugar was analyzed by HPLC, and proved that the residual reducing sugar was some unfermentable oligosaccharide，but the components were more complex than that fermentation by Saccharomyces cerevisiae.

非粮原料燃料乙醇发酵关键技术的研究

生物质燃料乙醇是一种高度清洁的交通液体燃料，是减少温室气体排放，缓解大气污染的最佳技术选择。以非粮原料生产燃料乙醇可以在进行能源生产的同时保证粮食安全，有利于产业的可持续发展。在众多的非粮原料中，甘薯是我国开发潜力最大的生物质能源作物之一。我国占世界甘薯种植总面积和产量的90％。同时，甘薯的单位面积燃料乙醇产量远大于玉米和小麦。其成本是目前酒精中最低廉的，因此利用甘薯生产乙醇是发展生物质燃料乙醇的首要选择。目前采用薯类全原料主要采用分批发酵生产乙醇，其技术水平低，发酵强度低，一般在0.7－2.5g/（L•h），乙醇浓度低，甘薯发酵乙醇为6－8％(v/v)，能耗高，环境负荷大，污染严重。针对上述问题，本文从菌株选育、原料预处理、中试放大、残糖成分分析等方面进行研究。 为了研究乙醇发酵生产规模扩大过程中，大型发酵罐底部高压条件下，CO2对酵母乙醇发酵的影响，我们通过CO2 加压的方法进行模拟试验，研究结果表明，发酵时间随压强的升高而逐渐延长，高压CO2 对乙醇发酵效率影响不大，在0.3 MPa 以下时，发酵效率均可达到90%以上。高压CO2 对发酵的抑制作用是高压和CO2 这两个因素联合作用的结果。高压CO2 条件下，酵母胞外酶和胞内重要酶类的酶活均表现出特征性。0.2 MPa 下，酶活性的变化趋势和0.1 MPa 条件下的较为一致。而0.3 MPa 下的酶活变化趋势与0.4 MPa 下的酶活更为接近。通过全基因表达分析发现在CO2 压力为0.3 MPa 下，乙醇发酵途径中多个基因表达量下调，同时海藻糖合成酶和热激蛋白基因表达量上调。 筛选耐高温的乙醇酵母菌株能够解决糖化温度和发酵温度不协调的矛盾，实现真正意义上的边糖化边发酵。高温发酵还能够降低发酵时的冷却成本，实现乙醇的周年生产。本研究筛选出一株高温发酵菌株Y-H1，进而我们对该菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性进行了分析。结果表明Y-H1 能够在40 ℃条件下正常进行乙醇发酵，发酵33h，最终乙醇浓度达到10.7%（w/w），发酵效率达到90%以上。同时发酵液最终pH 在3.5 左右，显示菌株具有一定的耐酸性能力。同时观察到40 ℃下，菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性发生了变化，乙醇发酵途径中关键酶基因表达下调，而海藻糖合成酶与热激蛋白基因表达量上调，这些结果为进一步研究酵母菌耐热调控机理提供了依据。 糖蜜是一种大规模工业生产乙醇的理想原料，本研究利用选育高浓度乙醇发酵菌株结合配套的发酵稳定剂，研究了糖蜜高浓度乙醇发酵情况。结果表明采用冷酸沉淀预处理糖蜜溶液，采用分批补料的发酵方式，乙醇浓度最高达到了10.26% (w/w)，发酵时间为42 h。同时观察到在糖蜜发酵中，乙醛含量与乙醇浓度存在一定的相关性。 快速乙醇发酵对于缩短乙醇生产周期、降低乙醇生产成本、减少原料腐烂损失具有重要意义。本研究诱变和筛选得到了一株快速乙醇发酵菌株10232B。在优化后的发酵条件下，采用10L 发酵罐进行分批乙醇发酵，经过18h,乙醇的最终浓度达到88.5g/L,发酵效率93.6%，平均乙醇生产速度达到4.92 g/L/h。此菌株在保持较高乙醇生产浓度的同时，拥有快速生产乙醇的能力，适合作为快速乙醇发酵生产菌种。 由于鲜甘薯具有粘度大的特点，传统液化糖化处理很难在短时间内充分糖化原料；高粘度的醪液也难以进行管道输送，容易堵塞管路；同时，也会降低后续的乙醇发酵效率。 本文采用了快速粘度分析法对鲜甘薯糊化粘度特性进行了分析，进而对预处理条件进行了研究，在最佳预处理条件下，糖化2h 后，醪液葡萄糖值最高可达99.3，粘度4.5×104 mPa.s，而采用传统糖化工艺，醪液DE 值仅为85.8，粘度大于1.0×105 mPa.s。 此预处理方法也可用于快速糖化不加水的醪液。后续的乙醇发酵试验表明，通过此预处理方法获得的糖化醪液对乙醇发酵无负面影响。 在前期已实现了实验室水平的鲜甘薯燃料乙醇快速乙醇发酵基础上，进一步将发酵规模扩大到500L，在中试水平上对甘薯乙醇发酵进行了研究。结果表明在500L 中试规模，采用边糖化边发酵（SSF）工艺，在料液比为3∶1，发酵醪液最高粘度为6×104mPa.s 条件下，发酵37h，乙醇浓度达到了12.7%（v/v），发酵效率91%，发酵强度为2.7 g/（L•h）。与目前国内的薯类乙醇发酵生产技术水平具有明显的优越性。 为研究甘薯、木薯乙醇发酵中残糖的组成，采用了高效液相色谱—蒸发光散射检测法，对乙醇发酵残糖进行了分析。结果表明，甘薯、木薯乙醇发酵残糖均为寡聚糖，主要由葡萄糖、木糖、半乳糖、阿拉伯糖和甘露糖构成。随着发酵时间延长，寡聚糖中的葡萄糖、半乳糖、甘露糖可被缓慢的水解释放。提高糖化酶量仅在一定程度上降低残糖，过量的糖化酶反而会导致残糖增加。同时发现3, 5-二硝基水杨酸法不能准确测定甘薯、木薯乙醇发酵中的残总糖含量。进一步筛选了两株残糖降解菌株，对甘薯乙醇发酵残糖的降解利用率均达到了40%以上，而且还能显著降低发酵醪液粘度。经形态学和rRNA ITS 序列分析，确定这两株菌分别属于为木霉属和曲霉属黑曲霉组。 通过对以甘薯原料为代表的非粮原料发酵技术研究开发，以期形成乙醇转化率高，能耗低，生产效率高、季节适应性好，原料适应性广，经济性强，符合清洁生产机制的燃料乙醇高效转化技术，为具有我国特色的燃料乙醇发展模式提供技术支持。 Sweet potato is one of the major feedstock for the fuel ethanol production in China. The planting area and the yield in China take 90% of the world. Sweet potato is an efficient kind of energy crops. The energy outcome per area is higher than corn or wheat. And the manufacture cost of ethanol is the lowest, compared with corn and wheat. So sweet potato is the favorable crop for the bioethanol production in China. However, the low-level fermentation technology restricts the development of ethanol production by sweet potato, including slow ethanol production rate, low ethanol concentration and high energy cost. To solve these problems, we conducted research on the strain breeding, pretreatment, pilot fermentation test and residual saccharides analysis. To study the impact of hyperbaric condition at bottom of the large fermentor on yeast fermentation, high pressure carbon dioxide (CO2) was adopted to simulate the situation. The results showed that the fermentation was prolonged with the increasing pressure. The pressure of CO2 had little impact on the ethanol yield which could reach 90% under the pressure below 0.3 MPa. The inhibition was combined by the high pressure and CO2. Under the high CO2 pressure, the extracellular and important intracellular enzyme activities were different from those under normal state. The changes under 0.1 MPa and 0.2 MPa were similar. The changes under 0.3 MPa were closer to those under 0.4 MPa. The application of thermotolerance yeast could solve the problem of the inconsistent temperature between fermentation and saccharificaton and fulfill the real simultaneous saccharification and fermentation. And it could reduce the cooling cost. A thermotolerance strain Y-H1 was isolated in our research. It gave high ethanol concentration of 10.7%（w/w）at 40 ℃ for 33 h. The ethanol yield efficiency was over 90%. At 40 ℃, the extracellular and important intracellular enzyme activities of Y-H1 showed the difference with normal state, which may indicate its physiological changes at the high temperature. Molasses is another feedstock for industrial ethanol production. By our ethanol-tolerance strain and the regulation reagents, the fermentation with high ethanol concentration was investigated. In fed-batch mode combined with cold acid deposition, the highest ethanol concentration was 10.26% (w/w) for 42h. The aldehyde concentration in fermentation was found to be related to ethanol concentration. The development of a rapid ethanol fermentation strain of Zymomonas mobilis is essential for reducing the cost of ethanol production and for the timely utilization of fresh material that is easily decayed in the Chinese bioethanol industry. A mutant Z. mobilis strain, 10232B, was generated by UV mutagenesis. Under these optimized conditions, fermentation of the mutant Z. mobilis 10232B strain was completed in just 18 h with a high ethanol production rate, at an average of 4.92 gL-1h-1 per batch. The final maximum ethanol concentration was 88.5 gL-1, with an ethanol yield efficiency of 93.6%. This result illustrated the potential use of the mutant Z. mobilis 10232B strain in rapid ethanol fermentation in order to help reduce the cost of industrial ethanol production. As fresh sweet potato syrup shows high viscosity, it is hard to be fully converted to glucose by enzymes in the traditional saccharification process. The high-viscosity syrup is difficult to be transmitted in pipes, which may be easily blocked. Meanwhile it could also reduce the later ethanol fermentation efficiency. To solve these problems, effects of the pretreatment conditions were investigated. The highest dextrose equivalent value of 99.3 and the lowest viscosity of 4.5×104 mPa.s were obtained by the most favorable pretreatment conditions, while those of 85.8 and over 1.0×105 mPa.s was produced by traditional treatment conditions. The pretreatment could also be applied on the material syrup without adding water. The later experiments showed that the pretreated syrup had no negative effect on the ethanol fermentation and exhibited lower viscosity. The fuel ethanol rapid production from fresh sweet potato was enlarged in the 500L pilot scale after its fulfillment on the laboratory level. The optimal ratio of material to water was 3 to 1 in 500L fermentor. With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg for 37h, which reached 92% of theoretical yield. The average ethanol production rate was 4.06 g/kg/h. And the maximum viscosity of syrup reached 6×104mPa.s. The results showed its superiority over current industrial ethanol fermentation. The compositions of the residual saccharides in the ethanol fermentation by sweet potato and cassava were analyzed by high performance liquid chromatography coupled with evaporative light-scattering detector. The results showed that all the residual saccharides were oligosaccharides, mainly composed of glucose, xylose, galactose, arabinose and mannose. The glucose, galactose and mannose could be slowly hydrolyzed from oligosaccharides in syrup during a long period. To increase the glucoamylase dosage could lower the residual saccharides to a certain extent. However, excess glucoamylase dosage led to more residual saccharides. And the method of 3, 5-dinitrosalicylic acid could not accurately quantify the residual total saccharides content. Two residual saccharides degrading strains were isolated, which could utilize 40% of total residual saccharide and lower the syrup viscosity. With the analysis of morphology and internal transcribed spacer sequence, they were finally identified as species of Trichoderma and Aspergillus niger.

组合白腐真菌预处理木质纤维素原料的研究

木质纤维素原料种类多、分布广、数量巨大，通过燃料乙醇生产技术、厌氧沼气发酵技术将其转化成乙醇、沼气等二次能源，一定程度上可以缓解化石能源的不断消耗所带来的能源危机，也解决了农林废弃物引起的环境污染问题。其中以木质纤维素原料生产燃料乙醇，还可以避免以淀粉类和糖类原料生产燃料乙醇时带来的“与人争粮”等一系列问题。因此具有重要的经济效益、环境效益和社会效益。 然而，木质纤维素原料结构致密，木质素包裹在纤维素、半纤维素外围，导致其很难被降解利用，必须进行适当的预处理，去除木质素，打破原有的致密结构，利于原料的后续利用。因此，预处理成为木质纤维素原料能源化利用的关键。而目前预处理环节的费用过于昂贵，于是寻找一种高效、低成本的预处理方法是当今研究的热点。 本论文采用组合白腐真菌对木质纤维素原料进行生物预处理研究，与其他物理化学法相比，该法有着专一性较强、反应温和、不造成环境污染、成本低等优势。白腐真菌主要通过分泌木质素降解酶对木质素进行降解，从而破坏原料的致密结构，提高后续利用效率。所以木质素降解酶酶活的高低是影响原料预处理效果的一个关键因素。于是本论文首先通过将白腐真菌进行组合的方式提高木质素降解酶（漆酶，Lac）酶活；接着对组合菌的菌株相互作用机理进行研究，阐明组合菌Lac 酶活提高的原因，为菌株组合提高Lac 酶活这种方法的应用提供理论依据，同时也为后续组合白腐真菌预处理木质纤维素原料提供指导；进一步采用固态发酵和木质素降解酶两种方式对木质纤维素原料进行预处理研究，最大化去除木质素成分，破坏原料的致密结构；最终对预处理后原料的酶解糖化进行初步研究，为原料后续的能源化应用奠定基础。具体研究结果如下： （1） 以实验室保存的三株主要分泌Lac 的白腐真菌为出发菌株，筛选得到一组Lac 酶活明显提高的组合菌55+m-6，其中菌株55 为Trametes trogii sp.，m-6 为Trametes versicolor sp.，组合后Lac 酶活较单菌株分别提高24.13倍和4.07 倍。组合菌的最适产酶条件为pH 6.5、C/N 16:1、Tween 80 添加量为0.01%，在该条件下组合菌的Lac 酶活峰值比未优化时提高4.11倍。 （2） 对组合菌55+m-6 菌株间相互作用机理进行研究，发现菌株之间不存在抑制作用；平板培养时，菌丝交界处Lac 酶活最高并分泌棕色色素；液体培养时，菌株m-6 对组合后Lac 酶活的提高起着更为重要的作用：菌株m-6的菌块、过滤灭菌胞外物以及高温灭菌胞外物均能明显刺激菌株55 的Lac产生；菌株55、m-6 进行组合后，同工酶种类未发生增减，但有三种Lac同工酶浓度有所提高；对菌株胞外物进行薄层层析和质谱分析，结果表明组合前后菌株胞外物中各物质在浓度上存在较大的变化。推测组合菌Lac酶活的明显提高，主要是由于菌株m-6 胞外物中的一些物质能刺激菌株55 分泌大量Lac 进行代谢，且这些刺激物质并非菌株m-6 特有，菌株55自身也可以代谢生成，但是适当的浓度才能刺激Lac 的大量分泌。 （3） 将组合菌55+m-6 用于固态发酵预处理木质纤维素原料，发现其对玉米秆的降解程度最大，在粉碎度40 目、含水率65%的最优处理条件下，处理至第15d，秸秆失重率为41.24%，其中木质素、纤维素、半纤维素均有降解，且Lac 和纤维素酶（CMC）酶活以及还原糖量均达到峰值。 （4） 对玉米秆进行木质素降解酶预处理，发现Lac/1-羟基苯并三唑（HBT）系统对玉米秆木质素的降解效果最好，在最优处理条件时，即HBT 用量0.2%、处理时间1d、Lac 用量50U/g，木质素降解率可达12.60%。预处理后玉米秆的致密结构被破坏，比表面积增大，利于后续酶与纤维素、半纤维素成分的结合。 （5） 对预处理后的玉米秆进行酶解糖化，其中组合菌固态发酵预处理后玉米秆的糖化率比对照高4.33 倍；Lac/HBT 系统预处理后玉米秆的糖化率比对照高2.99％，糖化液中主要含有木糖、葡萄糖两种单糖。 There are many kinds and large quantities of lignocellulosic biomass widely distributed on the earth. They can be converted into secondary energy such as fuel ethanol, biogas, et al., which can relieve the energy crisis caused by consumption of fossil energy resources and solve the problem of environmental pollution caused by agriculture and forestry waste. Meanwhile, the production of fuel ethanol from lignocellulosic biomass can ensure food supply to human kind instead of starch- and sugar-containing raw materials. So the energy conversion of lignocellulosic biomass contributes considerable economic, environment and social benefits. However, lignocellulosic biomass has the compact structure, in which lignin surrounds cellulose and hemicellulose, so it must be pretreated before energy usage and pretreatment is one of the most critical steps in the energy conversion of lignocellulosic biomass. At present, the cost of pretreatment is too expensive, so looking for an efficient and low-cost pre-treatment method is one of recent research hot spots. In this research, combined white rot fungi pretreatment method was used, which had some advantages in low cost, high specificity, mild reacting conditions and friendly environmental effects compared with the other physical and chemical methods. White rot fungi secrete lignin degrading enzymes to degrade the content of lignin and damage the contact structure of lignocellulosic biomass, so the activity of the lignin degrading enzymes is the key factor to the degradation effect of raw materials. Firstly, the combined fungi with high laccase activity were screened; secondly, the interaction mechanism between strains was studied, and the cause of higher laccase activity after strains combination was also preliminary clarified; under the guidance of the mechanism, lignocellulosic biomass was pretreated by the combined fungi; lastly, the enzymatic hydrolysis of pretreated lignocellulosic biomass was also preliminary studied; all of the researches could lay the foundation for the energy application of lignocellulosic biomass. The specific research results were as follows: (1) The combined fungi 55+m-6 with significant higher laccase activity were screened from the three white rot fungi stored in our lab which mainly secreted laccase. Strain 55 and strain m-6 were Trametes trogii sp. and Trametes versicolor sp., respectively. The laccase activity of combined fungi was 24.13 and 4.07-fold than strain 55 and strain m-6, respectively. The optimized condition for laccase production of the combined fungi in liquid medium was pH 6.5, C/N 16:1 and Tween 80 0.01%. In this optimized condition, the laccase activity of combined fungi was 4.11-fold higher comparing with which in non-optimized medium. (2) The interaction mechanism between strain 55 and strain m-6 was further studied, and no inhibition effect was observed. Brown pigment was secreted on the junction of the two strains on the plate, where the highest laccase activity was detected. Strain m-6 was much important to boost laccase activity of combined fungi in liquid medium, and strain 55 was stimulated by fungal plug, filter sterilized extracellular substances and high temperature sterilized extracellular substances of strain m-6 to produce laccase. The types of laccase isozymes did not change after combining strain 55 and strain m-6, but the concentrations of three types increased. Mass Spectrometry and TLC analysis of extracellular substances of each strain showed that concentration of some substances considerably changed after strains were combined. It was supposed that the cause of higher laccase activity of combined fungi was mainly due to some extracellular substances of strain m-6 with the appropriate concentration which stimulated laccase secretion of strain 55 and generated not only by strain m-6 but also by strain 55. (3) Combined fungi 55+m-6 were used to lignocellulosic biomass pretreatment with the type of solid-state fermentation. The highest degree of degradation of corn straw was obtained, including the rate of weight loss was 41.24% and the lignin, cellulose and hemicellulose were degraded partially under the optimized condition of 40 mesh, 65% water content on 15th day. Laccase, CMCase activities and content of reducing sugar reached the maximum value on that day. (4) Lignin degrading enzymes from combined fungi 55+m-6 were used for corn straw pretreatment. The most remarkable degradation of lignin in corn straw with Lac/1-hydroxybenzotriazole (HBT) system was observed, and the 12.60% lignin degradation was obtained under the optimized condition of 0.2% HBT, 50 U/g laccase for 1 d. After pretreated by Lac/HBT, the tight structure of corn straw was demolished and specific surface area increased, which had advantages for accessible of enzyme to cellulose and hemicellulose. (5) The corn straws pretreated by combined fungi 55+m-6 with the type of solid-state fermentation and Lac/HBT were used for enzymatic hydrolysis, and the saccharification rates of each pretreatment type were 4.33 times and 2.99% higher than CK, respectively. The enzymatic hydrolysis liquid of corn straw pretreated by Lac/HBT mainly contained xylose and glucose.

Valorization of an Industrial Organosolv-Sugarcane Bagasse Lignin: Characterization and Use as a Matrix in Biobased Composites Reinforced With Sisal Fibers

In the present study, the main focus was the characterization and application of the by-product lignin isolated through an industrial organosolv acid hydrolysis process from sugarcane bagasse, aiming at the production of bioethanol. The sugarcane lignin was characterized and used to prepare phenolic-type resins. The analysis confirmed that the industrial sugarcane lignin is of HGS type, with a high proportion of the less substituted aromatic ring p-hydroxyphenyl units, which favors further reaction with formaldehyde. The lignin-formaldehyde resins were used to produce biobased composites reinforced with different proportions of randomly distributed sisal fibers. The presence of lignin moieties in both the fiber and matrix increases their mutual affinity, as confirmed by SEM images, which showed good adhesion at the biocomposite fiber/matrix interface. This in turn allowed good load transference from the matrix to the fiber, leading to biobased composites with good impact strength (near 500 J m(-1) for a 40 wt% sisal fiber-reinforced composite). The study demonstrates that sugarcane bagasse lignin obtained from a bioethanol plant can be used without excessive purification in the preparation of lignocellulosic fiber-reinforced biobased composites displaying high mechanical properties. Biotechnol. Bioeng. 2010;107: 612-621. (C) 2010 Wiley Periodicals, Inc.

Estudo prospectivo da internacionalização do etanol combustível.

Esta dissertação tem como objetivo elaborar cenários para a internacionalização do etanol combustível através da metodologia da prospectiva estratégica. Após a descrição da história do desenvolvimento do etanol combustível na matriz energética brasileira, descreveram-se, além do atual mercado de etanol no Brasil e no Mundo, os principais aspectos que influenciarão no desenvolvimento de sua comercialização como commodity internacional. Para isto, é aplicada a metodologia de prospecção de cenários para a utilização do etanol no âmbito mundial. Para os cenários identificados, após a revisão das literaturas teórica e empírica sobre o tema, é elaborado um estudo que descreve estratégias de internacionalização possíveis para Petrobras Biocombustível S.A., subsidiária da Petrobras Petróleo Brasileiro S.A, responsável pelos negócios de etanol da companhia, tanto no Brasil quanto no exterior.

Uso de operações estruturadas para financiamento de projetos no setor sucroenergético brasileiro

O setor sucroenergético brasileiro passou por um intenso ciclo de crescimento nos últimos anos. Ainda assim, a demanda de etanol no país continua a superar a capacidade de produção, principalmente devido ao sucesso dos carros flex-fuel. As usinas voltaram também a atenção à bioeletricidade, aos alcoolquímicos e a diversos outros subprodutos e coprodutos do processo de produção de açúcar e etanol. Por estes motivos, se faz necessária a realização de maiores investimentos em ampliação e modernização das atuais usinas, e no aumento do número de unidades de produção. Para que tais investimentos sejam realizados da maneira mais eficiente, é necessário conhecer todas as opções de linhas de crédito disponíveis. Uma delas, pouco usada e com bons resultados é a Structured Project Finance. O presente trabalho tem como objetivo discutir o modelo de Structured Project Finance e apresentá-lo como uma possível alternativa de financiamento de projetos de usinas de açúcar e etanol no Brasil. Sua principal contribuição é apresentar uma alternativa de financiamento que agregue mais valor ao negócio.

Patentes e etanol lignocelulósico

Produção intelectual e desenvolvimento tecnológico podem diferenciar países e regiões no processo de desenvolvimento socioeconômico. No caso do Brasil, observa-se o papel energético do etanol combustível para veículos motorizados leves como um resultado importante do avanço tecnológico do país, que vai além da aptidão agroclimática. A contínua busca pela especialização tecnológica verticalizada do setor sucroenergético poderia levar o Brasil a uma posição, se não autônoma, mais confortável, não só de produtor de matéria-prima, mas de processos agregadores de valor no que diz respeito aos processos de produção de etanol de segunda geração, produzido a partir de biomassa lignocelulósica. O objetivo desta dissertação é analisar os esforços de P&D que resultaram em Depósitos e Publicações de patentes em órgãos oficiais como o United States Patent and Trademark Office (USPTO), o European Patent Office (EPO), e o Instituto Nacional de Propriedade Industrial (INPI), no tema etanol de segunda geração. Ainda, verifica-se se esses esforços impactam no poder concorrencial de países e firmas depositantes de patentes. Além das coletas e observações dos dados dos órgãos acima mencionados, foram calculados para os dados de depósitos e publicações de patentes no tema bioetanol lignocelulósico os índices de Herfindahl Hirschman (HHI) e a razão de concentração (Concentration Ratio) CR4, tradicionalmente utilizados para que órgãos reguladores de defesa do consumidor autorizem fusões e aquisições entre participantes de um determinado mercado. Esse método permite a observação do grau de competitividade entre as firmas depositantes de patentes no tema e a possível tendência sobre a detenção do controle em futuro próximo e a corrida para venda de royalties dos processos desenvolvidos em diferentes áreas tecnológicas para incrementar a produção industrial de etanol avançado. Os resultados indicam uma concentração elevada dos esforços de pesquisa, medidos pelos depósitos de patentes, referentes a etanol de segunda geração, em um número muito reduzido de empresas norte-americanas, quando analisada a base de dados dos EUA. O sucesso desses esforços, mensurados pela publicação de patentes, contudo, não se mostra concentrado nem nos EUA nem na União Europeia. No caso do Brasil, ainda não são encontradas publicações de patentes no tema Lignocellulosic Bioethanol, bem como apenas uma empresa brasileira possui uma patente publicada nos Estados Unidos. Esses resultados sugerem que investimentos em pesquisa científica no Brasil podem produzir mais artigos publicados e titulação acadêmica/científica que propriamente o registro de patentes em órgãos especializados em qualificar a invenção de métodos, processos ou fórmulas, dentro e fora do país. Isso pode significar tanto baixo esforço em pesquisa no assunto quanto à perda pelo autor e/ou sua instituição da oportunidade de ter seu esforço de pesquisa recompensado por meio de royalties, como compensação pela criatividade, dedicação intelectual e de recursos econômicos. Os resultados deste estudo contribuem para o debate a respeito da crescente necessidade de produção e abastecimento de fontes renováveis de energia, como o biocombustível etanol avançado à base do derivado bagaço de cana-de-açúcar, a custos mais competitivos como matéria-prima adicional e para produção incremental de etanol em futuro próximo. As conclusões do estudo indicam a necessidade do aumento na produção de conhecimento aplicado e em esforços para garantir sua propriedade intelectual, permitindo o retorno patrimonial com royalties.

Criação de mercado de créditos de biocombustível avançado para geração de economia financeira e ambiental no comércio de etanol entre Brasil e Estados Unidos

O comércio de etanol entre Brasil e Estados Unidos é fortemente guiado por uma assimetria regulatória, que agrega valor ao etanol brasileiro ao gerar uma de-manda americana pelo produto derivado da cana de açúcar. A demanda advém dos mandatos de consumo de biocombustíveis estabelecidos no programa americano conhecido como Renewable Fuel Standard (RFS). A assimetria emerge pelo fato de RFS dar ao etanol brasileiro a classificação de Biocombustível Avançado, tornando-o para os Estados Unidos um produto mais nobre que o etanol derivado do amido de milho produzido domesticamente. Apesar dos processos produtivos tornarem os dois produtos diferenciados quanto ao teor de emissão de CO2 em seus ciclos de vida, o produto final é o mesmo: etanol anidro combustível. Portanto, quando a aquisição de etanol brasileiro pelos Estados Unidos é tal que torna o balanço do-méstico de oferta e demanda deficitário no Brasil, faz-se necessária a importação brasileira de etanol americano. Esse vai e vem do produto ocorre de forma simultâ-nea e, por isso, considera-se a existência de um fluxo redundante de etanol, geran-do custos logísticos, transacionais e emissões de CO2 que poderiam ser evitados. Dado que o objeto do interesse americano pelo produto brasileiro é o baixo índice de emissões de CO2 no ciclo de vida do etanol de cana de açúcar, o fluxo redun-dante de etanol pode ser substituído por transações de Créditos de Biocombustível Avançado, uma nova ideia que é desenvolvida ao longo deste trabalho. Para cada Crédito transacionado, os Estados Unidos contabilizarão em seu balanço a emissão de CO2 correspondente ao volume equivalente de etanol de cana de açúcar, trans-ferindo para o Brasil as emissões correspondentes ao mesmo volume de etanol de-rivado do amido de milho. A substituição do fluxo físico pela transação de contratos é a fonte de economia proposta no título deste trabalho.