Progress 09/01/12 to 08/31/17

Outputs
Target Audience:The target audiences will include academic researchers and scientists and engineers in food, ennery and agricultural industries. The PI gave a seminar of biotechnolgy for biofuels and bioproducts to a group of high school teachers (10 people) at Frontiers in Science Anaerobic Digestion Workshop at MSU Anaerobic Digestion and Education Center (ADREC) on April 14th, 2014. The PI presented the fungal cultivations for bioproduct and biofuel production that the PI's research group is developing to a group of faculties fromTuskegee University (5 people) at MSU Anaerobic Digestion and Education Center on May 24th, 2016. The PI also presented our algal research on Bridge Artist in Residency seminar to a group of artists (100 people) at MSU Planetarium on Septmber 14th, 2016 . Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We attended international conference and presented our research results which contributed to our professional development. Liu, Y., Liao, W., Pavlik, D., Clary, W. 2017. Microalgae cultivation using a high-efficiency photobioreactor system for flue gas carbon dioxide sequestration. Poster presentation at the 2017 ASABE Annual International Meeting, Spokane, WA. July 16-19. How have the results been disseminated to communities of interest?Website, conferences, meetings, and tours have been used to dissemiate the studied technologies to different groups of interests.Several press releases on algal cutlivaton and wastewater/water treatment have been published on MSU and CANR websites to let general pulic know what the project has achieved. In addition, some specific meetings were hosted for targeted audience. For instance, I communicated with Paris-based artist Helen Evans who is known for the large-scale social and environmental interventions on our algal research and made a presentation on 2016/17 Bridge Artist in Resident seminar at MSU Planetarium on Sept. 14th, 2016. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Biobased fuels and chemicals are making important contributions to U.S. energy security, rural economic development, and environmental quality. Currently the majority of biobased fuels and chemicals are produced from crop-based feedstock, which significantly influences food production and leads to a food vs. fuel dilemma. Replacing crop-based feedstock with non-food-based feedstock must be achieved to realize sustainable biobased fuel and chemical production. Abundant cellulosic biomass as agricultural residues has been recognized as an attractive feedstock for production of biofuels and chemicals. Microalgae, with its relatively high lipid and carbohydrate contents, represent another most promising non-food based feedstock for biofuels production. Compared to terrestrial plants, algae have an enormous potential for biomass production without severe impacts on arable, food producing land. Accelerating cellulosic and algal fuel and chemical research and development is a key to realizing such a sustainable production system. This project developed a biorefinery concept thatimproves the cost-effectiveness of cellulosic biomass conversion by chemical and biological means, and algal cultivations that generate diversebiobased products. 1. Enhancing lipid accumulation in fungal biomass Fungi as one of key industrial microbes have been widely used to produce many value-added chemicals such as organic acids, enzymes, lipids, plant growth regulators, and antibiotics due to their metabolic versatility. Thus, I started my research program with fungal cultivation of lignocellulosic residues for biodiesel production. A wild type fungal strain, Umbelopsis isabellina (also called Mortierella isabellina), was screened and selected, which exhibits unique characteristics of multiple carbon utilization capacity (co-utilization of C5/C6 sugars and organic acids), better lipid content (>50% ) and high tolerance to inhibitors (phenolic compounds, hydroxymethylfurfural, and furfural) during the fermentation of lignocellulosic hydrolysates. Corresponding to the selected fungal strain, we developed a novel lignocellulosic co-hydrolysis process and a combined acid-alkali pretreatment process that maximize biomass conversion of C5/C6 sugars and acetic acid, and eliminates the need of a large amount of water for washing and detoxification. In addition, the fuel quality analysis also concludes that fungal biodiesel is a very good diesel alternative. These research outcomes indicate that combining the selected fungal strain, pretreatment and hydrolysis process creates a new route of lignocellulosic biodiesel production. 2. Enhancing production of chitin as a high value product using fungal cultivation Chitin is a natural amino polysaccharide widely distributed in the animal and plant kingdom. The structure of chitin is a linear polysaccharide made up of unbranched β-(1,4)-2-acetamido-2-deoxy-D-glucopyranosyl residues which is also called N-acetyl- D-glucosamine. The structural characteristics make chitin a very constructive biopolymer that can be used as coagulating agents in wastewater treatments, plant seed coating agents and potential biopesticide in agricultural industry, and biomaterials (e.g. absorbable sutures) in biomedical industry. Traditionally, chitin and chitosan are extracted from crustacean insects and shellfishes. Compared to the chitin from shellfishes, fungal chitin has advantages of lower level of inorganic materials, no geographic or seasonal limitations, better effectiveness in inducing the plant immune response while utilized as a fertilizer. Fungi can utilize diverse agricultural residues for growth. In Michigan, sugar beet pulp was treated as an agricultural waste that can be used as a cheap feedstock for fungal chitin production. We studied the conversion of sugar beet pulp into a value added product, chitin via a fungal fermentation. Sugar beet pulp residues (SBP) were used as a feedstock for a filamentous fungus Rhizopus oryzae fermentation to produce chitin. Enzymatic conditions for effective liberation of fermentable sugars from SBP were identified. Nitrogen sources, nutritional salts, and plant hormones were investigated as a means to improve the chitosan yield. Optimum conditions identified were applied to a 2L flask culture. Fungal chitosan yields reached 3.6% (per gram of SBP input). The economic potential of a chitin fermentation system sized for 2,000 metric tons of SBP was analyzed based on the results achieved. Compared to the current application of SBP as animal feed, the high value of the chitin significantly enhanced the economic performance. 3. Investigating enzyme production using pelletized fungal culture One problem associated with the fungi culture is the formation of undesired cotton-like (filamentous) mycelia, which will eventually hinder the application of large-scale fermenter culture due to heterogeneous growth conditions, oxygen/nutrient transfer limitation, and high energy consumption. To alleviate this problem, small pellets of fungal cells are preferred. We studied the lipase production from biodiesel waste stream glycerol using pelletized fungus Rhizopus oryzae. At the fixed glycerol concentration, lipase activity was significantly increased from 41 units/L to 700 units/L when changing fungal morphology from big clumps to small pellets. 4. Developing a culture strategy to enhance lipid/starch accumulation Microalgae have been considered as one of the most promising biological CO2 utilization systems due to their characteristics of high photosynthetic efficiency, fast growth, carbon-concentrating mechanism, and accumulation of high-energy-content cell components (lipids and carbohydrates). Microalgal cultivation was then adopted by my research group to utilize CO2. Microalgal strain development was carried out to convert CO2 into chemicals/fuels. One of the main barriers during algal chemical and fuel production is that the algal cell wall must be degraded to release components such as lipids, carbohydrates and other metabolites for target chemical production. Energy and chemical intensive processes such as acid hydrolysis and thermal treatment are currently used to release those components, and economic and environmental concerns limit the application of these processes. In order to address this problem, a novel concept of in vivo enzymatic hydrolysis to release algal carbohydrates has been developed by my research group. A thermophilic amylase gene (arAmyBH) from hyperthermophilic bacterium T. neaploitana was successfully expressed into Chlamydonomas reinhardtii chloroplast. The transgenic C. reinhardtii is able to efficiently accumulate the enzyme along with algal starch during the culture, and possesses the capability to digest intracellular starch at elevated temperature (50°C) to release mono-sugars in the absence of cell disintegration. The released mono-sugars without undergoing any chemical/thermal treatment can then be easily utilized by other microbial processes for chemicals/fuels production. In addition, a two-stage strategy was developed to improve the microalgal carbohydrate production for advanced biofuel production. In the first stage, Chlamydomonas. reinhardtii CC125 was cultivated in a photo-bioreactor with high CO2 loading of 5%. In the second stage, reducing CO2 content to 0.04 % led to a high carbohydrate content of 71 %, showing approximately 9 times improvement compared to that in the biomass from the first stage culture. These results suggested that photoautotrophic two-stage cultivation was an effective approach to accumulate microalgal carbohydrate as a feedstock for biofuel production.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Zanotti, M., Ruan, Z., Bustamante-Roman, M., Liu, Y., Liao, W. 2016. A sustainable lignocellulosic biodiesel production integrating solar- and bio-power generation. Green Chemistry 18, 5059-5068
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Liu, Z., Liao, W., Liu, Y. 2016. A sustainable biorefinery concept to convert agricultural residues into value-added chemicals. Biotechnology for Biofuels 9, 197
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhong Y., Liu, Z., Isaguirre, C., Liu, Y., Liao, W. 2016. Fungal fermentation on anaerobic digestate for lipid-based biofuel production. Biotechnology for Biofuels 9, 253
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Liu, Z., Liu, Y., Oyetunde, T., Hollinshead, W. D., Hermanns, A., Tang, Y., Liao, W. 2017. Exploring eukaryotic formate metabolisms to enhance microbial growth and lipid accumulation. Biotechnology for Biofuels 10, 22
• Type: Journal Articles Status: Published Year Published: 2017 Citation: MacLellan, J., Chen, R., Yue, Z.B., Kraemer, R., Liu, Y., Liao, W. 2017. Effects of protein and lignin on cellulose and xylan analyses of lignocellulosic biomass. Journal of Integrative Agriculture 16(6), 1268-1275
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Li, Q., Liu, Y., Liao, W., Powers, W. 2017. Microalgal cultivation using animal production exhaust air: technical and economic feasibility. CLEAN - Soil, Air, Water 45(4) 1500309
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Pavlik, D., Zhong, Y., Elizabeth, C., Liao, W., Morgan, R., Clary, W., Liu, Y. 2017 Microalgae cultivation of carbon dioxide sequestration and protein production using a high-efficiency photobioreactor system. Algal Research 25, 413-420

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:We worked with potato farmers in Michigan to develop a chitin rich fertilzer from cull potatoes for plant disease control. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We attended three international conferences and presented our research results which contributed to our professional development. Liu, Z., Vanderberg, M., Liao, W., Liu, Y. 2015. An integrated process to clean up biogas, reclaim water and utilize solid residues - magnifying sustainability of Anaerobic Digestion. Oral presentation at the 2015 AICHE Annual International Meeting, Salt Lake City, UT. November 6-9. Liu, Z., Liao, W., Liu, Y. 2016.A Sustainable Biorefining Concept to Convert Agricultural Residues into High-Value Chemicals. Post presentation at the 2016 Society of Industrial Microbiology and Biotechnology (SIMB) Annual Internation Meeting, New Orleans New Orleans, LA. July 24-28. Zhong, Y., Liu, Z., Isaguirre, C., Liu, Y., Liao, W. 2016. Fungal fermentation on anaerobic digestate for lipid-based biofuel production. Oral presentation at the 2016 ASABE Annual International Meeting, Orlando, FL. July 17-20. Liu, Z., Vanderberg, M., Sanyal, O., Lee, I., Liao, W., Liu, Y. 2016. A novel approach to simultaneously clean up biogas and reclaim water from anaerobic digestion. Oral presentation at the 2016 ASABE Annual International Meeting, Orlando, FL. July 17-20. How have the results been disseminated to communities of interest?We worked with Michigan potato farmers to convert cull potatoes into chitin rich fertilizer for potato pest control. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will continue working on 1) the fungal lipid production from lignocellulosic biomass and C1 substrates; 2) fungal fermentation on cull potatoes to produce chitin rich fertilizer for potato pest control; 3) algal cultivation on power plant flue gas for CO2 sequestration.

Impacts
What was accomplished under these goals? We integrated electrocoagulation and algal cultivation to treat a high strength wastewater - anaerobic digestion liquid effluent for reclaimed water and value-added algal biomass production. The integrated system synergistically takes advantages of both electrocoagulation and algal cultivation to enhance the efficiencies of wastewater treatment. The electrocoagulation treated wastewater had low turbidity with better light penetration (108 NTU) to enable algal growth. The algal cultivation had high-efficiency removal of phosphorus (99.4%) and nitrogen (88.2%). The dissolved iron in the electrocoagulation treated wastewater enhanced lipid accumulation of the algae. This study concluded a new route for agricultural wastewater treatment that turns wastewater from an environmental liability into a valuable asset. We designed an integrated Water Curtain-Microalgal Culture (WCMC) system to mitigate air emissions and recover nutrients to culture microalgae from Animal Feeding Operations (AFOs). The WCMC system included a water curtain, a windbreak wall and a raceway pond bioreactor. The water curtain was formed by continuously pumping water into a perforated water tank that flowed into the raceway pond bioreactor. The exhaust pollutants (NH3 and particulate matter (PM)) from a poultry house were dissolved and absorbed by the water droplets. The microalgal culture in the bioreactor was run in batch mode and the average growth rate was 12.7 ± 2.9 g/m2/day. Exhaust ammonia (NH3) and total suspended particle (TSP) concentrations were reduced by 74.9% and 89.2%, respectively. The harvested microalgal biomass had a protein content of 41.3%. The amino acid profile of microalgae demonstrated comparable quality to the WHO/FAO reference. Omega-3 fatty acids accounted for about 23% of total fat acids. Heavy metals (Pb, Cu, Fe and Zn) did not exceed the maximum tolerance levels. The adaptation of this new technology will reduce AFOs environmental impact, create a win-win situation for future animal agriculture and microalgal cultivation, and generate new alternate protein sources as food and feed supplements. We established a biorefinery concept to treat animal wastes and enhance the sustainability of animal wastes management. The biorefinery includes an anaerobic digestion (AD), electrocoagulation (EC) treatment of the liquid digestate, and fungal conversion of the solid fiber into a fine chemical - chitin. Animal wastes were first treated by an AD to produce methane gas for energy generation to power the entire biorefinery. The resulting liquid digestate was treated by EC to reclaim water. Enzymatic hydrolysis and fungal fermentation were then applied on the cellulose-rich solid digestate to produce chitin. EC water was used as the processing water for the fungal fermentation. This study demonstrates an energy positive and fresh-water-free biorefinery to simultaneously treat animal wastes and produce a fine chemical - chitin. The sustainable biorefinery concept provides a win-win solution for agricultural waste management and biorefining of value-added chemical production.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wang, D., Zhu, Z., Wang, X., Bustamante,Wang M., Xu, Y., Liu, Y., Liao, W. 2015. Improving mycelium-bound lipase production by aggregating Rhizopus chinensis on a draft tube in a modified stirred tank fermentor. Process Biochemistry 50, 2019-2028. Liu, Z., Liu, Y. 2016. Synergistic Integration of Electrocoagulation and Algal cultivation to Treat Liquid Anaerobic Digestion Effluent and Accumulate Algal Biomass. Process Biochemistry 51, 89-94. Chen, R., Liu, Y., Liao, W. 2016. Using environmentally friendly process combining electrocoagulation and algal cultivation to treat high strength wastewater. Algal Research 16, 330-337. Li, Y., Powers, W., Rozeboom, D., Liu, Y., Liao, W. 2016. An integrated Water Curtain-Microalgal Culture system (WCMC) to mitigate air emissions and recover nutrients from animal feeding operations. Algal Research-Biomass Biofuels and Bioproducts 18, 166-174. Zanotti, M., Ruan, Z., Bustamante, M., Liu, Y., Liao, W. 2016. A Sustainable Lignocellulosic Biodiesel Production Integrating Solar- and Bio-power Generation. Green Chemistry 18, 5059-5068. Liu, Z., Liao, W., Liu, Y. 2016. A sustainable bioreifinery concept to convert agricultural residues into value-added chemicals. Biotechnology for Biofuels 9, 197-206.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We attended two conferences and presented our research results which contributed to our professional development. Liao, W., Chen, R., Roman, M., Liu, Z., Sanyal, O., Liu, Y., Lee. I. 2015. Integrating solar-bio-nano-technologies to develop a self-sustaining organic waste utilization system. Oral presentation at the 2015 ASABE Annual International Meeting, New Orleans, LA. July 26-29. Zhong, Y., Ruan, Z., Zhong, Y., Archer, S., Liu, Y., Liao, W. 2015. A self-sustaining advanced lignocellulosic biofuel production by integration of anaerobic digestion and aerobic fungal fermentation. Oral presentation at the 2015 ASABE Annual International Meeting, New Orleans, LA. July 26-29. Li, Q., Powers, W., Rozeboom, D., Liu, Y., Liao, W. 2015. Mitigate air emissions from animal feeding operations and recover nutrients using an integrated water curtain-microalgal culture system. Oral presentation at the 2015 ASABE Annual International Meeting, New Orleans, LA. July 26-29. Liu, Z., Liao, W., Liu. Y. 2015. Integrating electrocoagulation (EC) and biological routes to convert organic residues into value-added chemicals. Poster presentation at the 37th Symposium on Biotechnology for Fuels and Chemicals, Sand Diego, CA. April 27-30. I was the Secretory of PRS-707 Food and Organic Waste Management and Utilization committee and a Moderator of Session "Anaerobic digestion and advances in manure, food and organic waste" at 2015 ASABE Annual Meeting, New Orleans, LA. July 26-29. How have the results been disseminated to communities of interest?We worked with Michigan Sugar Company to convert sugar beet pulp into chitosan based biopesticides for sugar beet pathogen control. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will continue working on 1) the fungal lipid production from lignocellulosic biomass; 2) production of fructooligosaccharides from sugar beets; 3) biological treatment and electrocoagulation treatment to reclaim agricultural wastewater for water reuse; and 4) novel eukaryote formatotrophic pathways to improve one-carbon metabolism for microbial processes.

Impacts
What was accomplished under these goals? We investigated the effects of inhibitory compounds (furfural, hydroxymethylfurfural, and ferulic and coumaric acids) in lignocellulosic hydrolysate on M. isabellina growth and lipid production. M. isabellina showed excellent tolerance to those inhibitors. The inhibition only delayed the substrate consumption and cell growth at the beginning of the fermentation, once the strain adapted to the toxic environment, its cell growth and lipid accumulation were significantly improved. 13C-carbon tracing indicated that M. isabellina could simultaneously utilize both glucose and acetate for cell mass growth and lipid accumulation with the existence of inhibitors. These results indicated that M. isabellina has potential to serve as a robust platform for producing value-added chemicals from lignocellulosic resources. This paper was highlighted on Renewable Energy Global Innovations Series as a key scientific article contributing to the excellence in energy research. This paper was published online on February, 2015 and cited by 2 articles. We developed a novel heterologous protein expression platform to produce a thermophilic amylase in algal chloroplast for direct hydrolysis of starch at elevated temperature after algal cultivation. The expression of amylase in algal chloroplast provided a solution towards effective utilization of microalgal starch for biofuel production. The new algal expression system could also be applied for other recombinant proteins (such as enzyme, therapeutic proteins) production which extends value-added application of algal cultivation. We applied a two-stage strategy to improve the microalgal carbohydrate production for advanced biofuel production. In the first stage, C. reinhardtii was cultivated in a photo-bioreactor on two different operation modes to improve the biomass production. Optimal culture conditions achieved in the batch operation was applied into semi-continuous operation, and biomass productivity was increased from 0.23 g L-1 d-1 to 0.47 g L-1 d-1 with 70 % medium replacement ratio and 5 % CO2 under continuous light of 135 µmol m-2 s-1. In the second stage, reducing CO2 content to 0.04 % led to a high carbohydrate content of 71 %, showing approximately 9 times improvement compared to that in the biomass from the first stage culture. These results suggested that photoautotrophic two-stage cultivation was an effective approach to accumulate microalgal carbohydrate as a feedstock for biofuel production. We developed a new multiple-stage treatment process by integrating electrocoagulation with biogas pumping to simultaneously reclaim anaerobic digestion effluent and clean up biogas. The 1st stage of electrocoagulation treatment under the preferred reaction condition led to removal efficiencies of 30%, 81%, 37% and 100% for total solids, chemical oxygen demand (COD), total nitrogen and total phosphorus, respectively. Raw biogas was then used as a reactant and pumped into the effluent to simultaneously neutralize pH of the effluent and remove H2S in the biogas. The 2nd stage of electrocoagulation treatment on the neutralized effluent showed that under the selected reaction condition, additional 60% and 10% of turbidity and chemical oxygen demand were further removed. The study concluded a dual-purpose approach for the first time to synergistically combine biogas purification and water reclamation for anaerobic digestion system, which well addresses the downstream challenges of anaerobic digestion technology. We also investigated glycerol utilization by a filamentous fungus, Rhizopus oryzae 9363 for lactic acid accumulation. There was no lactic acid acumulated from glycerol media at 30°C in contrast to glucose media. Increasing temperature from 30 °C to 37 °C led to a 63% decrease of the average growth rate of R. oryzae in glycerol media and a 61% increase of the average cell mass yield, and the cultures on glycerol media at 37 °C were able to generate lactic acid of 0.6 g/L. Whereas lactic acid accumulation on glucose media dropped to half of that at 30 °C. Moreover, supplementing 40 mM sodium pyruvate to cultures every day significantly improved the lactic acid synthesis of R. oryzae on glycerol media, which the lactic acid concentrations reached 1.33 g/L at 37 °C and 0.67 g/L at 30 °C, respectively. Our results indicated that glycerol utilization for lactic acid accumulation by Rhizopus sp. is limited by the availability of intracellular pyruvate, and controlling pyruvate flow is a key to enhance the lactic acid accumulation.

Publications

• Type: Book Chapters Status: Published Year Published: 2014 Citation: Liao, W., Liu, Y., Hodge, D. 2014. Biorefineries Integrated Biochemical Processes for Liquid Biofuels: Chapter 13 Integrated Farm-Based Biorefinery. Qureshi, N., Hodge, D., Vertes, A. (Ed.), Elsevier, New York, NY. ISBN: 978-0-444-59498-3.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wang, X., Ruan, Z., Guan, W., Kraemer, R., Zhong, Y., Liu, Y.2015. Glycerol utilization by Rhizopus oryzae for lactic acid accumulation. Biotechnology and Bioprocess Engineering 20, 389-395. Selke, S., Auras, R., Nguyen, T., Castro A., Cheruvathur, R., Liu, Y. 2015. Evaluation of Biodegradation-Promoting Additives for Plastics. Environmental Science & Technology. 49(6):3769-77 Ruan, Z., Hollinshead, W., Isaguirre, C., Tang, Y. J., Liao, W., Liu, Y. 2015. Effects of inhibitory compounds in lignocellulosic hydrolysates on Mortierella isabellina growth and carbon utilization. Bioresource Technology . 183, 18-24. Wang, X., Ruan, Z., Sheridan, P., Boileau, D., Liu, Y., Liao, W. 2015. Two-stage photautotrophic cultivation to improve carbohydrate production in Chlamydomonas reinhardtii. Biomass and Bioenergy. 74, 280-287 Wang, X., Ruan, Z., Sears, B. B., Liu, Y., Liao, W. 2015. Transgenic expression of a bacterial thermophilic amylase in Chlamydomonas reinhardtii chloroplast. BioEnergy Research. 8, 527-536 Liu, Z., Stromberg, D., Liu, X., Liao, W., Liu, Y. 2015. A new multiple-stage electrocoagulation process on anaerobic digestion effluent to simultaneously reclaim water and clean up biogas. Journal of Hazardous Materials 285, 483-490 Zhong, Y., Ruan, Z., Zhong, Y., Archer, S., Liu, Y., Liao, W. 2015. A self-sustaining advanced lignocellulosic biofuel production by integration of anaerobic digestion and aerobic fungal fermentation. Bioresource Technology 179, 173-179 Chen, R., Thomas, B. D., Liu, Y., Mulbry, W., Liao, W. 2014. Effects of algal hydrolysate as reaction medium on enzymatic hydrolysis of lignocelluloses. Biomass and Bioenergy 67,72-78

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We attended two conferences and presented our research results which contributed to our professional development. Li, Q., Liu, Y., Liao, W., Powers, W. 2014. Integrated wet scrubbers-microalgae culture system: design and evaluation. Oral presentation at the 2014 ASABE Annual International Meeting, Montreal, Canada. July 13-17. Li, Q., Liu, Y., Liao, W., Powers, W. 2014. Cultivating microalgae using animal production exhanst air as a nutrient source – a laboratory scale study. Oral presentation at the 2014 ASABE Annual International Meeting, Montreal, Canada. July 13-17. Liu, Z., Liao, W., Liu, Y. 2014. A new anaerobic digestion effluent utilization system integrating electrocoagulation and algal accumulation. Oral presentation at the 2014 ASABE Annual International Meeting, Montreal, Canada. July 13-17. Ruan, Z.H., Zanotti, M., Liao, W., Liu, Y., 2014. Development of an advanced biological process to convert lignocellulose into lipid-based fuel. Oral presentation at the 2014 ASABE Annual International Meeting, Montreal, Canada. July 13-17. Wang, X.Q., Ruan, Z.H., Sheridan, P., Boileau, D., Liu, Y., Liao, W., 2014Strategies of two-stage photoautotrophic cultivation for improving carbohydrate production in Chlamydomonas reinhardtii, 36th Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, Florida, April 28-May 1. Ruan, Z.H., Zanotti, M., Liao, W., Liu, Y., 2014. Development of an advanced biological process to convert lignocellulose into lipid-based fuel, 36th Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, Florida, April 28-May 1,. How have the results been disseminated to communities of interest? We are working with Michigan Sugar Company to convert sugar beet pulp into chitosan based biopesticides for sugar beet pathogen control. What do you plan to do during the next reporting period to accomplish the goals? For the next reporting period, we will continue working on 1) the fungal lipid production from lignocellulosic biomass; 2) production of chitosan based biopesticides for agricultural crop disease and pest control; 3) biological treatment and electrocoagulation treatment to reclaim agricultural wastewater for water reuse.

Impacts
What was accomplished under these goals? we developed a combined-hydrolysis and fungal lipid fermentation process that applied dilute acid pretreatment with alkaline pretreatment, directly followed by enzymatic saccharification and fungal fermentation without detoxification and liquid–solid separation to convert multiple lignocellulosic biomass into lipids. The process can significantly enhance chemical utilization and minimize processing steps. The lipids can be used to product biodiesel, biolubricants and biosolvents. The better inhibitor tolerance and fast growth rate of the selected fungus and simplified pretreatment process has potential for process scale-up and industrial commercialization. In addition, the results have been published on a key journal in our research field (see the publications). We developed an integrated system to culture algae using animal production exhaust air as a nutrient source to alleviate air pollution caused by animal operations. We applied biological treatment (algae cultiivation ) and physical-chemical treatment (electrocoagulation) to reclaim wastewater( anaerobic digestion effluent) which could extend the application of anaerobic digestion.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Ruan, Z., Zanotti, M., Archer, S., Liao, W., Liu, Y. 2014. Oleaginous fungal lipid fermentation on combined acid- and alkali-pretreated corn stover hydrolysate for advanced biofuel production. Bioresource Technology Bioresource Technology 163:12-17. Hussain, J., Ruan, Z.H., Nascimento, I.A., Liu, Y., Liao, W. 2014. Lipid profiling and corresponding biodiesel quality of Mortierella isabellina using different drying and extraction methods. Bioresource Technology 169:768-772 Ji, S., Lu, J., Liu, Z., Srivastava, D., Song, A., Liu, Y., Lee, I. 2014. Dynamic encapsulation of hydrophilic nisin in hydrophobic poly (lactic acid) particles with controlled morphology by a single emulsion process. Journal of Colloid and Interface Science 423, 85-93. Zhang, L., Wang, X.Q., Ruan, Z.H., Liu, Y., Niu, X.R., Yue, Z.B., Li, Z. M., Liao, W., Liu, Y., 2014 Fungal cellulase/xylanase production and corresponding hydrolysis using pretreated corn stover. Applied Biochemistry and Biotechnology 172, 1045-1054.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We attended the conference of ASABE to present our research results which contributed to our professional development. Liu, Z., Liao, W., Liu, Y. 2013. Reclaiming Water from Anaerobic Digestion Effluent using Electrocoagulation Flotation (ECF) Treatment. Oral presentation at ASABE 2013. Kansas City, Missouri. July 21 – July 24. Zanotti, M., Ruan, Z., Liu, Y., Liao, W. 2013 Conceptual mass and energy balance for solar-powered fungal biodiesel refinery on lignocellulosic feedstock. Poster presentation at ASABE 2013. Kansas City, Missouri. July 21 – July 24. How have the results been disseminated to communities of interest? Recently, Nandan Cleantec Limited, one of the largest biodiesel companies in India, found out the fungal lipid technology through our publication, and would like to collaborate with us for process commercialization. What do you plan to do during the next reporting period to accomplish the goals? For the next reporting period, we will continue working on the fungal lipid production from cellulosic biomass and transgenic expression of a thermophilic amylase in the Chlamydomonas reinhardtii chloroplast for biofuel production.We will also work on biological treatment of agricultural wastewater to reclaim water. .

Impacts
What was accomplished under these goals? During the reporting period, we focused on the biofuel production from lignocellulosic biomass using fungal fermentation. Even though lignocellulosic fuels have been intensively researched in the past decades, significant challenges still exist in successful realization of lignocellulosic biomass for biofuels and chemical production. The recalcitrant structure, dispersed nature of energy crops and agricultural residues, limited capacity of current available industrial strains to co-utilize C5 and C6 sugars, and tolerance of generated inhibitors during pretreatments are main barriers for biological conversion. In response to researching and developing new routes towards effective and sustainable biofuels/chemical production systems, our strategy is to use fungal fermentation to co-utilize C5/C6 sugars for lipid accumulation for biodiesel/biojet production, and the investigation is mainly being focused on two aspects: strain selection and optimization to increase efficiency of substance utilization towards target production; and development of multiple products to make the process more economic feasible. The first aspect has been achieved by applying advance systems biology techniques along with process optimization. An oleaginous fungus M. isabellina, which exhibits unique capacities of high lipid content, co-utilizing glucose, xylose and acetic acid, and tolerating relatively high concentrations of inhibition products, has been screened and selected as the strain to carry on fungal lipid accumulation on lignocellulosic hydrolysates. Corresponding to the selected fungal strain, we developed a novel lignocellulosic hydrolysis process called combined hydrolysis that maximizes biomass conversion of C5/ C6 sugars and acetic acid production, and eliminates the need of a large amount of water for washing and detoxification. Integrating the combined hydrolysis and oleaginous fungal fermentation led to a new route to utilize fungi to convert lignocellulosic materials into advanced fuels.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: 1.Xiao, Y., Ruan, Z., Liu, Z., Wu, S., Varman, A., Liu, Y., Tang, Y. 2013. Engineering Escherichia coli to convert acetic acid to free fatty acids. Biochemical Engineering Journal. 76, 60-69. 2. Lu, J., Weerasiri, R., Liu, Y., Wang, W., Ji, S., Lee, I. 2013. Enzyme Production by the Mixed Fungal Culture with Nano-Pretreated Biomass and Enzymatic Hydrolysis. Biotechnology and Bioengineering. 110,2123-2130. 3. Runrun Shi, Zhimin Li, Qin Ye, Jianhe Xu, Yan Liu. 2013. Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresource Technology 142, 338-44. 4. Liu, Z., Ruan, Z., Xiao, Y., Yi, Y., Tang, Y. Liao, W., Liu, Y. 2013. Integration of sewage sludge digestion with advanced biofuel synthesis. Bioresource Technology. 132, 166-172. 5. Ruan, Z. H., Zanotti, M., Ducey, C., Liao, W., Liu, Y. 2013. Co-hydrolysis of lignocellulosic biomass for microbial lipid accumulation. Biotechnology and Bioengineering. 110(4),1039-49. 6. Maclellan, J., Chen, R., Kraemer, R., Zhong, Y., Liu, Y., Liao, W. 2013. Anaerobic Treatment of Lignocellulosic Material to Co-produce Methane and Digested Fiber for Ethanol Biorefining. Bioresource Technology. 130,418-23. 7. Yue, Z. B., Chen, R., Yang, F., MacLellan, J., Marsh, T., Liu, Y., Liao, W. 2013. Effects of dairy manure and corn stover co-digestion on anaerobic microbes and corresponding digestion performance. Bioresource Technology. 128,65-71.

Progress 09/01/12 to 12/31/12

Outputs
OUTPUTS: I attended the 2012 AIChE Annual meeting which was held October 28th-November 2nd in Pittsburgh,Pennsylvania. My graduate students and I made three oral presentations: 1) Effects of farm wastes co-digestion on microbial community, methane production, and fiber quality as biorefining feedstock; 2)Transgenic expression of a bacterial exo-acting intracellular amylase in the Chlamydomonas reinhardtii chloroplast; 3) Comparison of fungal lipid accumulation on different lignocellulosic feedstocks. In addition, Nandan Cleantec Limited, one of the largest biodiesel companies in India, found out the technology through our publication, and would like to collaborate with us for applying the fungal process for biofuel and other valued-added chemical production. I am currently working on a proposal to Nandan Cleantec (they invited me to develop one). With Nandan's support and other external funding sources my research group is targeting on establishing a commercially feasible lignocellulosic fungal fuel/chemical production system. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In response to researching and developing new routes towards effective and sustainable biofuels/chemical production systems, our strategy is to use fungal fermentation to co-utilize C5/C6 sugars for lipid accumulation for biodiesel/biojet production. An oleaginous fungus M. isabellina, which exhibits unique capacities of high lipid content, co-utilizing glucose, xylose and acetic acid, and tolerating relatively high concentrations of inhibition products, has been screened and selected as the strain to carry on fungal lipid accumulation on lignocellulosic hydrolysates. Corresponding to the selected fungal strain, we also developed a novel lignocellulosic hydrolysis process called combined co-hydrolysis that maximizes biomass conversion of C5/ C6 sugars and acetic acid production, and eliminates the need of a large amount of water for washing and detoxification (See the following publication). Integrating the combined co-hydrolysis and oleaginous fungal fermentation led to a new route to utilize fungi to convert lignocellulosic materials into advanced fuels, which has demonstrated better production efficiency and economic performance than lignocellulosic ethanol production.

Publications

• Liu, Z., Ruan, Z., Xiao, Y., Yi, Y., Tang, Y. Liao, W., Liu, Y. (2012) Integration of sewage sludge digestion with advanced biofuel synthesis. Bioresource Technology (In press).
• Ruan, Z. H., Zanotti, M., Ducey, C., Liao, W., Liu, Y. (2012) Co-hydrolysis of lignocellulosic biomass for microbial lipid accumulation. Biotechnology and Bioengineering (In press).
• Maclellan, J., Chen, R., Kraemer, R., Zhong, Y., Liu, Y., Liao, W. (2012) Anaerobic Treatment of Lignocellulosic Material to Co-produce Methane and Digested Fiber for Ethanol Biorefining. Bioresource Technology (In press).