Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
THE SCIENCE AND ENGINEERING FOR A BIOBASED INDUSTRY AND ECONOMY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1001730
Grant No.
(N/A)
Project No.
CA-D-BAE-2230-RR
Proposal No.
(N/A)
Multistate No.
S-1041
Program Code
(N/A)
Project Start Date
Oct 22, 2013
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Project Director
Fan, ZH.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Biological and Agricultural Engineering
Non Technical Summary
The objectives of this project are to address research relating directly to biobased products and processing agricultural coproducts; research will influence rural community development and revitalizing rural economies indirectly. Increasing the breath of renewable energy production systems includes the production of power and second-generation liquid biofuels, including biomass-derived power generation. The objectives of this multistate project include (1) develop deployable biomass feedstock supply knowledge, processes and logistics systems that economically deliver timely and sufficient quantities of biomass with predictable specifications to meet conversion process-dictated feedstock tolerances; (2) investigate and develop sustainable technologies to convert biomass resources into chemicals, energy, materials and other value added products; (3) develop modeling and systems approaches to support development of sustainable biomass production and conversion to bioenergy and bioproducts; and (4) identify and develop needed educational resources, expand distance-based delivery methods, and grow a trained work force for the biobased economy. Most of this research is application oriented and will be useful to develop pilot projects, demonstrations and commercialization of biomass conversion to biobased products. Other outputs include educational materials that could be used in traditional classroom settings or for distance education and web based distribution, publications in peer reviewed journals, trade journals and popular magazines, development of intellectual property, and [resentations to economic development groups, legislative groups, and to the general public.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5111430202050%
5112010202050%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
1430 - Greens and leafy vegetables; 2010 - Sugar beet;

Field Of Science
2020 - Engineering;
Goals / Objectives
Develop deployable biomass feedstock supply knowledge, processes and logistics systems that economically deliver timely and sufficient quantities of biomass with predictable specifications to meet conversion process-dictated feedstock tolerances. Investigate and develop sustainable technologies to convert biomass resources into chemicals, energy, materials and other value added products. Develop modeling and systems approaches to support development of sustainable biomass production and conversion to bioenergy and bioproducts. Identify and develop needed educational resources, expand distance-based delivery methods, and grow a trained work force for the biobased economy
Project Methods
Objective B: Investigate and develop sustainable technologies to convert biomass resources into chemicals, energy, materials and other value added products. Despite the efforts that have been made in conversion technologies, deployment of commercial facilities, using either biochemical or thermochemical platforms, is still lacking. Improving sugar, bioenergy or bioproduct yields would be valuable in enabling these bioprocesses. Integration between feedstock supply and conversion could be one step in improving yields. The multi-state community can facilitate the integration between feedstock logistics and conversion by providing insight between conversion and biomass biological and physical properties. B.1. Biological conversion technologies Task 1: Develop pretreatment methods for biological conversion processes. Pretreatments are essential to deconstruct biomass, releasing sugars that can be converted to bioenergy and bioproducts. We will evaluate different pretreatment technologies (diluted acid, steam, alkaline pretreatment) on different feedstocks such as rice and wheat straw, poplar or food processing residuals. Task 2: Develop conversion processes.Biofuels, including advanced liquid transportation fuels, cellulosic ethanol, butanol will be investigated, integrating feedstock pretreatment to fermentation. Platform chemicals, food additives or animal feeds (single cell protein) will be researched. Task 3: Develop value-added, bio-based products from fractionated biomass. Fractionated biomass will be examined for value-added products development including lignin-based chemicals and materials, nontraditional oilseeds and other fine chemicals.

Progress 10/22/13 to 09/30/18

Outputs
Target Audience:Students are target audience in this project since this project provides a training and research platform for them. In a larger scale, the emerging biorefinery industries and the whole biofuels community are our target audience as we are generating new technology and new knowledge which lay the foundation for the bio-conversion science and engineering. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant provided training opportunities for two graduate students, five undergraduate students, and one postdoctoral researcher. How have the results been disseminated to communities of interest?We dissembled results through publication of manuscripts in peer-reviewed journal, presentations and posters in international conferences and commodity board meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The goal of this project is to increase the breath of renewable energy production systems which include the production of power and second-generation liquid biofuels. During the project period, our work was mainly focused on: Objective 2 Investigate and develop sustainable technologies to convert biomass resources into chemicals, energy, materials and other value added products We worked on developing new technologies for fuels and chemicals production from cellulosic biomass. We have engineered a strain which is able to convert cellulose to cellobionate without any enzyme addition. Starting from the wild type strain N. crassa 2489, which produces a full array of cellulases, we deleted six of seven of its β-glucosidase (bgl) genes, two transcription factor genes (cre-1 and ace-1) involved in carbon catabolite repression, and a gene encoding a cellobionic acid phosphorylase gene (ndvB) needed for cellobionic acid utilization, and over-expressed a laccase from Botrytis aclada under a copper metallothionein promoter. The resulting strain (N. crassa strain HL10 ) was able to produce 47.4 mM of cellobionic acid from 20g/L cellulose without the addition of any enzymes. The yield of cellobionic acid from hydrolyzed cellulose was about 94 %. To our knowledge, this is the first example of production of an organic acid from cellulose at high yield without any enzyme addition. Conversion of cellobiose to cellobionic acid was found to be able to improve cellulose conversion and increase product yield. We also developed a new tool for gene editing in N. crassa. We tested a new a bacterial recombination system employing β-recombinase acting on six recognition sequences (β-rec/six) which allowed repetitive site-specific gene deletion and marker recycling in N. crassa. The advantage of this new method is that it needs only one transformation per deletion, while the widely used Cre/LoxP system needed three transformations. Another big advantage of this system is the strict cis action of the -recombinase, which eliminates the danger of undesired and uncontrolled chromosomal rearrangements. We demonstrated the functionality of the beta-recombinase based gene knockout system in N. crassa for the first time. In this report period, we studied the inhibition of cellulase by a novel substrate cellobionic acid. While inhibition of cellulases by cellobiose has been extensively studied, cellobionic acid inhibition of cellulases has not received much attention. We investigated the inhibition of cellobiohydrolase I by cellobiose and cellobionic acid using p-nitrophenyl-β-D-lactopyranoside as a model cellulosic substrate. Additionally, cellobionic acid inhibition of CDH was evaluated. The kinetic properties and mechanism of cellobionic acid inhibition were studied and compared to that of cellobiose inhibition. To our knowledge, this study provided the first direct comparison of cellobiose and cellobionate inhibition of CBHI and the first kinetic evaluation of cellobionate inhibition of CDH. We investigated the alcohol consumption and tolerance of N. crassa. N. crassa was found to be able to consume both a native alcohol-ethanol and a non-native alcohol isobutanol as the carbon source. The rate of ethanol consumption is faster than that of isobutanol. The deletion of the one of two major alcohol dehydrogenase gene (adh1) from the genome can efficiently prohibit both ethanol and isobutanol consumption by this strain, confirming this gene encodes an ADH responsible for alcohol consumption. The deletion of the adh3 does not have any obvious effect on preventing alcohol consumption. N. crassa Δadh1 can tolerate up to 50 g/L ethanol and 8.8 g/L isobutanol when grown on glucose or Avicel as the carbon source. Such information are very useful for the biofuels community especially for those who are interested in using N. crassa as consolidating bioprocessing microorganism for alcohol production (Fan). We also investigated the conversion of lactose in cheese whey to a high value chemical lactobionic acid (LBA). LBA is a versatile, high-value, low-volume chemical with numerous applications in the food, pharmaceutical, cosmetic, and chemical industries. We have engineered a N. crass strains which heterologous expressing laccase and a strain heterologously expressing cellobiose dehydrogenase, LBA was able to produced by a mixture of the strain using lactose or cheese whey as the substrate. We investigated beneficial effect of gluconate on Escherichia coli fermentation using lean medium. In a previous study, E. coli AH003, a derivative of E. coli KO11 was constructed by deleting L-lactatedehydrogenase (ldh) and pyruvate formate lyase (pfl) genes. This strain was found to convert gluconate to ethanol at higher yield (97.5%) as compared to its parent strain in rich media. E. coli AH003 and its parent strain E. coli KO11 were used as the ethanologen to convert glucose and gluconate to ethanol in M9 minimal medium. E. coli AH003 grew very poorly on glucose in M9 medium. However, it achieved rapid growth when gluconate was used as the carbon source. The addition of gluconate to medium containing glucose improved the rate of glucose utilization. In contrast, E. coli KO11 grew well on both glucose and gluconate in M9 medium. The addition of gluconate to medium containing glucose did not improve the rate of glucose utilization. We believe that the deletion of the pfl gene in E. coli AH003 led to the different fermentation results. The co-fermentation of gluconate and glucose could be a useful strategy to improve the rate of glucose fermentation and decrease nutrient requirements for engineered strains lacking the pfl gene and grown under anaerobic conditions.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Lin, H., A. Hildebrand, T. Kasuga, Z. Fan. 2017 Engineering Neurospora crassa for cellobionate production directly from cellulose without any enzyme addition. Enzyme and Microbial technology, 99: 25-31.
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: ao W., Y. Wang, E. W. Walters, H. Lin, S, Li, H. Huang, T. Kasuga, Z. Fan. 2018 Homoethanol Production from Glycerol and Gluconate Using Recombinant Klebsiella oxytoca Strains. Applied and Environmental Microbiology.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Tao, W., Z. Fan, H. Lin, H. Huang, 2017 Conversion of Gluconate and Glycerol to Ethanol Using the Recombinant Klebsiella Oxytoca Strains, AICHE meeting, Salt Lake City, UT.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The scientific community, policy makers, industry and other stakeholders interested in bioenergy production and biomass conversion. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant provided training opportunities for one graduate student, one undergraduate students, and one postdoctoral researcher. How have the results been disseminated to communities of interest?We have published one paper in a peer-reviewed journal. We also present our work in international conferences such as American Institute of Chemical engineers annual meeting. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Lactobionic acid (LBA) is a versatile, high-value, low-volume chemical with numerous applications in the food, pharmaceutical, cosmetic, and chemical industries. Microbial production of LBA has the advantages of high selectivity and the ability to use cheese whey, a waste stream from the dairy industry, which is rich in lactose. However, microbial LBA production has not yet been developed as successfully as other organic acids, despite attempts being made to use wild type bacteria, fungi and yeast strains. There is no commercial implementation of microbial LBA production technologies due to the following factors: 1) The most robust strains are pathogenic, which prevents their industrial implementation. 2) The enzyme (cellobiose dehydrogenase (CDH) or lactose dehydrogenase) which oxidizes lactose to LBA is produced only in the stationary phase. The highest level production of LBA was only achievable under resting cell conditions, which are not usually used in industrial fermentations. 3) The microorganism consumed LBA at the later stage of the fermentation. We report engineering a Neurospora crass strain for cellobionic acid production from cheese whey and solving the problems associated with LBA fermentation using metabolic engineering efforts. We have engineered a strain which heterologous expressing laccase and a strain heterologously expressing cellobisoe dehydrogenase, LBA was able to produced by a mixture of the strain using lactose or chesse whey as the substrate.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Lin, H., A. Hildebrand, T. Kasuga, Z. Fan. Engineering Neurospora crassa for cellobionate production directly from cellulose without any enzyme addition. Enzyme and Microbial technology. 99: 25-31
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Lin, H, X. Zhou, T, Kasuga, and Z. Fan:: Conversion of cheese whey to lactobionic acid, 2016 AICHE meeting, San Francisco.


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

Outputs
Target Audience:The scientific community, policy makers, industry and other stakeholders interested in bioenergy production and biomass conversion. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant provided training opportunities for two graduate student, one undergraduate students, and one postdoctoral researcher. How have the results been disseminated to communities of interest?We have published one paper in a peer-reviewed journal. We also present our work in international conferences such as American Institute of Chemical engineers annual meeting. What do you plan to do during the next reporting period to accomplish the goals?We will work on testing production of cellobionic acid from pretreated cellulosic substrates.

Impacts
What was accomplished under these goals? Our research group has been working on engineering Neurospora crassa for cellobionic acid production without any enzyme addition. Starting from the wild type strain N. crassa 2489, which produces a full array of cellulases, we deleted six of seven of its β-glucosidase (bgl) genes, two transcription factor genes (cre-1 and ace-1) involved in carbon catabolite repression, and a gene encoding a cellobionic acid phosphorylase gene (ndvB) needed for cellobionic acid utilization. With these modifications, cellulose degradation by the engineered fungus N. crassa F5?ace-1?cre-1?ndvB will be stopped at the cellobiose stage, while some glucose generated via the side reaction of cellulases could still support fungal growth and enzyme production. With the presence of cellobiose dehydrogenase (CDH) naturally produced by the fungus, part of the cellobiose is dehydrogenized to produce cellobionate (Publication #19). We found that the conversion of cellobiose to cellobionate was limited by the slow re-oxidation of CDH by molecular oxygen. As a result, cellobiose and cellobionate were produced at a ratio of 2 to 1. The yield of cellobionate from hydrolyzed cellulose was about 25%. We adopted a CDH-redox mediator-laccase system to accelerate the electron transfer from the reduced CDH to molecular oxygen. Adding catalytic amount of laccase and redox mediator to the fermentation system, cellobiose can quickly be converted to cellobionate. The yield of cellobionate from cellulose can be improved to 90% as compared to 25%, which was the case without laccase and redox mediator addition (. Since exogenous laccase addition still represents a substantial cost, we looked at heterologously expressing laccase in N. crassa. We have tried different promoters and different sources of laccases. We successfully expressed a laccase from Botrytis aclada under a copper metallothionein promoter in F5?mus-51?ace-1?cre-1?ndvB, resulting N. crassa strain HL10. N. crassa HL10 was able to produce 47.4 mM of cellobionic acid from cellulose without the addition of any enzymes. The yield of cellobionic acid from hydrolyzed cellulose was about 94 %. To my knowledge, this is the first example of production of an organic acid from cellulose at high yield without any enzyme addition. Conversion of cellobiose to cellobionic acid was found to improve cellulose conversion and increase product yield (Submitted #1). In this report period, we studied the inhibition of cellulase by a novel substrate cellobionic acid. While inhibition of cellulases by cellobiose has been extensively studied, cellobionic acid inhibition of cellulases has not received much attention. We investigated the inhibition of cellobiohydrolase I by cellobiose and cellobionic acid using p-nitrophenyl-β-D-lactopyranoside as a model cellulosic substrate. Additionally, cellobionic acid inhibition of CDH was evaluated. The kinetic properties and mechanism of cellobionic acid inhibition were studied and compared to that of cellobiose inhibition. To our knowledge, this study provided the first direct comparison of cellobiose and cellobionate inhibition of CBHI and the first kinetic evaluation of cellobionate inhibition of CDH.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hildebrand, A., J.B. Addison, T. Kasuga, Z, Fan. (2015) Cellobionic acid inhibition of cellobiohydrolase I and cellobiose dehydrogenase. Biochemical Engineering Journal, 109: 236-242
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Hildebrand, A., J.B. Addison, T. Kasuga, Z, Fan.: Cellobionic acid inhibition of cellobiohydrolase I and cellobiose dehydrogenase., 2015 AICHE meeting, Salt lake City.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Lin, H, T, Kasuga, and Z. Fan: Engineering Neurospora crassa for cellobionic acid production from cellulose, 2015 AICHE meeting, Salt Lake City.
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Lin, H., A. Hildebrand, T. Kasuga, Z. Fan. Engineering Neurospora crassa for cellobionate production directly from cellulose without any enzyme addition. Enzyme and Microbial technology. *


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

Outputs
Target Audience:The scientific community, policy makers, industry and other stakeholders interested in bioenergy production and biomass conversion. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant provided training opportunities for two graduate student, one undergraduate students, and one postdoctoral researcher. How have the results been disseminated to communities of interest?We have published two papers in a peer-reviewed journal and one thesis. We also present our work in international conferences and regional conferences. What do you plan to do during the next reporting period to accomplish the goals?We will engineer Neurospora crassa for heterologously laccase expression to enable cellobionate production from cellulose without any enzyme addition.

Impacts
What was accomplished under these goals? Neurospora crassa has been engineered for direct cellobionate production from cellulose. In our previous effort, N. crassa was deleted six out of seven beta-glucosidase genes (bgl), the resulting strain F5 was bale to produce cellobiose as the major product and produce cellobionate as a minor product. We found that the regeneration of cellobiose dehydrogenase (CDH) by oxygen is the rate-limiting step in converting cellobiose to cellobionate. The rate and yield of cellobionate from cellulose can be improved by adding low concentrations of laccase and a redox mediator to the fermentation system to speed up cellobiose conversion to cellobionate. The cellobionate yield was optimized by varying fermentation conditions such as pH, buffer strength, and laccase and redox mediator addition time. Mass and material balances were performed. It was found that cellobionate yield from consumed Avicel was improved to 91% with the laccase and redox mediator addition (Fan). The alcohol consumption and tolerance of N. crassa were also investigated. N. crassa was found to be able to consume both a native alcohol-ethanol and a non-native alcohol isobutanol as the carbon source. The rate of ethanol consumption is faster than that of isobutanol. The deletion of the one of two major alcohol dehydrogenase gene (adh1) from the genome can efficiently prohibit both ethanol and isobutanol consumption by this strain, confirming this gene encodes an ADH responsible for alcohol consumption. The deletion of the adh3 does not have any obvious effect on preventing alcohol consumption. N. crassa Δadh1 can tolerate up to 50 g/L ethanol and 8.8 g/L isobutanol when grown on glucose or Avicel as the carbon source. Such information are very useful for the biofuels community especially for those who are interested in using N. crassa as consolidating bioprocessing microorganism for alcohol production (Fan).

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hildebrand, A.,�Kasuga, T., and Fan, Z �2015.�Production of cellobionate using an engineered Neurospora crassa strain with laccase and redox mediator addition. PLoS ONE , 10: e0123006
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Desai, S., Rabinovitch-Deere, C.A., Fan, Z., and Atsumi, S. 2015�Isobutanol production from cellobionic acid in Escherichia coli. �Microbial Cell Factory, 14: 52
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hildebrand, A., Szewczyk, E., Lin, H.,�Kasuga, T., and Fan, Z. 2015. Engineering Neurospora crassa for improved cellobiose and cellobionate production.�Applied and Environmental Microbiology, 81:595-603
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Amanda Hildebrand, A biochemicxal route for fuels and chemicals production from cellulosic biomass, Ph.D. thesis University of California , Davis. Advisor: Zhiliang Fan
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Hildebrand, A, Szewczyk, E., Kasuga,�T., Fan, Z. ��Engineering Neurospora crassa for improved cellobionate production from cellulose,�presented at the 2015 AICHE annual meeting, Atlanta, GA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Fan, Z., Sun, Y., Han, S., Ma, D., Conversion of glucose and gluconate mixture to ethanol by E coli. AH003,�presented at the 2015 AICHE annual meeting, Atlanta, GA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Hildebrand, A, Szewczyk, E., Kasuga,�T., Fan, Z. �Engineering Neurospora crassa for improved cellobionate production from cellulose, accepted for oral presentation, at the 36th Symposium on Biotechnology for Fuels and Chemicals, at the Hilton Clearwater Beach, Clearwater Beach, FL USA.


Progress 10/22/13 to 09/30/14

Outputs
Target Audience: The scientific community, policy makers, industry and other stakeholders interested in bioenergy production and biomass conversion. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project contributes to the development of knowledge and human resource skills. We are training many graduate students and post doctoral scholars on this project. Their training is unique in that it has a focus in both engineering and cell biology with application to sustainable agriculture and energy production. Graduate students are also trained in the mentoring of undergraduates offering multiple opportunities for human resource development. Partner organizations include government agencies and private companies, such as US Department of Agriculture, California Energy Commission, Chevron Technology Ventures, Mendota Bioenergy, CleanWorld, and others. How have the results been disseminated to communities of interest? The research results have been published in scientific articles, conference papers, presentations, lecture notes and research reports. What do you plan to do during the next reporting period to accomplish the goals? As part of the work on developing new pretreatment methods for biological conversion process, work is being done to characterize other microorganisms and microbial communities tolerant to new ionic liquids. As part of the work on developing new conversion technology, work is being done to further develop sugar beet to ethanol conversion technology, complete the commercial demonstration of beet ethanol plant in the Central Valley in California, and developcost effective technologies for producing biofertilizer products from anaerobic digestate. Work is also being done on developing a new biochemical conversion process through producing sugar aldonic acids. We will continue to improve cellobonate yield from cellulose by further engineering the Neurospora crassa strain.

Impacts
What was accomplished under these goals? Work continued on the development of an integrated geospatial optimization model to evaluate hybrid poplar feedstock production across the Pacific Northwest. The model is used to assess sustainability metrics on both a site-specific and system-wide basis and is spatially explicit and flexible to the desired resolution. The integration framework includes poplar growth models (3PG-Coppice, EPIC), bioenergy crop adoption (BCAM), and statewide agricultural production (SWAP) models to examine crop substitution effects, and a geospatial bioenergy systems model (GBSM) to determine optimal siting for biorefineries based on the desired regional outcomes. Environmental lifecycle assessment and socioeconomic impacts are also modeled. A major objective is the development of interactive quantitative tools to support decision processes by landowners and others involved in the development of new biomass resources for biofuels and bioenergy. A web-based application was developed for general public access that can be used to predict poplar growth and production on a site specific basis using default or user specifications for soil, climate, and other inputs. Larger scale applications have been developed to evaluate economic, environmental, and social impacts for optimized biorefinery siting in the region. Research was done to develop pretreatment methods for biological conversion processes. High-solids incubations were performed to enrich for microbial communities and enzymes that deconstruct rice straw under mesophilic (35 ?C) and thermophilic (55 ?C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome and metatranscriptome sequencing were performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Sugar beets were investigated as an extremely efficient, high-yielding industrial crop for renewable biofuel production. A non-traditional approach employing enzymatic liquefaction and fermentation of whole sugar beets for bioethanol combined with anaerobic digestion of stillage was developed and tested in the lab. A pilot demonstration of this process was conducted at the UC Davis Biogas Energy Project facility using approximately 40 tons of beets. To achieve rapid scale-up objectives, low severity pretreatment, readily available process equipment, commercial enzymes, industrial unmodified S. cerevisiea and minimal fermentation controls were employed. Triplicate 5-ton batch SSF fermentations averaged 0.36 gram-ethanol per gram-initial total solids, which was approximately 90% of that achieved in the lab under similar conditions. Biogas production from stillage at both lab and pilot operations indicate specific biogas production rates over 350ml CH4/gVS are achievable and could be sufficient to offset a majority of facility fuel requirements at industrial scale. The optimum conditions for beet liquefaction and fermentation were determined. Research is underway to determine the best approach and equipment for enzyme recycling in the beet conversion system. A commercial demonstration beet ethanol plant has been designed and is currently under construction. The major accomplishment in the area of anaerobic digestion was the completion of UC Davis Renewable Energy Anaerobic Digestion (READ) facility in January 2014. The READ facility has been in operation since then, converting approximately 50 tons of food and animal waste each day into electricity and heat. It uses a three-stage high solids anaerobic digestion technology developed at UC Davis. The performance data of digesters have been collected and reported. Research has also been carried out to process the digestate into biofertilizer products. Advanced solid-liquid separation technologies were studied and optimized to achieve the effective nutrient separation and concentration. We also worked on developing a new biochemical route for fuels and chemicals production from cellulosic biomass. We further engineered Neuropsora crassa strain F5 for higher yield cellobiose and cellobionate production from cellulose. The effects of two catabolite repression genes cre-1 and ace-1 on cellulase production were investigated. The F5?ace-1 mutant showed no improvement over the wild type. The F5?cre-1 and F5?ace-1?cre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5?ace-1?cre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5?ace-1?cre-1?ndvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, as compared to 19% by the strain F5?ace-1?cre-1. We also improved the yield of cellobionate from cellulose by adding low concentrations of laccase and a redox mediator to the fermentation system The cellobionate yield was optimized by varying fermentation conditions such as pH, buffer strength, and laccase and redox mediator addition time . Mass and material balances were performed. It was found that cellobionate yield from consumed Avicel was improved to 91% with the laccase and redox mediator addition.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Xiao, X., R. Zhang, Y. He, Y. Li, L. Feng, C. Chen and G. Liu. Influence of particle size and alkaline pretreatment on the anareobic digestion of corn stover. BioResources, 2013, 8(4): 5850-5860.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhang, J., Y. Wang, L. Zhang, R. Zhang, G.Liu and G. Cheng. Understanding changes in cellulose crystalline structure of lignocelluosic biomass during ionic liquid pretreatment by XRD [J]. Bioresource Technology, 2014, 151(2014):402-405.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, J., L. Feng, D. Wang, R. Zhang, G. Liu and G. Cheng.. Theomogravimetric analysis of lignocellulosic biomass with ionic liquid pretreatment. Bioresource Technology, 2014, 153(2014):379-382.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Li, Y. R. Zhang, Y. He, C. Zhang, X. Liu and C. Chen. Anaerobic co-digestion of chicken manure and corn stover in batch and continuously stirred tank reactor (CSTR). 2014. Bioresource Technology (2014):342-347.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Li, J., S.M. Zicari, Z. Cui and R. Zhang. Processing anaerobic sludge for extended storage as anaerobic digester inoculum. Bioresource Technology. 2014. 166(2014):201-210.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Prilepova, O., Q. Hart, J. Merz, N. Parker, V. Bandaru and B. Jenkins.� 2014.� Design of a GIS-based web application for simulating biofuel feedstock yields.� ISPRS Int. J. Geo-Inf. 3:929-941.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Steenwerth, K.L., A.K. Hodson, A.J. Bloom, M.R. Carter, A. Cattaneo, C.J. Chartres, J.L. Hatfield, K. Henry, J.W. Hopmans, W.R. Horwath, B.M. Jenkins, E. Kebreab, R. Leemans, L. Lipper, M.N. Lubell, S. Msangi, R. Prabhu, M.P. Reynolds, S.S. Solis, W.M. Sischo, M. Springborn, P. Tittonell, S.M. Wheeler, S.J. Vermeulen, E.K. Wollenberg, L.S. Jarvis and L.E. Jackson.� 2014.� Climate-smart agriculture global research agenda: scientific basis for action.� Agriculture & Food Security 3:11,
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Asato, C. J. Li, S. Zicari and R. Zhang: Anaerobic digestion of bioethanol stillage for biogas energy production and nutrient and water recovery, Presentation at 2014 ASABE and CSBE/SCGAB Annual International Meeting. Montreal, Canada.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Szewczyk, E., T. Kasuga, and Z. Fan. 2014 A new variant of self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa. Journal of Microbiological Methods, 100: 17-23.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hildebrand, A.,E. Szewczyk, H. Lin,T. Kasuga, Z. Fan. 2015 Engineering Neurospora crassa for improved cellobiose and cellobionate production. Applied and Environmental Microbiology, 81: 595-603.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Simmons C, Reddy A, D'Haeseleer P, Khudyakov J, Billis K, Pati A, Simmons B, Singer S, Thelen M, VanderGheynst J. 2014. Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw. Biotechnology for Biofuels 7(1):495
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Simmons CW, Reddy AP, Simmons BA, Singer SW, VanderGheynst JS. 2014. Bacillus coagulans tolerance to 1-ethyl-3-methylimidazolium-based ionic liquids in aqueous and solid-state thermophilic culture. Biotechnology Progress 30(2):311-316.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Yu C, Thy P, Wang L, Anderson SN, VanderGheynst JS, Upadhyaya SK, Jenkins BM. 2014. Influence of leaching pretreatment on fuel properties of biomass. Fuel Processing Technology 128:43-53.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fan, Z., Y. Sun, S. Han, D. Ma: Conversion of glucose and gluconate mixture to ethanol by E coli. AH003, AICHE meeting, AICHE annual meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Hildebrand, A, E. Szewczyk, T. Kasuga, Z. Fan: Engineering Neurospora crassa for improved cellobionate production from cellulose, accepted for oral presentation, at the 36th Symposium on Biotechnology for Fuels and Chemicals, at the Hilton Clearwater Beach, Clearwater Beach, FL USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zicari, S. N. Aramrueang, C. Asato, C. Chen and R. Zhang: Integrated processing of sugar beets at the lab and pilot scale for bioethanol and biogas production, Abstract and Poster presentation at 36th Symposium on Biotechnology for Fuels and Chemicals. April 29, 2014. Clearwater Beach, FL.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Simmons CW, Reddy AP, Simmons BA, Singer SW, VanderGheynst JS. 2014. Effect of inoculum source on the enrichment of microbial communities on lignocellulosic biofuel feedstocks under thermophilic and high-solids conditions. Journal of Applied Microbiology. 117(4) 1025-1034.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zhang, M. and R. Zhang: Integrated biological treatment and membrane separation for nutrient recovery from the effluent of anaerobic digester, Presentation at 2014 ASABE and CSBE/SCGAB Annual International Meeting. Montreal, Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zicari, S., N. Aramrueang and R. Zhang: Pilot scale demonstration of bioethanol production from sugar beet, Presentation at 2014 ASABE and CSBE/SCGAB Annual International Meeting. Montreal, Canada.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Tittmann, P., Q. Hart, C. W. Murphy, N. Parker and B. Jenkins.� 2013.� Heuristic biorefinery location analysis in the US Pacific Northwest.� Association of American Geographers Annual Meeting, Los Angeles, California.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Bandaru, V. 2014. Systems for advanced biofuels production for woody biomass in the Pacific Northwest Incorporating. Bioenergy into Sustainable Landscape Designs Workshop. New Bern, North Carolina, March.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Yeo, B.-L., N. Parker, M. Jenner, S. Kaffka. 2014. The Bioenergy Crop Adoption Model (BCAM): Economics of sustainably producing hybrid poplars as a short-rotation woody biomass feedstock in the Pacific Northwest. Northwest Wood-based Biofuels + Co-Products Conference, Seattle, Washington, April.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Bandaru. V., Q. Hart, O. Prilepova, J. Mertz, N. Parker, B. Jenkins. 2014. Can Short Rotation Hybrid Poplar Be a Promising Sustainable Energy Supply Source in Pacific Northwest Region? Northwest Wood-based Biofuels + Co-Products Conference, Seattle, Washington, April.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Hart. Q., O. Prelipova, N. Parker, J. Merz, P. Tittmann, B. Jenkins. 2014. Modeling poplar growth as a short-rotation woody crop for biofuels in the Pacific Northwest. Northwest Wood-based Biofuels + Co-Products Conference, Seattle, Washington, April.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Bandaru. V., N. Parker, Q. Hart, O. Prilepova, J. Mertz, S. Kaffka, B. Jenkins. 2014. Developing an integrated sustainability framework for the assessment of poplar based bioenergy systems. International Bioenergy & Bioproducts Conference, Tacoma, Washington, September.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Hart. Q., O. Prelipova, N. Parker, J. Merz, P. Tittmann, B. Jenkins. 2014. Modeling poplar growth as a short-rotation woody crop for biofuels in the Pacific Northwest. 22nd European Biomass Conference and Exhibition, Hamburg, Germany, June.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Understanding the Economics of Poplar-based Jet Fuels in the Pacific Northwest. 22nd European Biomass Conference and Exhibition, Hamburg, Germany, June.Parker. N., M. Jenner, J. Crawford, Q. Hart, S. Boucarum, Y. Li, P. Tittmann, S. Kaffka, B. Jenkins. 2014. Understanding the Economics of Poplar-based Jet Fuels in the Pacific Northwest. 22nd European Biomass Conference and Exhibition, Hamburg, Germany, June.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Bandaru. V., Q. Hart, O. Prilepova, J. Mertz, N. Parker, B. Jenkins. 2014. Net Energy and Water Implications Associated with Regional Poplar Production for Biofuels on Marginal Lands. 22nd European Biomass Conference and Exhibition, Hamburg, Germany, June.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Bandaru. V., N. Parker, Q. Hart, O. Prilepova, J. Mertz, Y. Li, S. Boucarum, S. Kaffka, B. Jenkins. 2014. Integrated Modeling Framework for Bioenergy Sustainability Assessment; A Case Study Determining Sustainable Biorefinery Systems in the State of California, USA. 22nd European Biomass Conference and Exhibition, Hamburg, Germany, June.