Team of Biomass Engineering

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1 74 Team of Biomass Engineering

2 Team of Biomass Engineering Brief Introduction The team of biomass engineering focuses on the key scientific problems in the production of bio-based energy, materials and chemicals from non-grain biomass, especially from lignocellulosic biomass. The target is to establish green technology platforms for biomass utilization, provide technical support and theoretical basis for industrialization, and consequently improve the international competitiveness in biomass engineering research. The group is engaged in the major research areas including: 1) component fractionation and directional conversion technologies of lignocellulosic biomass, 2) process engineering for the integrated production of bio-based energy, materials and chemicals from biomass, 3) new process on natural medicine production from medicinal plants, and 4) biodenitrification, bio-desulfurization and bio-removal of heavy metal with petroleum microbe. Research Activities 1. Key Technologies and Demonstration for Straw Ethanol Fermentation Coupled with Solid State Enzymatic Hydrolysis Controversies on bioethanol production from straw have mainly surrounded the economical feasibility and environmental impacts for decades. This is primarily caused by lack of efficient systematic integration of the related specialties regarding to the characteristics of straw. To achieve an economical and environmentally-friend bioethanol production system from straw, a number of breakthroughs are needed, not only in individual process, but also in the balance and integration of these processes. An assembled pretreatment technology by coupling steam explosion with mechanism carding and alkaline hydrogen peroxide was invented which can degrade 85.08% and 97.6% of hemicellulose and lignin, respectively. The cost of cellulase in the straw ethanol production was decreased from 2350 to 1500 RMB/t. Finally, a demonstration production line with annual production of 3000 tons of straw ethanol was established at the cost of straw ethanol of 5500 RMB/t. 2. Industrialized Bio-refining Line for Straw with the Multi-production of Biobased Products The ultimate aim of biomass-economy is to replace the products coming from oil refineries by biomass refineries. Lignocellulosic biomass is an integrator of multifarious functionalized macromolecules coupled with complex chemical bonds. It is difficult to be economically degraded into monosaccharides and used as the raw material for biotransformation. In our group a t/a straw refining industrial production line was established at Jilin Province in In this straw refining industrialization line, butanol, acetone and ethanol are produced from the fermentation of hemicelluloses, long fibers used for paper making, short fibers and lignin used for preparing polyether, phenolic resins and other products. It has the annual capacity of tons of butanol, tons of acetone and ethanol, tons of high purity cellulose. This project could receive total profit of 660 million RMB with the poly-production of solvent (butanol, acetone, and ethanol), phenolic resin and polyether polyols. This production line creates a new industrial refining model using straw as raw material and breaks the technically and economically long-standing problems that bothers the biomass industry. 75

3. Process Intensification and Integration on Gas Bio-desulfurization Bio-desulfurization is a novel technology for deeply removing hydrogen sulfide and recovering sulfur, which has many advantages

Identification on the key factors restricting the development of bio-desulfurization in order to overcome the deficiency of existing technology has been one of the most important focuses of our

The mechanism of the sulfur oxidases system in them by identifying the coding genes of key enzyme and the regulation of these genes expression has been illustrated.

Infrastructure From 1986, IPE has been carrying out the research on biomass engineering.

magnetic resonance, near infrared spectroscope.

3 3. Process Intensification and Integration on Gas Bio-desulfurization Bio-desulfurization is a novel technology for deeply removing hydrogen sulfide and recovering sulfur, which has many advantages over chemical method, such as simple process, easy operation and low cost. Identification on the key factors restricting the development of bio-desulfurization in order to overcome the deficiency of existing technology has been one of the most important focuses of our research. So far, we have obtained several halo-alkaline sulfide oxidizing bacteria. The mechanism of the sulfur oxidases system in them by identifying the coding genes of key enzyme and the regulation of these genes expression has been illustrated. A novel bio-desulfurization bioreactor has been designed for biogas cleaning. Owing to these achievements, a 1,000 Nm 3 /d pilot plant for biogas bio-desulfurization is going to be built. Infrastructure From 1986, IPE has been carrying out the research on biomass engineering. After development for nearly thirty years, the biomass engineering group has possessed various basic equipments and advanced analytical instruments, such as HPLC, GC, LC-MS, low field nuclear magnetic resonance, near infrared spectroscope. Moreover, several apparatus platforms for biomass engineering have been designed with the completely independent intellectual property rights, including steam explosion apparatus, liquid hot water and microwave apparatus, gas double dynamic solid-phase Fig. 1 Apparatus for biomass engineering designed by biomass engineering group [(A) Low pressure steam explosion apparatus (50 L); (B) Continuous solid-state simultaneous enzymatic hydrolysis, ethanol fermentation and ethanol separation apparatus (500 L); (C) Ultrasound extraction device for the active ingredients in plants (200 L); (D) Gas double dynamic solidstate fermentation reactor (200 L); (E) Continuous microwave pretreatment apparatus for b fermentation reactor, etc. (Fig. 1). Many types of equipment have been scaled up and used in the industrialization project of biomass. For example, the low pressure steam explosion apparatus was designed to realize the effective pretreatment of biomass without adding any chemical reagents and scaled up to 1, 5, 20, 30 and 50 m 3 in size. Gas double dynamic solid-state fermentation reactor was devised to improve the heat and mass transfer during the solid-state fermentation process and consequently enhance the fermentation efficiency. So far, it has been scaled up to 5, 50 and 100 m 3 which can realize the online parameter monitoring. The group has 27 staff members, including 6 professors, 7 associate professors, 7 assistant professors, 5 engineers, and 2 administrators. Many of them are the leading talents in the field of biomass. These entire infrastructures guarantee the successful completion of the research projects and the international advanced level of the research achievements in the biomass engineering group. 76

characterized with complex porous media, such as porosity, surface area, and permeability.

$resistance of porous plant biomass to mass transfer. It is determined by porous structure parameters including pore size distribution, specific surface area, tortuosity, fractal dimension and so on.$

4 Research Progress 1. Intrinsically Porous Characteristics of Lignocellulosic Biomass and the Concept of Seepage Recalcitrance in Terms of Process Engineering As solid phase substrates, lignocellulosic biomass is characterized with complex porous media, such as porosity, surface area, and permeability. The heat and mass transfer of porous media are involved in a few major operation units of lignocellulose bioconversion. These pores are used for the transfer of fluids (Fig. 2). Based on the cognition of intrinsically porous characteristics of lignocellulosic biomass, the concept of seepage recalcitrance in terms of process engineering was proposed, and defined as the resistance of porous plant biomass to mass transfer. It is determined by porous structure parameters including pore size distribution, specific surface area, tortuosity, fractal dimension and so on. The proposed biomass seepage recalcitrance is distinct from biomass recalcitrance which would advance our understanding about the relationships between structure and enzymatic hydrolysis of lignocellulosic biomass. Moreover, for plant biomass refining, pretreatment technology would be guided with an engineering index (threshold pressure) and enzymatic hydrolysis would be intensified by overcoming the threshold pressure. (a) (b) (c) Fig. 2 SEM images of the transverse section of corn stalk pith [(a) The corner space between parenchyma cells (b), and the fiber cell around vascular tissue (c). Where, (a) can represent the pores on organ level, (b) and (c) can represent the pores on cell level. By illustrating the pore size distribution of corn stalks on different levels, we can reveal the porous media nature of plant biomass.] 2. Metabolic Engineering and Fermentation Regulation for Succinic Acid Production Succinic acid, a 4-carbon dicarboxylic acid, is an important platform chemical. The fermentative production of succinic acid by microbial organisms as a green technology has the advantages of CO2 fixation and using biomass as the feedstock. However, all natural succinic acid-producing organisms have the problems of lower titers, lower yields and other byproducts from mixed-acid fermentations. In our group, a series of engineering strains with higher succinic acid yield were constructed with the strategy of metabolic engineering (Fig. 3). The highest succinic acid yield can reach 1.47 (mol/mol). The fermentation with corn stalk hydrolysate was also Fig. 3 Metabolic flux in the engineered E. coli with enhancement of HCO3 supply 77

carried out, which laid the basis for the development of a biorefinery process of succinic acid.

of succinic acid (Fig. 4). The cost of the novel coupled process was much lower than traditional processes in which the fermentation and purification were separated.

4 Efficient succinic acid production via an integrated fermentation process coupled with in situ separation. 3.

5 carried out, which laid the basis for the development of a biorefinery process of succinic acid. On the other hand, ISPR (in situ product removal) strategy through a coupled expanded bed adsorption system was applied to eliminate the product inhibition and consequently enhance the productivity of succinic acid (Fig. 4). The cost of the novel coupled process was much lower than traditional processes in which the fermentation and purification were separated. Take operational cost as an example, it was 60%~70% at a discount. Succinic acid production cost was reduced to $/kg. Fig. 4 Efficient succinic acid production via an integrated fermentation process coupled with in situ separation. 3. Identification on Factors Affecting Biomass Utilization and New Pretreatment Technology Development Based on Liquid Hot Water and Microwave Irradiation The group focuses on biomass utilization including new pretreatment technology development, physical/chemical structure change in pretreatment process, and biomass pretreatment reactor design and lignin conversion to high value-added products. Microwave-assisted CaCl 2, coupling of microwave irradiation and ball milling, and ph value precorrected liquid hot water have been invented to realize the high temperature pretreatment under atmospheric pressure. Through investigations on the correlation between enzymatic hydrolysis and physical/chemical structure changes of biomass during pretreatment process, we found that high concentration of alkali or high temperature was necessary in dissolving or decomposing the cellulose. Appropriate pretreatment severity eliminated the effects of structural diversities in feedstocks, which led to convergence in the ethanol fermentation. Meanwhile, the continuous microwave pretreatment reactor and continuous hot water pretreatment reactor were invented. Perfect efficiency was achieved when they were used for the pretreatment of corn straw, rice straw, and Miscanthus, etc. 4. Key Technologies for Sustainable Use of Elite Medicinal Plants Fig. 5 The raw material (a) and ITS sequence of Maca (b) The increased interest in natural remedies brings great challenge of maintaining a balance between the demand of expanding market for plant-based medicines and the need to protect medicinal biodiversity. Key technologies for germplasm conservation and mass micropropagation of elite medicinal plants, and scale-up production of high-value plant-derived secondary metabolites and proteins in bioreactor systems were developed in biomass engineering group. The development of effective biotechnologies that define plant yield in terms of both biomass and medicinally active phytochemicals is, therefore, extremely important for long-term, sustainable use of medicinal plants. Taking Maca (Lepidium meyenii Walp.), a crop originated from Peru, as an example, a method based on internal transcribed spacer (ITS) sequence was built to identify Maca (Fig. 5), and the HPLC determination method of macamides was established. Based on the molecular and chemical methods, we achieved the authenticity and quality evaluation of Maca, which was useful to regulate the quality of Maca merchandises and to form the Maca industry standard. 78

5. Industrialized Biorefinery Lines of Biomass with Economic and Social Benefits By using the technologies achieved in our group several industrialized biorefinery lines have been established

production line (Shandong Province), etc. The 3 10 5 t/a straw refining industrial production line was established in 2010 with annual capacity of 3.5 10 4 tons of butanol, 1.

2 10 5 tons of high purity cellulose (producing for 5 10 4 tons bio-based polyetherpolyol), and 3 10 4 tons of high purity lignin (producing for 2 10 4 tons of bio-based phenolic resin) (Fig. 6).

Therefore, the problem of low hydrolysis efficiency of cellulose and high cost of cellulase in the traditional technology could be resolved.

This project could receive total profit of 660 million RMB per year with the co-production of solvent (butanol, acetone, and ethanol), phenolic resin and polyether polyols. Fig.

6 5. Industrialized Biorefinery Lines of Biomass with Economic and Social Benefits By using the technologies achieved in our group several industrialized biorefinery lines have been established successfully in China, such as 3000 t/a straw ethanol production line (Shandong Province, China), t/a straw refining industrial production line (Jilin Province), t/a straw butanol production line (Shandong Province), etc. The t/a straw refining industrial production line was established in 2010 with annual capacity of tons of butanol, tons of acetone and ethanol, tons of high purity cellulose (producing for tons bio-based polyetherpolyol), and tons of high purity lignin (producing for tons of bio-based phenolic resin) (Fig. 6). In this project, hemicellulose hydrolysate (mainly xylose) was used for butanol fermentation instead of cellulose. Therefore, the problem of low hydrolysis efficiency of cellulose and high cost of cellulase in the traditional technology could be resolved. Meanwhile, cellulose and lignin were further converted to the bio-based polyether-polyol and phenolic resin, respectively. This project could receive total profit of 660 million RMB per year with the co-production of solvent (butanol, acetone, and ethanol), phenolic resin and polyether polyols. Fig. 6 Main equipment for t/a straw refining industrial production line [(a) Steam explosion tanks (6.5 m 3 16); (b) Acid hydrolysis tanks (80 m 3 4); (c) Butanol fermentation tanks (400 m 3 32); (d) Distillation tower for butanol; (e) Polyether polyol production workshop] Selected Publications and Achievements Hongzhang Chen. Biotechnology of Lignocellulose: Theory and Practice. Springer-Verlag, Hongzhang Chen. Modern Solid State Fermentation: Theory and Practice. Springer-Verlag, Hongzhang Chen, Weihua Qiu. Key Technologies for Bioethanol Production from Lignocellulose. Biotechnol. Adv., 2010, 28: Hongzhang Chen, Liying Liu. Unpolluted Fractionation of Wheat Straw by Steam Explosion and Ethanol Extraction. Bioresour. Technol., 2007, 98: Hongzhang Chen, Guanhua Li, Hongqiang Li. Novel Pretreatment of Steam Explosion Associated with Ammonium Chloride Preimpregnation. Bioresour. Technol., 2014, 153: Qin He, Hongzhang Chen. Pilot-scale Gas Double-dynamic Solid-state Fermentation for the Production of Industrial Enzymes. Food and Bioprocess Technology, 2013, 10(6): Junying Zhao, Hongzhang Chen. Correlation of Porous Structure, Mass Transfer and Enzymatic Hydrolysis of Steam Exploded Corn Stover. Chem. Eng. Sci., 2013, 104: Yongshui Qu, Quanyuan Wei, Hongqiang Li, et al. Microwave-assisted Conversion of Microcrystalline Cellulose to 5-Hydroxymethylfurfural Catalyzed by Ionic Liquids. Bioresour. Technol., 2014, 162: Caixia Wang, Wei Ming, Daojiang Yan, et al. Novel Membrane-based Biotechnological Alternative Process for Succinic Acid Production and Chemical Synthesis of Bio-based Poly(butylenes succinate). Bioresour. Technol., 2014, 156:

10. Caixia Wang, Anders Thygesen, Yilan Liu, et al. Bio-oil Based Biorefinery Strategy for the Production of Succinic Acid. Biotechnology for Biofuels, 2013, 6: 74-85. 11.

7 10. Caixia Wang, Anders Thygesen, Yilan Liu, et al. Bio-oil Based Biorefinery Strategy for the Production of Succinic Acid. Biotechnology for Biofuels, 2013, 6: Sustainable Control of Forest Pests and Fungal Insecticides Industrialization. National Science and Technology Progress Award, Gas Dual-dynamic Solid State Fermentation Technique and Application. WIPO-SIPO Award Chinese Outstanding Patented Invention, Research Professors Prof. Hongzhang Chen (Team Leader), born in 1961, got his B.S. degree of Agronomy in 1983 from Nan Forest University, received his M.S. degree of Microbiology in 1991 from Shandong University, and obtained his PhD in 1998 from Institute of Process Engineering (IPE), CAS. He has been professor of biochemical engineering at IPE, CAS since He is mainly engaged in the bioprocess engineering of biomass refinery. He has revealed that the chemical and structural heterogeneity is one of the key reasons for the low-value utilization of lignocellulosic biomass and proposed the concept of selectively structural destruction and functionalized conversion of biomass. He also proposed the new design theory to strengthen the biological reaction and mass transfer of solid state fermentation process taking the external normal periodic force as power source. As the technology director, he has established more than ten industrialized biorefinery projects of biomass. He has published more than 150 journal papers and 23 monographs. His work was positively cited in Biotechnology Advances, Bioresource Technology, Chemical Engineering, etc. He has applied 210 patents, including 117 authorized patents and 8 international patents. Affiliation Member, Biomass energy industry technology innovation strategic alliance Member, Energy Research Council, Chinese Academy of Science Member, Biomass energy professional committee of Chinese Solar Energy Society Editorial Board member, Journal of Chongqing University (English Edition) Editorial Board member, Chinese Journal of Process engineering Biotechnology of Lignocellulosic biomass; Bio-based products refining technology; Solid-state fermentation technology. 80

Prof. Jianmin Xing, born in 1967, got his B. Sc in 1989 from Shandong University, and Ph.D. in 1998 from Chinese Academy of Sciences.

He developed an in situ product removal process for succinic acid production, which integrated membrane filtration with fermentation.

He has published more than 60 papers in international journals and acquired 18 practical technology-based patents. His work was positively cited by Chem. Rev., Angew. Chem. Int. Ed., Crit. Rev. Biotechnol.

8 Prof. Jianmin Xing, born in 1967, got his B. Sc in 1989 from Shandong University, and Ph.D. in 1998 from Chinese Academy of Sciences. He has been professor of chemical engineering at Institute of Process Engineering, CAS, since He is mainly engaged in biocatalysis and biotransformation. He developed an in situ product removal process for succinic acid production, which integrated membrane filtration with fermentation. He also constructed a novel process for the biodesulfurization of natural gas and biogas with haloalkaliphilic microorganisms with the recovery of elemental sulfur. He has published more than 60 papers in international journals and acquired 18 practical technology-based patents. His work was positively cited by Chem. Rev., Angew. Chem. Int. Ed., Crit. Rev. Biotechnol., etc. Affiliations Editorial board member, Chinese Journal of Biotechnology Editorial board member, Chinese Journal of Process Engineering Metabolic engineering of biocatalysts; Bioprocess monitoring and control; Separation and purification of biochemicals. Prof. Bing Zhao was born in 1966 and received his Ph.D. degree in Biochemical Engineering from Institute of Process Engineering (IPE), Chinese Academy of Sciences (CAS) in He was awarded the first Dean scholarship award of CAS. He has won 7 Provincial and ministerial Science and technology achievements, published more than 120 papers and held more than 26 patents. There have been a number of scientific and technological achievements applied in the scale of production. Affiliation Member of Degree Committee of IPE, CAS Member of Engineering Technical Committee of IPE, CAS Special biological resources, especially in the special plant resources and biorefinery: molecular breeding, large-scale breeding, quality evaluation and control, new technology and equipment for extraction and separation of natural product, structural modification, relationship between composition and bioactivity, new commercial product development. Prof. Chunzhao Liu was born in 1969 and received his Ph.D. degree in biochemical engineering from Institute of Process Engineering (IPE), CAS in He was a postdoctoral fellow at Nagoya University ( ), Salem-Teikyo University ( ), and University of Guelph ( ). He has been a professor of IPE since His current research focuses on bio-manufacturing engineering related to metabolic engineering, bioreactor design and scale up, bioprocess intensification and bioseparation. Affiliations Editor, Chinese Journal of Process Engineering Editor, Chinese Science Bulletin Editor, Journal of Tropical Organisms Biochemical Engineering; Enzyme Engineering; Industrial Microbiology and Biotechnology. 81

In 09/2010, he was employed as a professor at IPE-CAS supported by 100 Talents Program of Chinese Academy of Sciences, where he headed a research group of Biomass Pretreatment and Product Engineering

9 Prof. Jian Xu, born in 1975, received his PhD in 2006 from the Institute of Process Engineering, Chinese Academy of Sciences (IPE-CAS). Between 09/2006 and 12/2007, he was a postdoctoral fellow in SUNY-ESF (USA). Afterwards, he continued his postdoctoral research on biomass utilization at Technical University of Denmark. In 09/2010, he was employed as a professor at IPE-CAS supported by 100 Talents Program of Chinese Academy of Sciences, where he headed a research group of Biomass Pretreatment and Product Engineering (BPPE). His study is devoted to establishing new eco-friendly biomass utilization technologies, especially on technical innovation of pretreatment and lignin-based conversion process. He has invented continuous microwave irradiation pretreatment reactor used for catalytic production of 5-HMF which has been proved effective. He also developed microwave irradiation assisted biphasic reaction system used for efficient conversion of industrial lignin into phenolic compounds. He is author of over 20 journal papers. His work was positively cited by Bioresource Technology, Energy and Fuels, RSC Advances and Applied Energy, etc. Macro/microstructure changes on biomass before and after different pretreatments; Relationship between structural parameters of biomass and its utilization efficiency; Green biomass pretreatment technology development; Catalytic conversion of lignin to value-added products. Prof. Yejun Han, born in 1978, obtained his B.S. degree in Food Science and Engineering from Qingdao Agricultural University in 2001, received his M.S. degree in Microbiology 2004 from Nanjing Agricultural University. He received his PhD from Chinese Academy of Sciences in 2008, and completed his postdoctoral training at University of Illinois at Urbana-Champaign, North Carolina State University from 2008 to He specializes in enzymology, microbiology, and biochemical engineering, and was appointed as principal investigator in 2013 through 100 Talents Program. He has authored more than 40 original manuscripts, patents, book chapters, and conference abstracts. Genetic and fermentation engineering; Thermophiles; Enzymology; Especifically, development of novel gene engineering strain, industrial enzymes and fermentation process for chemicals and biofuels production. 82

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