Polymer Biocatalysis and Biomaterials - American Chemical Society

Chapter 2

Biotechnology: Key to Developing Sustainable Technology for the 21 Century: Illustrated in Three Case Studies st

Downloaded by COLUMBIA UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: February 15, 2005 | doi: 10.1021/bk-2005-0900.ch002

L o r i A . Henderson Novozymes North America, Inc., 77 Perry Chapel Church Road, Franklinton, NC 27525

The maturation of technology from scientific breakthroughs to commercial applications is driven by the symbiotic relationship that exists between society, academia, industry and government. With vast improvements and increasing availability of new bioengineering techniques today, the future in developing enzyme technologies to meet the demands of many industrial applications holds great promise. From textiles to ethanol production, the unique advantage of enzymes serves as the impetus for increasing R&D projects and their commercialization. Herein, describes the emergence of three technologies related to BioPreparation™, Bioethanol and Biocatalysis, whose commercial success is contingent on the discovery and bioprocess engineering of enzymes for industrial applications. The research activities and strategies in meeting the needs of all 3 industries is discussed via 3 case studies - I. Discovery and Exploratory of BioPreparation™, II. Development of Low Cost Technology for Biomass to Ethanol Production and, III. Discovery of Next Generation Biocatalysis for Chemical Synthesis.

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© 2005 American Chemical Society In Polymer Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Case Study 1: The Discovery of Biopreparation


Introduction In textiles, one of the most negative impacts on the environment originates from traditional processes used to prepare cotton fiber, yarn and fabric. Fabric preparation consists of a series of various treatments and rinsing steps critical to obtaining good results in subsequent textile finishing processes. These waterintensive wet processing steps contribute large volumes of wastes, particularly from alkaline scouring and continuous/batch dyeing. Such treatments generate large amounts of salts, acids and alkali. Scouring is a cleaning process that removes impurities from cotton substrates during textile processing. In view, of the 40 billion pounds of cottonfiberthat are prepared annually on an international level, it becomes clear that the preparation process is a major source of environmentally harsh chemical contribution to the effluent, with the major offender being sodium hydroxide and its salts. Conventional chemical preparation processes involve treatment of the cotton substrate with hot solutions of sodium hydroxide, chelating agents and surface active agents, often followed by neutralization step with acetic acid. The scouring process is designed to break down or release natural waxes, oils and contaminants and emulsify or suspend these impurities in the scouring bath. Typically, scouring wastes contribute high BOD loads during cotton textile preparation. According to the EPA (/), -50% of the total BOD in preparing knitted fabrics is due to scouring chemicals. Cotton preparation in the textile mill is a sequence of events that define all of the industrial steps leading from fiber to fabric. Cloth is created by weaving or knitting mostly raw cotton yarn that is then processes for dyeing. In this case, greige fabric is converted to a fully dyeable yarn or fabric using different wet processing steps. Many of the processes represent aggressive chemical treatments that incorporate high concentrations of harsh, corrosive chemicals like sodium hydroxide, hydrogen peroxide, lubricants, séquestrants, etc. Moreover, the textile processing industries are driven by time-consuming production processes, which consume much energy and resources. Thus, Novozymes initiated an innovative research project with the intent of developing biological alternatives to chemical preparative routes in cotton textile preparation. The goal of this project was to develop a technology that would significantly reduce pollution and resources without increasing capital expenses. This technology referred to as BioPreparation™ has been reduced to practice via several field and industrial trials conducted across the globe (2). The idea originated from a technology review of plant cell wall physiology, cotton fiber morphology and its' composition to determine the different structureproperty relationships that exists within cotton fibers. Pioneering studies were subsequently pursued in the laboratory to develop test methods and protocols for evaluating the effect of various enzymes on woven cotton fabrics. Several

In Polymer Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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exhaustive empirical screening tests were carried out in a labomat to simulate the scouring of fabric. In summary, BioPreparation™ is an enzymatic process for treating cotton textiles that meets the performance characteristics of alkaline scour systems while reducing chemical and effluent load.


Results and Discussion The goal of cotton preparation is primarily to improve the wettability of cotton fiber. A cotton fiber is the seed hair of plants of the genus Gossypium where each fiber is generated from a single cell. A mature fiber is composed of three main layers: a primary cell wall, secondary wall and lumen. Cotton wax, located in die outer layer of cotton fibers called the cuticle, is regarded as a major obstacle influencing the quality of dyeing as well as die "dynamics" of wetting in wet textile preparation of fabric. It was believed for quite some time that these hydrophobic substances (long chain fatty acids, esters, alcohols) and other impurities formed a distinct protective layer that surrounds the primary cell wall based on the morphology of cotton fibers (2, 3). The composition of the primary cell wall consists of mainly cellulose and xylogucan located within die fibrils. Part of die primary cell wall also contains a small percentage of impurities like pectin, protein and waxes. The secondary wall constitutes the bulk of a maturefiberin fibril from spirally arranged around the fibril axis. The fibrils consist of bundles of cellulosic microfibrils that are 0.025 μιη thick and at least 10 μπι long. The lumen is the central canal and contains residual proteins. As the mysteries in this project started to unravel, it was realized that only the primary cell plays a vital role in textile preparation. In this investigation, it was postulated that the cotton waxes are oriented in a 3-dimensional structure within the primary cell wall architecture and can be removed by degradation of polymeric materials. The objective was to examine the physco-chemical nature of die interlinked networks within a cotton cell wall. As shown in Figure 1, the architecture of a plant cell wall consist of a series of polymer networks that when superimposed upon one another givesriseto a very complex structure. The model is a simplified view of the specific interactions between three networks: 1 - cellulose-hemicellulose characterized by the hydrogen bonding of xyloglucan to the cellulose, 2 - the pectic polymers held in the matrix through ionic bonding of the egg-box type and 3 - glycoprotein (extensin) that are structurally independent of the polysaccharides. Moreover, the cellulose/xyloglycan network described above is embedded by the outer pectin netting. The junction zone of pectin network is held together by ionic, hydrophobic or hydrogen bonds depending on the degree of methylation for the different pectic polymers. This research project investigated the interactions between polymers and substrates like waxes by experimenting with enzymes and studying their effect



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Figure 1. The top drawing is a proposed model simplified to demonstrate the various interpenetrating networks within the primary cell wall of cotton . The reaction scheme belowt illustrates the products formed by the enzymatic hydrolysis ofpectic polymers via a β-elimination reaction. Note the complexity between the interactions-degradation ofthe polysaccharides. 3



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on cotton textiles. Enzymes such as glucanases, cellulases, hemicellulases, and pectinases were screened in simulated scouring tests and the absorbency measured. The results consistently proved that enzymes, referred to as pectinases, would degrade polymeric substrates like pectin from the surface of cotton and produce a highly absorbent material. This success led to cloning, purifying and isolating a bacterial pectate lyase at high yields with one proteolytic activity. The results also support the proposed mechanism of action. The mechanism of polymer degradation (Figure 1) is a β-elimination reaction which cleaves the a-1,4 links between galacturonosyl residues to produce two products: an unsaturated oligosaccharide (C4-C5 bond) and a hydroxy-terminated chain end oligomer (referred to as the reducing chain). The conclusions from this research are consistent with the proposed substrate model and reaction mechanism postulated in Figure 1. The BioPreparation™ of cotton takes place by: i) cleaving polyglacturonic acid and methoxylated derivatives to break the outer pectin network and hence alter the morphology of the cell wall and ii) removing the waxes, proteins and other components within this matrix via solubility and emulsification with the aid of surfactants and chelants. The enzyme is also compatible with other enzymatic preparations (amylases, cellulases) used to improve the performance properties of cotton fabrics (4). The process also decreases both effluent load and water usage to the extent that the new technology becomes an economically viable alternative. A reduced need for sodium hydroxide significantly reduces BOD and COD in the effluent, as was determined by analyzing spent scouring baths from numerous field trials with cotton knits and yarn. When similar process auxiliaries (e.g. surfactants) were used, the BioPreparation™ process decreased BOD and COD load by 25 and 40%, respectively, relative to conventional sodium hydroxide treatments. To appreciate the significance of these reductions, consider the cost savings to a woven processing mill (desize-dyeing) that produces -2.3 million lbs of goods per month. Assuming the charges for BOD and COD is $0.20 and $0.122/lb for amounts over the permissible limits , the annual cost to the mill for water and waste charges is $637,000 per year when calculated with a water consumption of 12 gal/lb. fabric. If the waste values are decreased according to the above field data, the cost is $424, 980/yr which represents -33% savings to the mill Similar cost savings were documented at a few targeted mills. Additional field trials indicated a 30% reduction in water use by elimination of both the acetic acid neutralization step and several rinse cycles. Even further opportunities to reduce effluent load and water use were realized when BioPreparation™ was used to combine treatment steps. Results from a 1-step scour-dyeing process combined in a single bath showed a 20 and 50% reduction in BOD and COD, respectively, compared to the conventional 2-step alkaline treatment. Assuming the same BOD/COD charges as before, such reductions would allow the mill to save 30% of their costs. 1


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Conclusions Application studies on both a pilot and industrial scale were explored using BioPrep 3000L as the main component in scouring knit and woven textiles. Variations in process variables such as enzyme concentration, pH and temperature were performed in pilot scale equipment designed for exhaust and pad operations. The "treated" fabrics were evaluated against the conventional alkaline processes based on standardize test methods including performance characteristics like dyeing (5). Successful full-size industrial trials in preparing yarn and knitted textiles were conducted and confirmed the processing advantages described herein. These advantages relative to chemical preparative routes are as follows: (i) ease of operation (no modification of existing equipment), (//) improve environmental effluents based on BOD, COD, and TDS, (///) selective degradation of components that enhance properties with minimal weight loss (maintaining the quality/integrity of the cotton fiber) and (iv) consumes significantly less water, time and energy. In conclusion, this biocompatible process provides an economical & an environmentally friendly alternative to alkaline scour systems or any combinations thereof, currently used in the textile industry today. The technology also emphasizes a novel strategy in the industry focusing on pollution prevention rather than innovative treatments.

Case Study 2: The Future of Bioethanol Production Introduction The US needs alternatives to foreign oil for transportation to wean the country from its dependency on imported oil. Using biomass as a feedstock for ethanol production could expand the domestic ethanol market, improve national security, create jobs, dispose of burdensome biomass waste and produce a clean transportation fiiel. From a biomass energy standpoint, unhealthy forests are only one of many sources that could eventually support a biomass energy industry. DOE's new technology for biomass conversion to ethanol could increase production efficiencies up to about 4:1. Using biofuels such as ethanol provides measurable air quality benefit by reducing vehicle emissions and abating field burning of some agriculture residues. It will reduce air pollution and the greenhouse gases that are implicated in the problems of global climate change. Estimates are that 40% of today's smog, 33% of annual C02 emissions and 67% of CO production come from automobiles and other forms of transportation.



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The market opportunity in the production of ethanol is expanding rapidly due to the elimination or significant reduction in die use of MTBE, an oxygenate that is traditionally blended with fuel. The potential for a new process technology referred to as bioethanol is receiving significant attention with vast number of government sponsored research programs emerging. It has the potential to match the features of petroleum at a low price. Bioethanol can be produced from domestically abundant sources of biomass including agricultural and forestry residues, wastepaper and other municipal solid waste and ultimately woody and herbaceous crops grown on underutilized land. Because the fossil fuel inputs in growing such materials and converting to ethanol is low, a high ratio of energy production is achieved and the net release of C02 that contributes to global climate changes is zero. When ethanol is added to gasoline it improves fuel combustion, thereby reducing tailpipe emissions of CO and unburned hydrocarbons that form smog. By applying the rapidly advancing sources of biotechnology to bioethanol production, the technology can be improved to make bioethanol competitive for blending with gasoline with that from corn in the US. This innovative technology is based on integrating chemoenzymatic routes and microbial conversion to the production of ethanol from renewable resources. Novozymes has two research projects related to the development of this process technology using biotechnology tools/applications to: i) Find more efficient enzymes through a combination of diversity mining, protein engineering and DNA discovery tools, and ii) Conduct lab-scale application tests for proof-ofconcept in biomass-ethanol production. Biomass Treatment Technologies The U.S. Department of Energy (DOE) and the National Renewable Energy Laboratories (NREL) have been working closely with state agencies, academic institutions and a wide range of industrial partners to accelerate the advancement of "new bioethanol technology". Researchers are working to demonstrate biochemical conversion processes in real-world applications with emphasis on improving the efficiency and economics of the process technologies by focusing their efforts on the most challenging steps in the process. Through the US government subsidized programs, the major thrust of the advanced R&D on biochemical conversion technologies is on pretreatment, cellulase enzymes and catalyst development for products from sugars (see Figure 2). More recent sponsorships are also directed at demonstrating process integration at a pilot plant scale to produce bioethanol and other chemicals - steps towards establishing a true "biorefinery" with value-added coproducts.



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Corn Stover Wheat straw Barley straw

Pretreatment • ChemicalDilute acid, AFEX, ARP, Hot wash • Physical Hydrothermal • Biologicalmicrobes

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Enzyme Hydrolysis Low Cost Cellulases & Hemicellula ses On-site enzyme production

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Fermentation Robust, pentose utilizing yeast/bacteria Novel microbial catalysts for direct conversion

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Process Engineering: SHF, SSF orSSCF Figure 2: An illustration of the various research activities underway to develop the next generation bioethanol plant The 3 key areas of R&D are in pretreatments, enzyme hydrolysis andfermentations. Significant efforts focus on process engineering and plant designs with the following at the core: SHF separate hydrolysis and fermentation, SSF - a simultaneous saccharificationfermentation, or SSCF - a simultaneous saccharification-cofermentation.


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Pretreatment Technologies for Lignocellulosic Conversion (Step 1) In the utilization of enzymes for the hydrolysis of the cellulose, a pretreatment of the lignocellulosic material is necessary to break up the barrier made by both lignin and hemicelluloses. The lignin is one of the major obstacles in enzymatic hydrolysis because it binds a large part of the enzymes. In some studies, only 40-50% of the total protein added can be recovered in solution. The remaining portion is irreversibly bound to lignin (6). Furthermore, the physical and/or chemical pretreatment may increase the accessible surface area and change the crystallinity of the cellulose, which results in increased digestibility of the cellulose. The efficiency of these pretreatments is normally evaluated by measuring the sugar release during the combined pretreatment-enzyme hydrolysis. Thus, an effective pretreatment technique renders the cellulose more digestible, avoids degradation of hemicellulose sugars and removes lignin. The barriers to develop a robust pretreatment process include a lack of fundamental understanding related to the chemistry at work in pretreatment of biomass and the hydrolysis of hemicellulose, reactor design fundamentals, equipment reliability, and materials of construction. During the past decade, the science of pretreatments has received increasing attention because of its significance in the first stage of processing biomass. Among the variety of available pretreatment technologies reported in the literature, there are at least 5 general classes: base-catalyzed, acid-catalyzed, non-catalyzed, solvent and chemical based systems. From the developmental work by NREL Advance Pretreatment Project (7,8% and the Biomass Refining Consortium of Applied Fundamentals and Innovations Team (CAFI) (7,8) a strong fundamental knowledge base of biomass pretreatment chemistries, kinetics and process economics is underway. The Advance Pretreatment Projects focus on clean fractionation, hemicellulases and accessory enzymes, biomass compositional analysis and the application of NREL's Bioethanol Pilot Development Unit. Together, their goal is to expand ongoing efforts in understanding the impact of reactor design and configuration on thermochemical cellulose hydrolysis and include a broader range of biomass pretreatments and fractionation approaches. This partnership continues to investigate 5 leading technologies based on performance and process economics using corn stover as the model substrate. The aim is to identify the strongest possibilities of achieving broad commercial applicability in an advanced technology sugar/lignin platform. Upon conclusion of the CAFI program at least four leading, potentially viable pretreatment processes were analyzed in detail: dilute acid, ammonia fiber explosion (AFEX), ammonia recycle percolation (modified ARP) and Pressurized Hot Wash treatment (PHW) (8-13\ No single pretreatment is currently a clear front-runner, however, for meeting the advanced technology targets. A l l have some process performance, economic or


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complexity issues that have not been adequately addressed. The different pretreatment methods solubilize primarily the hemicellulose in different lignocellulosic material - with marginal impact on lignin and cellulose and hemicellulose. The following is a brief description of those methods having the most commercial potential (8-13).


•

Dilute Sulfuric Acid - Depending on acid content and residence time, it can achieve near complete solubilization of hemicellulose resulting in high yields of xylose. The dilute process has a little to no impact on solubilizing lignin and cellulose crystallinity. Dilute acid (

Polymer Biocatalysis and Biomaterials - American Chemical Society

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