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A Perspective on Bioprocessing for Biofuels, Bio-Based Chemicals, and Bioproducts
Chapter 1
A Perspective on Bioprocessing for Biofuels, Bio-Based Chemicals, and Bioproducts Majid Hosseini Manufacturing and Industrial Engineering Department, The University of Texas Rio Grande Valley, Edinburg, TX, United States
1.1 INTRODUCTION Given the dire global consequences that higher temperatures and unstable climate changes could trigger, intensified in the developing countries, there is an urgent need for progressive policy amendments. The consensus is that this century’s energy goal, providing sufficient affordable energy to meet and exceed demand while mitigating undue environmental impacts, is not achievable without a massive innovative effort on a global level. Although it would be naive to believe that biofuels would resolve all of the current global energy problems and concerns, investing in their development could yield solutions to climate change while decreasing fossil fuel dependence for those countries that import energy. Thus, investing in novel energy technologies could be fruitful economically, environmentally, and in geopolitical terms. The most recent analysis conducted by the International Energy Agency in 2017 [1] projects that by 2040, global primary energy demand is on track to increase by 30% and oil demand continues to grow to 2040, while the share of direct and indirect renewable use in final energy consumption rises globally from 9% today to 16% in 2040. Still fossil fuels account for a large portion of the projected expansion in demand by 2040. Although modest in nature, the demand for natural gas is also expected to experience growth and natural gas use rises by 45% to 2040. The increasing global demand for energy will also impose a credible threat to the world’s energy security. Further exacerbating the interim security risks is the disproportionate amount of countries that produce oil compared to ever growing demand from those that rely on importing oil and gas. Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts. DOI: https://doi.org/10.1016/B978-0-12-817941-3.00001-2 © 2019 Elsevier Inc. All rights reserved.
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Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts
A plentiful supply of biomass is critical for a bio-based industry. Biofuels are defined as fuels which are derived from biomass, with the most prolific liquid biofuels being bioethanol and biodiesel. Ethanol, an alcohol, can fuel cars designed to operate on either pure ethanol or a blend of gasoline and ethanol dubbed “gasohol.” In its blended form, ethanol can become an octane booster, an additive that can reduce pollution when combined with unleaded gasoline replacing the more commonly used methyl tertiary-butyl ether additive. Another biofuel is biodiesel that can also be used in its pure or blended (with petroleum diesel) form. Although the focus of biofuels is typically within the transportation sector, their cooking capability could prove to hold great global significance, specifically in the rural communities of the developing countries. Comparatively, the amount of pollutants and emissions created by biofuels in cooking applications is drastically lower than that of its traditional solid-fuel counterparts. As such, biofuels as a whole could improve the quality of life for billions across the world. Future energy security and reduced greenhouse gas emissions may be achieved through advances in biofuels and bioproducts, freeing the global economy from the constraints of fossil fuel dependence. Specifically, increases in the forestry and agricultural sectors alongside a boost to rural economies are some of the benefits of adopting the use of biofuels and bioproducts while also creating new opportunities for growth via biorefineries and their production of various consumer products (e.g., bio-based chemicals, biofuels, and value-added products). In order to totally replace petroleum and its derived products, the production of biofuels requires a plentiful, renewable, and eco-friendly feedstock, such as biomass. A potential solution lies in the implementation of an integrated biorefinery where the main feedstock for biofuel and bio-based chemicals can have the nutritional components extracted while the byproducts are converted into profitable materials (i.e., animal fodder). Furthermore, by utilizing both macro- and microalgae, arable land usage concerns would be mitigated while global fuel demands are met [2]. By pursuing new technologies and discoveries within aquaculture, genetic engineering, energy crops, and conversion methodologies, sustainable eco-friendly biorefineries will significantly impact the world’s energy, biofuel, bioproducts, and renewable chemical supplies. Biorefineries also have no shortage of obstacles to overcome. Virgin fossil fuels may ultimately be replaced by the production of renewably sourced bio-based chemical, value-added bioproducts, and biofuels. Concurrently, attempts at making alternative fuels readily available in the market must be done along with ensuring environmental and economic sustainability. The integration of producing value-added bioproducts alongside energy outputs in a biorefinery may yield systematic improvements to both productivity and profitability. Economic success lures shareholders and corporations into investing in new biorefineries, thus increasing the domestic
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bioenergy supply. A biorefinery’s efficiency and productivity can be optimized by conserving energy usage as well as utilizing feedstocks, waste, and byproduct streams to their fullest extent while employing economies of scale to reduce operating expenditures. The economics of lignocellulosic biorefineries are not yet viable due to the difficulties and cost associated with cellulose’s processing, pretreatment, and enzymatic hydrolysis. In addition, microalgae-based processes also face hurdles associated with scale up, making the production process far from cost effective [3]. To surmount these challenges, advancements in biomass conversion via genetics, bioprocessing, and metabolic engineering will be needed in order to support an economical and sustainable bio-based future. As such, all elements of the biomass must be fully utilized so as to maximize profit while minimizing waste generation when producing biofuels and bioproducts. The incorporation of various improvements across a multitude of disciplines including less costly enzymes for hydrolysis, newly synthesized catalyst for biomass to biofuel conversion, and enhanced bioprocessing techniques will allow the goal of a bio-based future to become fully realized commercially and globally. Foreign petroleum dependence is anticipated to diminish in the wake of widespread adoption of biofuels and bioenergy production. In order to develop the necessary infrastructure and technology prior to implementation, renewable resource processing must be given scientific priority. Yet another significant opportunity lies in environmental protection via bioprocessing. An overall reduction in industrially generated and municipal waste streams could be realized through feedstock bioconversion to biofuels in place of toxic hydrocarbon production. The enduring goal of the scientific community is to create carbon neutral bioprocesses that efficiently use a wide range of renewable resources to produce energy and chemicals with the intent to make technological and scientific gains in meeting the emergent bioeconomy’s demands. Incremental efficiency improvements as well as rapid analytical characterization of renewable fuel have led to developments in robust, stable, and even automated bioprocessing technologies and systems. Recent development of specially adapted separation and purification technologies for the recovery of bio-based products and the progress made in bioprocessing technologies will facilitate the economic conversion of renewable feedstocks into biofuels, bio-based chemicals, and value-added bioproducts. Microorganisms that have been genetically modified (GM) and are capable of transforming biomass into biofuels, bio-based chemicals, and value-added bioproducts along with development of bioproducts’ biomanufacturing processes that are highly regulated, controlled, and have foreseeable performance may provide means in an earlier adoption of biofuels production from renewable resources.
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Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts
1.2 CHALLENGES AND PROSPECT OF BIOPROCESSING FOR BIOFUELS AND BIOPRODUCTS The advanced technologies used for bioprocessing face obstacles not only in recognizing industry needs but also in furthering the transfer of technology while teaching the next generation of engineers and scientists that will support growth within the field. This includes supporting innovative alternative fuels technologies that will fully harness that potential of contemporary bioprocessing. Essentially, the tenants of industrial bioprocessing are understanding the biocatalysts or microorganisms employed, maintaining product quality and safety no matter the operation’s size, exercising environmental stewardship, and promoting process innovation to maintain a competitive edge within the industry as well as with consumers. Specific bioengineered processes of note include bioreactor design, concentrating and purifying diluted product streams, and applying cost-effective engineered solutions to biofacilities from cradle to grave [4]. In order to nurture growth in the understanding of how to produce a growing and wide array of biofuels, bio-based chemical, and bioproducts, a sustained and internationally agreed upon policy is paramount. A selected few of more than 72,000 algae species have been thoroughly evaluated [5], and even fewer have been used on an industrial scale. The latest attempts in laboratory and pilot scale phototrophic, heterotrophic, and mixotrophic microalgae cultivation have unveiled novel organisms that have yet to be exploited. While renewable energy is already being used in many different forms, microalgae-derived carbon-neutral biofuels are desirable candidates due to their sustainability, large carbon dioxide sequestration capacity, large lipid production, and their ability to grow in a multitude of environments (e.g., brine, brackish, and wastewaters) [6 9]. The cheap and economical extraction of lipids form microalgae continues to be a major hurdle in its commercial biofuel adoption. Fervent studies are currently ongoing to ascertain the capability of microalgae as both a biofuel and in carbon dioxide fixation. While a promising biofuel feedstock, microalgae are not yet utilized on an adequate industrial scale for bulk commodities. The final cost of the extraction process can also be aided by a comprehensive technoeconomic analysis, which will also provide guidance by means of a cost/benefit analysis for future process improvement (e.g., increased lipid yields). However, relevant breakthroughs in technology (e.g., genetic modification, metabolic engineering techniques, and biorefining) indicate that further developments will yield a suitable process that is both economical and sustainable in the near future. Future industrial processes may rely upon the novel chemicals yet to be unlocked within microalgae. Existing examples of modern bioprocessing technologies demonstrate the vast variety of manufacturing methodologies required to create bioproducts and biofuels. A singular step has the potential to greatly skew the cost,
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quality, and properties of the resulting product. Transforming microorganisms and creating new biocatalyst necessitate the use of many different types of advanced bioprocessing techniques. Something that is often neglected is the variety of engineering skills, specifically those for bioprocessing that are required when dealing with bio-based products. Specific bioengineering challenges in the field today are as follows: ensuring that regulatory and biological standards are met in equipment design while remaining economical; ensuring bioproducts processes are environment friendly and sustainable; and demanding consistent high-quality bioproducts by implementing robust and rapid purification processes. Highly specialized bioproducts are often the result of dilute solution fermentation, a process that can be optimized by improving energy usage and efficiency which can be done by driving down costs in handling, synthesis, and downstream processing. Other areas of improvement include bioreactor design and making conditions more conducive to cultivating and creating microorganisms and their products. Advanced bioprocessing techniques also can make advances in fermentation (i.e., submerged, solid substrate) while cellular and genetic manipulation can make strides in the field by changing the physical properties of microbe membranes to negate the toxicity caused by extracellular fermentation products. The three dominant products of biofuels and bioenergy are presently bioethanol, biodiesel, and biogas. Synthesized via the fermentation of soluble sugars or starches (e.g., sugarcane, corn), bioethanol is considered to be a first-generation biofuel. Within the scientific community, there is a push toward the development of second-generation bioethanol, derived from the lignocellulosic biomass of plants, with the initial results showing great promise. Comprising lignin polymers, hemicelluloses, and cellulose, lignocellulosic biomass is a renewable resource and can be used to manufacture biofuels and value-added bioproducts. Unfortunately, the molecular structure and heterogeneity of lignocellulose may prove to be problematic. The lignin’s resistance to enzymatic degradation, preventing the conversion of the plant’s available polysaccharides into sugars, is the overwhelming recalcitrant factor for biofuels production. Furthermore, as a nonlinear polymer that is inherently chemically diverse and composed of weak reactive linkages along with multiple monomer units, the lignin’s phenolic polymer within its cell wall component is to blame for overall biomass recalcitrance. As such, lignin degradation is cost prohibitive, requiring pretreatment to access the polysaccharide content required for biomass conversion to biofuel. Genetic manipulation techniques have attempted to facilitate plant biomass processing by employing a methodology that, while in its infancy, would create plant that either accumulates less lignin or yields lignin that is readily decomposable. Conventional techniques utilize genetic engineering in such a way that modifies the enzyme expression used for lignin biosynthesis.
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Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts
1.3 BIOPROCESSING ROUTES FOR THE PRODUCTION OF BIOFUELS AND BIOPRODUCTS The developments of advanced biotechnology and bioprocesses for the production of biofuels and bioproducts have been selected as topics to be discussed at length herein. Biotechnology was first used by Karl Ereky in 1919, in reference to the science and methodologies that create products from raw materials while aided by living organisms [10]. Genetic engineering (or recombinant DNA technology) utilizes biotechnology to manipulate the cellular structure genetically through the introduction or elimination of specific genes. The use of these techniques yields GM organisms (GMOs), organisms altered by inserting at least one transgene frequently from a different species than that of the recipient. The 1970s gave way to the first genetic modification experiments, combining synthetic human genes with bacterial genes. Ten years later, research could insert genes into fungi and yeast as well as otherwise foreign genes into plant and limited animal cells. In regards to the biofuels sector, both biotechnology and genetic engineering hold great promise in advancing agricultural production and converting biomass. Such gains may come in the form of energy crop production optimization, increasing crop yield per acre, modifying feedstock to aid in ease of fuel conversion, and the novel creation of enzymes that support downstream biofuel processing [11]. Although genetic engineering appears quite alluring at present, it is not clear if it will deliver on its promises, be overshadowed by new technologies, or become cost prohibitive in the future. Although selective breeding methods will continue to influence agricultural advancement, genetic engineering may focus efforts to enhance existing crops while expanding the catalog of plant varieties available for industrial use. Crops can be designed through genetic to aid in processing and subsequently are created for novel forms of raw materials. These plant varieties may be either altered or chosen for desirable traits that allow then to be easily converted into fuels. An example of this that is currently employed is the synthesis of bioethanol from the fermented sugars of corn via pretreatment processes that break up lignocellulose in order to eliminate the lignin, thereby promoting cellulose deconstruction by creating pathways for enzyme access. Presently, enzymatic pretreatments and production are costly. Genetic engineering could solve this impedance in biofuels progress by creating plants with helpful traits such as reduced lignin content, self-producers of cellulase or ligninase enzymes for cellulose or lignin degradation, respectively, or total yield boost to cellulose or biomass content. Modifying a plant’s genetic structure could also create plants that require less water, are able to fixate nitrogen, can be harvested easily, and biorefined into consumable protein, carbohydrates, and fibers for both the food energy industries. Bioengineered oilseed crops may also be utilized as the key ingredients in biodiesel as bio-based lubricants and esterified fatty acids. In
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addition to trait modification, biotechnology and bioprocessing could also be employed to increase yields. The past four decades have witnessed an incredible upward trend of annual rate of yield increases, the vast majority of which have been achieved through improved genetics via traditional gene recombination and recurrent selection. This trend has also been supplement by GMO introductions over the past decade but in a limited capacity. While genetic engineering has developed pests, weeds, and disease-resistant plants that tolerate drought and salinity stressors, the overall impact to the environment has yet to be determined. The production aspect of biofuels may also reap benefits through the use of genetic manipulation within the energy sector. Two major hurdles in the lignocelluloses conversion process that may be solved using genetic engineering are the expensive cellulose decomposing enzymes (cellulases) and the microbes’ limited fermentation ability of the breakdown products. This scope is also broadened by those scientists who wish to surpass the limited yeast diet of the microbes. Since yeast fermentation is limited to breaking down glucose and other hexoses, if researchers could also incorporate the decomposition of pentoses derived from hemicellulose, 15% 50% (plant species dependent) of previously unavailable lignocellulose can become available for biofuel production. Originating cellulose and hemicelluloses, the available lignocellulose sugars for fermentation cannot be accessed for enzymatic hydrolysis, thus necessitating an expensive pretreatment step which modifies the lignocellulosic structure. Pilot studies are currently underway that are evaluating other potential pretreatment technologies A variety of cellulases and hemicellulases are required for lignocellulose carbohydrate hydrolysis, a process that creates fermentable sugars. The hydrolysis process varies depending upon the feedstock: in cellulose, the linear glucose chains are broken down sequentially, whereas hemicellulases’ branched chains of varying sugars and functional groups are hydrolyzed. Currently, both pretreatment and hydrolysis technologies are on the brink of becoming commercially viable. With improved enzymatic functionality and a reduction in cost, high substrate containing lignocellulose is now able to be processed. Although these two technologies must still resolve technical and scientific issues, large-scale lignocellulosic substrate fermentation is possible in the near future albeit with the expectation of significant yield improvements and further cost reductions. While in its infancy, dedicating genetic research into energy crops and their manufacturing processes seems to be a worthwhile investment, for both the public and private sectors, as many countries are implementing strict biofuel targets while simultaneously addressing climate change. Capturing the attention of scientists across the world in the past 10 years, advanced bioprocessing applies science and engineering methodologies to microorganism and their derived products to create bioproducts with a small or neutral
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Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts
eco-footprint. Academics and industry leaders alike have yet to fully understand how indicative bioprocessing technologies will be in the commercial success of biofuels. What is certain is that developing and current bioproducts will need new, cost-effective, and efficient processes. As such, advanced bioprocessing will be in high demand in the bioindustries and the expanding biofuels, bio-based chemicals, and bioproducts marketplace. Although certain challenges exist in bioprocessing such as finding technologies that are scalable, low in cost, and minimizing waste creation, as these issues are adequately addressed, alternative fuels will become more viable.
1.4 RENEWABLE RESOURCES AND TRADE ISSUES Amidst all of these tantalizing opportunities, bioprocessing will need to be used on a grand scale, specifically for the cost-effective production of biofuels, bio-based chemical, and bioproducts using locally source feedstocks while developing new technologies. Carbon dioxide generated during combustion processes may be mitigated through engineering a large volume bioprocess. One potential answer to the issue of facilities that emit CO2 would be to incorporate either a photosynthetic or nonphotosynthetic organism into the stream to consume said emissions. Inventive engineering would be required to feasibly undertake such a large-scale problem while being sensitive to cost. Concurrently, biocatalyst can be used on a smaller scale in various applications to address similar issues facing energy and bioproducts production. Renewable feedstocks can be used to create a myriad of biobased chemicals. Although it has yet to become practical from an economic standpoint, bioprocessing may become more acceptable with raising petroleum prices, shortages in hydrocarbon reserves, or merely from an environmental stewardship perspective. Were this to occur, appropriate bioreactors and biocontrol systems would be necessary to implement advanced bioprocessing technologies in order to produce bio-based fuels, chemicals, and value-added product within stringent economic and environmental limits. Generally, current and future renewable resource applications occur on an industrial scale and create low-value products. To keep processing costs low, choosing to bioprocess certain feedstocks must be done with thoughtful regard. Bioprocessing technologies are continuing to find innovative applications in the manufacture of an assortment of bio-based products. Advancements must continue to deliver gains in hydrolysate fermentation, waste treatment, and the conversion of byproducts to value-added goods. The renewable bioconverting and fermentation industries stand to make large gains with the discovery of a cheap fermentable sugar source, which would pave the way to producing high-value specialty chemicals and additional technology advances. Some examples of these resulting products include alcohols, ketones, organic acids, microbial polysaccharides (oil well drilling), and amino acids (animal fodder) as well as high-value products such as
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flavors, fragrances, pharmaceuticals, and pigments. A plentiful renewable feedstock is agroderived (e.g., corn) starch which creates materials that utilize fermentation, enzymatic, and separating processes. A substantial amount of the liquid fuel market, specifically in the transportation sector, may be sourced in the future from cellulosic biomass used to create ethanol, further decreasing dependency on petroleum while reducing greenhouse gas emissions. Onstream reliability, productivity, and capital expenditures may be positively influenced by conceptual advanced bioreactors designed for high throughputs. Prospective research topics in bioreactor design and engineering are optimizing microbe to solids contact, continuous processing, large fermenter process control, and microbial metabolic manipulation. The economic implications of producing consumable from bioreactors will affect environmental, energy, and food sectors. An injection of bioprocessing theory will be needed to further develop the most appropriate biomasses types and conversions for reforming while fully utilizing any residual products. Biomass can even be thermally reformed into synthesis gas which can then be used in existing hydrocarbon plants. In order to create this revolutionary technology, a multidisciplinary knowledge must be used to their fullest extent in the generation of such a novel process. This cross-disciplinary approach spanning various scientific fields will facilitate advances in renewable resource bioprocessing. Much like biopharmaceuticals, the end consumer product typically originates from an organism. The key difference between these two industries lies in their production volumes, with bioprocesses requiring not only large facilities and feed rates but also consistent, cheap, and dynamic processes. Ultimately, the end objective when using renewable resources is to create bioproducts or biocatalyst so as to produce value-added consumables (e.g., fuel, chemicals, energy, etc.). Other important factors include the process economics, scalability, and quality consistency. In order to become economically feasible, researchers must continue to drive down processing costs of these bioproducts so as to meet a reasonable market price. As with any established process, for bioprocessing technologies to become widely adopted, improvements to efficiency, operation, equipment as well as gains in processing volumes and reliability must be realized. Researchers must capitalize on this opportunity afforded by renewable resources as the ramifications of such technologies will have far reaching effects on natural resources and arable land across the globe. There is opportunity for the developed and developing countries alike to enjoy the rewards of bioprocessing so long as an appropriate regulatory framework is established alongside the development of careful strategies. In this sense, the developed countries can strengthen their domestic energy security while avoid any social and economic disruptions caused by fossil fuel scarcities and inflation. On the other hand, the developing countries
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Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts
could reduce their foreign dependency on oil, tempering their exposure to oil price fluctuations while lessening their overall environmental footprint. Globally, rural areas could experience unemployment reductions and supplemental revenue with the introduction of a bio-based economy. Agricultural products could be introduced to new markets, increasing their overall worth. The international community is expected to see positive impacts by achieving these goals but none as much as the developing countries whose populations struggle with limited access to commercial energy, air pollution, and declining agricultural growth. There is a calculated amount of risk assumed when discussing bioprocessing and biotechnology despite the promise of biofuels, bio-based chemicals, and value-added bioproducts. Energy-specific GMO crops, while intended to boost yields increase and cultivate desirable traits, have raised concerns of their potential to threaten human and environmental health. While presently lacking definitive scientific evidence, it is postulated that the harm of genetic engineering may only be apparent in future studies. Although energy optimized crops would not be suitable for animal or human consumption, there are still cross-species pollination concerns, breeding between GM and nonGM or indigenous species that have yet to be alleviated. Crops intended as livestock feed or for human could become contaminated with the GM strains, thus elevating the fear level of the public. Societal reluctance to the adoption of GM crops for any reason, as well as any lingering or suspected environmental and sustainability impacts, must also be tackled prior to the widespread adoption of the technology.
1.5 CONCLUSION This perspective demonstrated novel systems that apply advanced bioprocessing technologies to produce biofuels, bio-based chemicals, and value-added bioproducts from renewable sources. Utilizing proven strategies and appropriate regulatory frameworks, expanding renewable fuels could potentially alleviate the serious issues presented before countries such as increasingly volatile petroleum cost and supply, ultimately aiding in the ease of access to cheap energy, especially for those in the developing countries. Biofuels could play a role in preserving the environment, lowering greenhouse gas emissions, presenting new opportunities to rural communities, and adding increased value to agricultural products. Genetic engineering could be the key factor in optimizing biofuels production, becoming a component of the global energy mix. Agricultural ventures that employ the use of genetic engineer practices are not without scrutiny or controversy. The emergent issue of global energy security has become the focus of the scientific community, where studies are being conducted to deliver environmentally feasible and economical alternative fuels. Biofuel is one such approach that may be able to meet future demand.
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REFERENCES [1] International Energy Agency, “World Energy Outlook 2017”, Executive Summary. ,http://www.iea.org/Textbase/npsum/WEO2017SUM.pdf., 2017, p. 1 (last accessed November 2018). [2] M. Hosseini, Sustainable Pretreatment/Upgrading of High Free Fatty Acid Feedstocks for Biodiesel Production, University of Akron, 2013. [3] M. Hosseini, H.A. Starvaggi, L.-K. Ju, Additive-free harvesting of oleaginous phagotrophic microalga by oil and air flotation, Bioprocess. Biosyst. Eng. 39 (7) (2016) 1181 1190. [4] N.R. Council, Putting Biotechnology to Work: Bioprocess Engineering., National Academies Press, 1992. [5] M.D. Guiry, How many species of algae are there? J. Phycol. 48 (5) (2012) 1057 1063. [6] M. Hosseini, L.-K. Ju, Use of phagotrophic microalga Ochromonas danica to pretreat waste cooking oil for biodiesel production, J. Am. Oil Chem. Soc. 92 (1) (2015) 29 35. [7] N. Moradi-kheibari, H. Ahmadzadeh, M. Hosseini, Use of solvent mixtures for total lipid extraction of Chlorella vulgaris and gas chromatography FAME analysis, Bioprocess. Biosyst. Eng. 40 (9) (2017) 1363 1373. [8] L.-K. Ju, M. Hosseini, Method and System for Reducing Free Fatty Acid Content of a Feedstock, US Patent App. 14/450,601, 2015. [9] L.-K. Ju, M. Hosseini, Treatment/Cleaning of Oily Water/Wastewater Using Algae, U.S. Patent Application No. 14/909,522, 2016. [10] K. Ereky, Biotechnologie der Fleisch-, Fett-, und Milcherzeugung im landwirtschaftlichen Grossbetriebe: fu¨r naturwissenschaftlich gebildete Landwirte verfasst, P. Parey, Berlin, 1919, p. 84. [11] C.E. Wyman, B.J. Goodman, Biotechnology for production of fuels, chemicals, and materials from biomass, Appl. Biochem. Biotechnol. 39 (1) (1993) 41.
A Perspective on Bioprocessing for Biofuels, Bio-Based Chemicals, and Bioproducts Majid Hosseini Manufacturing and Industrial Engineering Department, The University of Texas Rio Grande Valley, Edinburg, TX, United States
1.1 INTRODUCTION Given the dire global consequences that higher temperatures and unstable climate changes could trigger, intensified in the developing countries, there is an urgent need for progressive policy amendments. The consensus is that this century’s energy goal, providing sufficient affordable energy to meet and exceed demand while mitigating undue environmental impacts, is not achievable without a massive innovative effort on a global level. Although it would be naive to believe that biofuels would resolve all of the current global energy problems and concerns, investing in their development could yield solutions to climate change while decreasing fossil fuel dependence for those countries that import energy. Thus, investing in novel energy technologies could be fruitful economically, environmentally, and in geopolitical terms. The most recent analysis conducted by the International Energy Agency in 2017 [1] projects that by 2040, global primary energy demand is on track to increase by 30% and oil demand continues to grow to 2040, while the share of direct and indirect renewable use in final energy consumption rises globally from 9% today to 16% in 2040. Still fossil fuels account for a large portion of the projected expansion in demand by 2040. Although modest in nature, the demand for natural gas is also expected to experience growth and natural gas use rises by 45% to 2040. The increasing global demand for energy will also impose a credible threat to the world’s energy security. Further exacerbating the interim security risks is the disproportionate amount of countries that produce oil compared to ever growing demand from those that rely on importing oil and gas. Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts. DOI: https://doi.org/10.1016/B978-0-12-817941-3.00001-2 © 2019 Elsevier Inc. All rights reserved.
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Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts
A plentiful supply of biomass is critical for a bio-based industry. Biofuels are defined as fuels which are derived from biomass, with the most prolific liquid biofuels being bioethanol and biodiesel. Ethanol, an alcohol, can fuel cars designed to operate on either pure ethanol or a blend of gasoline and ethanol dubbed “gasohol.” In its blended form, ethanol can become an octane booster, an additive that can reduce pollution when combined with unleaded gasoline replacing the more commonly used methyl tertiary-butyl ether additive. Another biofuel is biodiesel that can also be used in its pure or blended (with petroleum diesel) form. Although the focus of biofuels is typically within the transportation sector, their cooking capability could prove to hold great global significance, specifically in the rural communities of the developing countries. Comparatively, the amount of pollutants and emissions created by biofuels in cooking applications is drastically lower than that of its traditional solid-fuel counterparts. As such, biofuels as a whole could improve the quality of life for billions across the world. Future energy security and reduced greenhouse gas emissions may be achieved through advances in biofuels and bioproducts, freeing the global economy from the constraints of fossil fuel dependence. Specifically, increases in the forestry and agricultural sectors alongside a boost to rural economies are some of the benefits of adopting the use of biofuels and bioproducts while also creating new opportunities for growth via biorefineries and their production of various consumer products (e.g., bio-based chemicals, biofuels, and value-added products). In order to totally replace petroleum and its derived products, the production of biofuels requires a plentiful, renewable, and eco-friendly feedstock, such as biomass. A potential solution lies in the implementation of an integrated biorefinery where the main feedstock for biofuel and bio-based chemicals can have the nutritional components extracted while the byproducts are converted into profitable materials (i.e., animal fodder). Furthermore, by utilizing both macro- and microalgae, arable land usage concerns would be mitigated while global fuel demands are met [2]. By pursuing new technologies and discoveries within aquaculture, genetic engineering, energy crops, and conversion methodologies, sustainable eco-friendly biorefineries will significantly impact the world’s energy, biofuel, bioproducts, and renewable chemical supplies. Biorefineries also have no shortage of obstacles to overcome. Virgin fossil fuels may ultimately be replaced by the production of renewably sourced bio-based chemical, value-added bioproducts, and biofuels. Concurrently, attempts at making alternative fuels readily available in the market must be done along with ensuring environmental and economic sustainability. The integration of producing value-added bioproducts alongside energy outputs in a biorefinery may yield systematic improvements to both productivity and profitability. Economic success lures shareholders and corporations into investing in new biorefineries, thus increasing the domestic
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bioenergy supply. A biorefinery’s efficiency and productivity can be optimized by conserving energy usage as well as utilizing feedstocks, waste, and byproduct streams to their fullest extent while employing economies of scale to reduce operating expenditures. The economics of lignocellulosic biorefineries are not yet viable due to the difficulties and cost associated with cellulose’s processing, pretreatment, and enzymatic hydrolysis. In addition, microalgae-based processes also face hurdles associated with scale up, making the production process far from cost effective [3]. To surmount these challenges, advancements in biomass conversion via genetics, bioprocessing, and metabolic engineering will be needed in order to support an economical and sustainable bio-based future. As such, all elements of the biomass must be fully utilized so as to maximize profit while minimizing waste generation when producing biofuels and bioproducts. The incorporation of various improvements across a multitude of disciplines including less costly enzymes for hydrolysis, newly synthesized catalyst for biomass to biofuel conversion, and enhanced bioprocessing techniques will allow the goal of a bio-based future to become fully realized commercially and globally. Foreign petroleum dependence is anticipated to diminish in the wake of widespread adoption of biofuels and bioenergy production. In order to develop the necessary infrastructure and technology prior to implementation, renewable resource processing must be given scientific priority. Yet another significant opportunity lies in environmental protection via bioprocessing. An overall reduction in industrially generated and municipal waste streams could be realized through feedstock bioconversion to biofuels in place of toxic hydrocarbon production. The enduring goal of the scientific community is to create carbon neutral bioprocesses that efficiently use a wide range of renewable resources to produce energy and chemicals with the intent to make technological and scientific gains in meeting the emergent bioeconomy’s demands. Incremental efficiency improvements as well as rapid analytical characterization of renewable fuel have led to developments in robust, stable, and even automated bioprocessing technologies and systems. Recent development of specially adapted separation and purification technologies for the recovery of bio-based products and the progress made in bioprocessing technologies will facilitate the economic conversion of renewable feedstocks into biofuels, bio-based chemicals, and value-added bioproducts. Microorganisms that have been genetically modified (GM) and are capable of transforming biomass into biofuels, bio-based chemicals, and value-added bioproducts along with development of bioproducts’ biomanufacturing processes that are highly regulated, controlled, and have foreseeable performance may provide means in an earlier adoption of biofuels production from renewable resources.
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Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts
1.2 CHALLENGES AND PROSPECT OF BIOPROCESSING FOR BIOFUELS AND BIOPRODUCTS The advanced technologies used for bioprocessing face obstacles not only in recognizing industry needs but also in furthering the transfer of technology while teaching the next generation of engineers and scientists that will support growth within the field. This includes supporting innovative alternative fuels technologies that will fully harness that potential of contemporary bioprocessing. Essentially, the tenants of industrial bioprocessing are understanding the biocatalysts or microorganisms employed, maintaining product quality and safety no matter the operation’s size, exercising environmental stewardship, and promoting process innovation to maintain a competitive edge within the industry as well as with consumers. Specific bioengineered processes of note include bioreactor design, concentrating and purifying diluted product streams, and applying cost-effective engineered solutions to biofacilities from cradle to grave [4]. In order to nurture growth in the understanding of how to produce a growing and wide array of biofuels, bio-based chemical, and bioproducts, a sustained and internationally agreed upon policy is paramount. A selected few of more than 72,000 algae species have been thoroughly evaluated [5], and even fewer have been used on an industrial scale. The latest attempts in laboratory and pilot scale phototrophic, heterotrophic, and mixotrophic microalgae cultivation have unveiled novel organisms that have yet to be exploited. While renewable energy is already being used in many different forms, microalgae-derived carbon-neutral biofuels are desirable candidates due to their sustainability, large carbon dioxide sequestration capacity, large lipid production, and their ability to grow in a multitude of environments (e.g., brine, brackish, and wastewaters) [6 9]. The cheap and economical extraction of lipids form microalgae continues to be a major hurdle in its commercial biofuel adoption. Fervent studies are currently ongoing to ascertain the capability of microalgae as both a biofuel and in carbon dioxide fixation. While a promising biofuel feedstock, microalgae are not yet utilized on an adequate industrial scale for bulk commodities. The final cost of the extraction process can also be aided by a comprehensive technoeconomic analysis, which will also provide guidance by means of a cost/benefit analysis for future process improvement (e.g., increased lipid yields). However, relevant breakthroughs in technology (e.g., genetic modification, metabolic engineering techniques, and biorefining) indicate that further developments will yield a suitable process that is both economical and sustainable in the near future. Future industrial processes may rely upon the novel chemicals yet to be unlocked within microalgae. Existing examples of modern bioprocessing technologies demonstrate the vast variety of manufacturing methodologies required to create bioproducts and biofuels. A singular step has the potential to greatly skew the cost,
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quality, and properties of the resulting product. Transforming microorganisms and creating new biocatalyst necessitate the use of many different types of advanced bioprocessing techniques. Something that is often neglected is the variety of engineering skills, specifically those for bioprocessing that are required when dealing with bio-based products. Specific bioengineering challenges in the field today are as follows: ensuring that regulatory and biological standards are met in equipment design while remaining economical; ensuring bioproducts processes are environment friendly and sustainable; and demanding consistent high-quality bioproducts by implementing robust and rapid purification processes. Highly specialized bioproducts are often the result of dilute solution fermentation, a process that can be optimized by improving energy usage and efficiency which can be done by driving down costs in handling, synthesis, and downstream processing. Other areas of improvement include bioreactor design and making conditions more conducive to cultivating and creating microorganisms and their products. Advanced bioprocessing techniques also can make advances in fermentation (i.e., submerged, solid substrate) while cellular and genetic manipulation can make strides in the field by changing the physical properties of microbe membranes to negate the toxicity caused by extracellular fermentation products. The three dominant products of biofuels and bioenergy are presently bioethanol, biodiesel, and biogas. Synthesized via the fermentation of soluble sugars or starches (e.g., sugarcane, corn), bioethanol is considered to be a first-generation biofuel. Within the scientific community, there is a push toward the development of second-generation bioethanol, derived from the lignocellulosic biomass of plants, with the initial results showing great promise. Comprising lignin polymers, hemicelluloses, and cellulose, lignocellulosic biomass is a renewable resource and can be used to manufacture biofuels and value-added bioproducts. Unfortunately, the molecular structure and heterogeneity of lignocellulose may prove to be problematic. The lignin’s resistance to enzymatic degradation, preventing the conversion of the plant’s available polysaccharides into sugars, is the overwhelming recalcitrant factor for biofuels production. Furthermore, as a nonlinear polymer that is inherently chemically diverse and composed of weak reactive linkages along with multiple monomer units, the lignin’s phenolic polymer within its cell wall component is to blame for overall biomass recalcitrance. As such, lignin degradation is cost prohibitive, requiring pretreatment to access the polysaccharide content required for biomass conversion to biofuel. Genetic manipulation techniques have attempted to facilitate plant biomass processing by employing a methodology that, while in its infancy, would create plant that either accumulates less lignin or yields lignin that is readily decomposable. Conventional techniques utilize genetic engineering in such a way that modifies the enzyme expression used for lignin biosynthesis.
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1.3 BIOPROCESSING ROUTES FOR THE PRODUCTION OF BIOFUELS AND BIOPRODUCTS The developments of advanced biotechnology and bioprocesses for the production of biofuels and bioproducts have been selected as topics to be discussed at length herein. Biotechnology was first used by Karl Ereky in 1919, in reference to the science and methodologies that create products from raw materials while aided by living organisms [10]. Genetic engineering (or recombinant DNA technology) utilizes biotechnology to manipulate the cellular structure genetically through the introduction or elimination of specific genes. The use of these techniques yields GM organisms (GMOs), organisms altered by inserting at least one transgene frequently from a different species than that of the recipient. The 1970s gave way to the first genetic modification experiments, combining synthetic human genes with bacterial genes. Ten years later, research could insert genes into fungi and yeast as well as otherwise foreign genes into plant and limited animal cells. In regards to the biofuels sector, both biotechnology and genetic engineering hold great promise in advancing agricultural production and converting biomass. Such gains may come in the form of energy crop production optimization, increasing crop yield per acre, modifying feedstock to aid in ease of fuel conversion, and the novel creation of enzymes that support downstream biofuel processing [11]. Although genetic engineering appears quite alluring at present, it is not clear if it will deliver on its promises, be overshadowed by new technologies, or become cost prohibitive in the future. Although selective breeding methods will continue to influence agricultural advancement, genetic engineering may focus efforts to enhance existing crops while expanding the catalog of plant varieties available for industrial use. Crops can be designed through genetic to aid in processing and subsequently are created for novel forms of raw materials. These plant varieties may be either altered or chosen for desirable traits that allow then to be easily converted into fuels. An example of this that is currently employed is the synthesis of bioethanol from the fermented sugars of corn via pretreatment processes that break up lignocellulose in order to eliminate the lignin, thereby promoting cellulose deconstruction by creating pathways for enzyme access. Presently, enzymatic pretreatments and production are costly. Genetic engineering could solve this impedance in biofuels progress by creating plants with helpful traits such as reduced lignin content, self-producers of cellulase or ligninase enzymes for cellulose or lignin degradation, respectively, or total yield boost to cellulose or biomass content. Modifying a plant’s genetic structure could also create plants that require less water, are able to fixate nitrogen, can be harvested easily, and biorefined into consumable protein, carbohydrates, and fibers for both the food energy industries. Bioengineered oilseed crops may also be utilized as the key ingredients in biodiesel as bio-based lubricants and esterified fatty acids. In
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addition to trait modification, biotechnology and bioprocessing could also be employed to increase yields. The past four decades have witnessed an incredible upward trend of annual rate of yield increases, the vast majority of which have been achieved through improved genetics via traditional gene recombination and recurrent selection. This trend has also been supplement by GMO introductions over the past decade but in a limited capacity. While genetic engineering has developed pests, weeds, and disease-resistant plants that tolerate drought and salinity stressors, the overall impact to the environment has yet to be determined. The production aspect of biofuels may also reap benefits through the use of genetic manipulation within the energy sector. Two major hurdles in the lignocelluloses conversion process that may be solved using genetic engineering are the expensive cellulose decomposing enzymes (cellulases) and the microbes’ limited fermentation ability of the breakdown products. This scope is also broadened by those scientists who wish to surpass the limited yeast diet of the microbes. Since yeast fermentation is limited to breaking down glucose and other hexoses, if researchers could also incorporate the decomposition of pentoses derived from hemicellulose, 15% 50% (plant species dependent) of previously unavailable lignocellulose can become available for biofuel production. Originating cellulose and hemicelluloses, the available lignocellulose sugars for fermentation cannot be accessed for enzymatic hydrolysis, thus necessitating an expensive pretreatment step which modifies the lignocellulosic structure. Pilot studies are currently underway that are evaluating other potential pretreatment technologies A variety of cellulases and hemicellulases are required for lignocellulose carbohydrate hydrolysis, a process that creates fermentable sugars. The hydrolysis process varies depending upon the feedstock: in cellulose, the linear glucose chains are broken down sequentially, whereas hemicellulases’ branched chains of varying sugars and functional groups are hydrolyzed. Currently, both pretreatment and hydrolysis technologies are on the brink of becoming commercially viable. With improved enzymatic functionality and a reduction in cost, high substrate containing lignocellulose is now able to be processed. Although these two technologies must still resolve technical and scientific issues, large-scale lignocellulosic substrate fermentation is possible in the near future albeit with the expectation of significant yield improvements and further cost reductions. While in its infancy, dedicating genetic research into energy crops and their manufacturing processes seems to be a worthwhile investment, for both the public and private sectors, as many countries are implementing strict biofuel targets while simultaneously addressing climate change. Capturing the attention of scientists across the world in the past 10 years, advanced bioprocessing applies science and engineering methodologies to microorganism and their derived products to create bioproducts with a small or neutral
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eco-footprint. Academics and industry leaders alike have yet to fully understand how indicative bioprocessing technologies will be in the commercial success of biofuels. What is certain is that developing and current bioproducts will need new, cost-effective, and efficient processes. As such, advanced bioprocessing will be in high demand in the bioindustries and the expanding biofuels, bio-based chemicals, and bioproducts marketplace. Although certain challenges exist in bioprocessing such as finding technologies that are scalable, low in cost, and minimizing waste creation, as these issues are adequately addressed, alternative fuels will become more viable.
1.4 RENEWABLE RESOURCES AND TRADE ISSUES Amidst all of these tantalizing opportunities, bioprocessing will need to be used on a grand scale, specifically for the cost-effective production of biofuels, bio-based chemical, and bioproducts using locally source feedstocks while developing new technologies. Carbon dioxide generated during combustion processes may be mitigated through engineering a large volume bioprocess. One potential answer to the issue of facilities that emit CO2 would be to incorporate either a photosynthetic or nonphotosynthetic organism into the stream to consume said emissions. Inventive engineering would be required to feasibly undertake such a large-scale problem while being sensitive to cost. Concurrently, biocatalyst can be used on a smaller scale in various applications to address similar issues facing energy and bioproducts production. Renewable feedstocks can be used to create a myriad of biobased chemicals. Although it has yet to become practical from an economic standpoint, bioprocessing may become more acceptable with raising petroleum prices, shortages in hydrocarbon reserves, or merely from an environmental stewardship perspective. Were this to occur, appropriate bioreactors and biocontrol systems would be necessary to implement advanced bioprocessing technologies in order to produce bio-based fuels, chemicals, and value-added product within stringent economic and environmental limits. Generally, current and future renewable resource applications occur on an industrial scale and create low-value products. To keep processing costs low, choosing to bioprocess certain feedstocks must be done with thoughtful regard. Bioprocessing technologies are continuing to find innovative applications in the manufacture of an assortment of bio-based products. Advancements must continue to deliver gains in hydrolysate fermentation, waste treatment, and the conversion of byproducts to value-added goods. The renewable bioconverting and fermentation industries stand to make large gains with the discovery of a cheap fermentable sugar source, which would pave the way to producing high-value specialty chemicals and additional technology advances. Some examples of these resulting products include alcohols, ketones, organic acids, microbial polysaccharides (oil well drilling), and amino acids (animal fodder) as well as high-value products such as
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flavors, fragrances, pharmaceuticals, and pigments. A plentiful renewable feedstock is agroderived (e.g., corn) starch which creates materials that utilize fermentation, enzymatic, and separating processes. A substantial amount of the liquid fuel market, specifically in the transportation sector, may be sourced in the future from cellulosic biomass used to create ethanol, further decreasing dependency on petroleum while reducing greenhouse gas emissions. Onstream reliability, productivity, and capital expenditures may be positively influenced by conceptual advanced bioreactors designed for high throughputs. Prospective research topics in bioreactor design and engineering are optimizing microbe to solids contact, continuous processing, large fermenter process control, and microbial metabolic manipulation. The economic implications of producing consumable from bioreactors will affect environmental, energy, and food sectors. An injection of bioprocessing theory will be needed to further develop the most appropriate biomasses types and conversions for reforming while fully utilizing any residual products. Biomass can even be thermally reformed into synthesis gas which can then be used in existing hydrocarbon plants. In order to create this revolutionary technology, a multidisciplinary knowledge must be used to their fullest extent in the generation of such a novel process. This cross-disciplinary approach spanning various scientific fields will facilitate advances in renewable resource bioprocessing. Much like biopharmaceuticals, the end consumer product typically originates from an organism. The key difference between these two industries lies in their production volumes, with bioprocesses requiring not only large facilities and feed rates but also consistent, cheap, and dynamic processes. Ultimately, the end objective when using renewable resources is to create bioproducts or biocatalyst so as to produce value-added consumables (e.g., fuel, chemicals, energy, etc.). Other important factors include the process economics, scalability, and quality consistency. In order to become economically feasible, researchers must continue to drive down processing costs of these bioproducts so as to meet a reasonable market price. As with any established process, for bioprocessing technologies to become widely adopted, improvements to efficiency, operation, equipment as well as gains in processing volumes and reliability must be realized. Researchers must capitalize on this opportunity afforded by renewable resources as the ramifications of such technologies will have far reaching effects on natural resources and arable land across the globe. There is opportunity for the developed and developing countries alike to enjoy the rewards of bioprocessing so long as an appropriate regulatory framework is established alongside the development of careful strategies. In this sense, the developed countries can strengthen their domestic energy security while avoid any social and economic disruptions caused by fossil fuel scarcities and inflation. On the other hand, the developing countries
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could reduce their foreign dependency on oil, tempering their exposure to oil price fluctuations while lessening their overall environmental footprint. Globally, rural areas could experience unemployment reductions and supplemental revenue with the introduction of a bio-based economy. Agricultural products could be introduced to new markets, increasing their overall worth. The international community is expected to see positive impacts by achieving these goals but none as much as the developing countries whose populations struggle with limited access to commercial energy, air pollution, and declining agricultural growth. There is a calculated amount of risk assumed when discussing bioprocessing and biotechnology despite the promise of biofuels, bio-based chemicals, and value-added bioproducts. Energy-specific GMO crops, while intended to boost yields increase and cultivate desirable traits, have raised concerns of their potential to threaten human and environmental health. While presently lacking definitive scientific evidence, it is postulated that the harm of genetic engineering may only be apparent in future studies. Although energy optimized crops would not be suitable for animal or human consumption, there are still cross-species pollination concerns, breeding between GM and nonGM or indigenous species that have yet to be alleviated. Crops intended as livestock feed or for human could become contaminated with the GM strains, thus elevating the fear level of the public. Societal reluctance to the adoption of GM crops for any reason, as well as any lingering or suspected environmental and sustainability impacts, must also be tackled prior to the widespread adoption of the technology.
1.5 CONCLUSION This perspective demonstrated novel systems that apply advanced bioprocessing technologies to produce biofuels, bio-based chemicals, and value-added bioproducts from renewable sources. Utilizing proven strategies and appropriate regulatory frameworks, expanding renewable fuels could potentially alleviate the serious issues presented before countries such as increasingly volatile petroleum cost and supply, ultimately aiding in the ease of access to cheap energy, especially for those in the developing countries. Biofuels could play a role in preserving the environment, lowering greenhouse gas emissions, presenting new opportunities to rural communities, and adding increased value to agricultural products. Genetic engineering could be the key factor in optimizing biofuels production, becoming a component of the global energy mix. Agricultural ventures that employ the use of genetic engineer practices are not without scrutiny or controversy. The emergent issue of global energy security has become the focus of the scientific community, where studies are being conducted to deliver environmentally feasible and economical alternative fuels. Biofuel is one such approach that may be able to meet future demand.
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REFERENCES [1] International Energy Agency, “World Energy Outlook 2017”, Executive Summary. ,http://www.iea.org/Textbase/npsum/WEO2017SUM.pdf., 2017, p. 1 (last accessed November 2018). [2] M. Hosseini, Sustainable Pretreatment/Upgrading of High Free Fatty Acid Feedstocks for Biodiesel Production, University of Akron, 2013. [3] M. Hosseini, H.A. Starvaggi, L.-K. Ju, Additive-free harvesting of oleaginous phagotrophic microalga by oil and air flotation, Bioprocess. Biosyst. Eng. 39 (7) (2016) 1181 1190. [4] N.R. Council, Putting Biotechnology to Work: Bioprocess Engineering., National Academies Press, 1992. [5] M.D. Guiry, How many species of algae are there? J. Phycol. 48 (5) (2012) 1057 1063. [6] M. Hosseini, L.-K. Ju, Use of phagotrophic microalga Ochromonas danica to pretreat waste cooking oil for biodiesel production, J. Am. Oil Chem. Soc. 92 (1) (2015) 29 35. [7] N. Moradi-kheibari, H. Ahmadzadeh, M. Hosseini, Use of solvent mixtures for total lipid extraction of Chlorella vulgaris and gas chromatography FAME analysis, Bioprocess. Biosyst. Eng. 40 (9) (2017) 1363 1373. [8] L.-K. Ju, M. Hosseini, Method and System for Reducing Free Fatty Acid Content of a Feedstock, US Patent App. 14/450,601, 2015. [9] L.-K. Ju, M. Hosseini, Treatment/Cleaning of Oily Water/Wastewater Using Algae, U.S. Patent Application No. 14/909,522, 2016. [10] K. Ereky, Biotechnologie der Fleisch-, Fett-, und Milcherzeugung im landwirtschaftlichen Grossbetriebe: fu¨r naturwissenschaftlich gebildete Landwirte verfasst, P. Parey, Berlin, 1919, p. 84. [11] C.E. Wyman, B.J. Goodman, Biotechnology for production of fuels, chemicals, and materials from biomass, Appl. Biochem. Biotechnol. 39 (1) (1993) 41.