Industrial Algae Measurements October 2017

Industrial Algae Measurements October 2017 | Version 8.0

A publication of ABO’s Technical Standards Committee

Founded in 2008, the Algae Biomass Organization (ABO) is a non-profit organization whose mission is to promote the development of viable commercial markets for renewable and sustainable products derived from algae. Our membership is comprised of people, companies, and organizations across the value chain. More information about the ABO, including membership, costs, benefits, members and their affiliations, is available at our website: www.algaebiomass.org.

The Technical Standards Committee is dedicated to the following functions: • Developing and advocating algal industry standards and best practices • Liaising with ABO members, other standards organizations and government • Facilitating information flow between industry stakeholders • Reviewing ABO technical positions and recommendations

For more information, please see: http://www.algaebiomass.org

Staff Committees Executive Director – Matthew Carr Events Committee General Counsel – Andrew Braff, Wilson Sonsini Goodrich & Rosati Chair – Craig Behnke, Sapphire Program Chair – Jason Quinn, Colorado State University Administrative Coordinator – Barb Scheevel Administrative Assistant – Nancy Byrne Member Development Committee John Benemann, MicroBio Engineering

Budget & Finance Committee Board of Directors Martin Sabarsky, Cellana Chair – Jacques Beaudry-Losique, Algenol Biofuels Bylaw & Governance Committee Vice Chair – David Hazlebeck, Global Algae Innovations Chair – Mark Allen, Accelergy Corporation Director Emeritus – Keith Cooksey Executive Director – Matt Carr Technical Standards Committee Chair – Lieve Laurens, National Renewable Energy Laboratory Secretary – Tom Byrne, Byrne & Company Deputy Chair – Keith Cooksey, Environmental Biotechnology Consultants Mark Allen, Accelergy Corporation Bob Avant, TAMU Director Recruitment Committee Chair - Xun Wang, Triton Algae Innovations Jacques Beaudry-Losique, Algenol Craig Behnke, Sapphire Energy Executive Policy Council Amha Belay, Earthrise Nutritionals Chair – Mark Allen, Accelergy and Jill Kauffman Johnson, Corbion John Benemann, MicroBio Engineering Tom Byrne, CarlsonSV Laurel Harmon, LanzaTech Dave Hazlebeck (Global Algae Innovations) Jill Kauffman Johnson – Corbion Biotech Stephen Mayfield, California Center for Algae Biotechnology, UC San Diego Martin Sabarsky, Cellana Inc. Leslie van der Meulen, BioProcess Algae Xun Wang, President, Triton Algae Innovations, Ltd. Rebecca White, Qualitas Health Ross Zirkle, DSM Nutritional Products

Algae Biomass Organization | 1 About IAM 8.0

This document, released in October 2017, reviews a set of minimum descriptive parameters and metrics required to fully characterize the economic, sustainability, and environmental inputs and outputs of an aquatic biomass processing operation. Voluntary adoption of a uniform common language and methodology will accelerate and allow the industry to grow.

The IAM 8.0, like the IAM 7.0 is dedicated to Mary Rosenthal (1958 - 2014) who, as first Executive Director of the ABO, championed the development of technical standards in our still nascent industry. When ABO started it was no more than a small group of like-minded engineers, entrepreneurs, and scientists who saw the need to have an organization promoting the use of algal biomass and the growth of an algae industry to produce it. Mary helped champion the Technical Standards Committee into what it is today. She understood why developing Standards and formally distributing elemental information is critical to spawning any industry. Mary was a charming and engaging contributor to the efforts of the committee and together with her, the Committee spent time in the trenches fighting for this work and we owe her gratitude for paving the way to help us get where we are today.

ABO Technical Standards Committee Authors Dr. Lieve M. L. Laurens, Committee chair, Senior research scientist, Dr. Casey Lippmeier, Principal Scientist, DSM Nutritional Products, National Renewable Energy Laboratory, Golden, CO, USA, Columbia, MD, USA Contact: [email protected] Steve Howell, President and founder of MARC-IV, Chairman of the Dr. Keith E. Cooksey, Deputy committee chair, Environmental ASTM task force on biodiesel standards, Kearney, MO, USA biotechnology consultant, Professor emeritus, Montana State Dr. Robert Gardner, Assistant professor, Department of University, Bozeman, MT, USA Bioproducts and Biosystems Engineering, University of Minnesota, Jim Sears, Co-founder Visual Exploration LLC, former ABO Morris, MN, USA committee chair and CTO, A2BE Carbon Capture LLC, Dr. Jose Olivares, Division leader Biosciences Division, Los Alamos Boulder, CO, USA National Laboratory, Los Alamos, USA Dr. Mark Edwards, Professor, Arizona State University Morrison Dr. Robert McCormick, Principal engineer, Fuels Performance, School of Agribusiness and Resource Management, Vice president at National Renewable Energy Laboratory, Golden, CO, USA Algae Biosciences Inc Dr. Amha Belay, Earthrise Nutritionals, Calipatria, CA, USA Dr. Tryg Lundquist, Associate professor, Department of Civil and Environmental Engineering, California Polytechnic State University, Chad Miller, Clearas Water Recovery, Missoula, MT, USA San Luis Obispo, CA, USA Dr. Craig Behnke, Vice president at Sapphire Energy, San Diego, CA, USA

Contributing Authors and Reviewers Ryan Davis, National Renewable Energy Laboratory; Dr. Jason Quinn, Utah State University; Amha Belay, Earthrise Nutritionals; Greg Sower, ENVIRON IntI.; William Hiscox, WSU Pullman; Don Scott, National Biodiesel Board; Ron Pate, Sandia National Labs; James Collett, Pacific Northwest National Laboratory; Mark Allen, Accelergy Corporation; David Glass, Joule Unlimited; Mike Johnson, DSM Nutritionals

Support Funding for editing and typesetting was provided by FedEx through a donation to the Algae Foundation. Printing was carried out by SCHOTT. Dr. Liesbeth Aerts helped with edits of IAM 7.0, which is carried forward in this version. Graphic design was provided by ClearVision Design, Minneapolis, MN.

Table of Contents About the IAM 8.0 Document Page 2 Executive Summary Page 3 Chapter 1: State of the Art Algal Product and Operations Measurements Page 5 Chapter 2: Life Cycle and Techno-Economic Analysis for the Uniform Definition of Algal Operations Page 13 Chapter 3: Regulations and Policy on Algal Production Operations Page 20 Chapter 4: Use of Wastewater in Algal Cultivation Page 24 Chapter 5: Regulatory and Process Considerations for Marketing Algal-Based Food, Feed, and Supplements Page 28 Chapter 6: Regulatory Considerations and Standards for Algal Biofuels Page 31 Chapter 7: Open and Closed Algal Cultivation Systems Page 33 ABO 2017 Corporate Members Page 39

Algae Biomass Organization | 2 Executive Summary

The goal of this document is to provide an nutrients required by the algae, as well as • Chapter 4 Use of wastewater in algal overview of the current state of the art of land requirements, process consumables, cultivation discusses the considerations of measurements or metrics, as well as policy and human resources required by the using wastewater as a nutrient and water and regulatory environments that are infrastructure. Green Box outputs include source for an algae farm and takes into pertinent to the development and growth the different classes of algal products account the regulations and permitting of a successful algal industry. The descriptive as well as industrial waste emissions involved in commercialization. Algal parameters listed in this document are including gas, liquid, and solid discharges. growth on wastewater is discussed in designed to provide the industry and Together, the measured inputs and outputs the context of the presence of pollutants academic groups with a common language generically carve out the total economic and in different production systems, and direct and objective parameters for the and environmental footprint of any algal and ultimately evaluation metrics for evaluation of technologies currently on track operation. Identifying this total footprint will wastewater treatment and recycling are to be commercialized. The methodologies, become increasingly central in the funding, listed. metrics, and discussions in this document regulatory, and sustainability review of an • Chapter 5 Regulatory and process continue to equally encompass autotrophic, expanding algae industry, and will ultimately considerations for marketing algal-based heterotrophic, open pond, photobioreactor, come to define the commercial viability of food, feed, and supplements outlines and open water production, as well as harvest specific ventures. regulatory process steps in obtaining and conversion processes for microalgae, In the content that follows, we present the approval from the respective overseeing macroalgae, and cyanobacteria, and are aimed metrics and language of algal measurements agencies for the inclusion of algae as at being process and pathway agnostic. to provide a guide to the regulatory novel dietary ingredients or food/feed Industrial Algae Measurements version 8.0 is a environment and other considerations additives. collaborative effort representing contributions applicable to the algae industry. of over 30 universities, private companies, • Chapter 6 Regulatory considerations and national laboratories over the past • Chapter 1 State-of-the-art-algal product and standards for algal biofuels describes seven years. This fully updated October 2017 and operations measurements discusses the process required to produce a legally version offers detailed recommendations on methodologies for assessing productivity marketable biofuel from algae, with links measurement methodologies for use across at the cellular level, along with the to comply with the new developments the industry and roadmaps of the regulatory detailed composition of the products. on the Renewable Fuel Standard that is environments surrounding different facets In this version of the document, we administered by the EPA. of the industry. This 2017 Industrial Algae include a discussion of available standard Measurements (IAM 8.0) supersedes the 2013 procedures for feedstock and product • Chapter 7 Open and closed algal and 2015 Industrial Algae Measurements characterization that have been made cultivation systems, including (IAM 6.0 and 7.0) and previous Minimum available through standards agencies such heterotrophic fermentation of algae, Descriptive Language documents (or MDL) as ASTM, AOAC, and AOCS. describes measurement parameters and reporting metrics that are particularly that the Technical Standards Committee has • Chapter 2 Life cycle and tcehno- published from 2010 through 2012. Overall, important in comparing algal growth economic analysis for the uniform systems. the industry guidance contained in IAM 8.0 definition of algal operations gives a has been broadened in scope, with increased rudimentary understanding of life cycle The industry will continue to face challenges depth and a new chapter layout for content. analyses specifically applicable to the now and in the future. In particular relating ABO’s “Green Box” approach discussed below, algae industry and increasingly important to algal operations that will vary in size describes the industry’s environmental, in the funding and government support of from individual bioreactor arrays producing economic, and carbon footprint via programs. quantifying the inputs and outputs specialty chemicals and nutraceuticals, of an installation. These input/output • Chapter 3 Regulations and policy on to expansive farm-scale production of measurements systematically allow for algal production operations reviews and food products and biofuels. Accurate economic projections (through techno- summarizes regulatory and permitting assessment of their future economic and economic analyses) and sustainability processes applicable to algae farming, environmental footprint will be critical to calculations (through life cycle assessments). and provides a framework overview of the financing development and performing Inputs include the carbon, water, energy, and siting approval process. environmental life cycle analysis (LCA). There

A. Total Infrastructure (Hectare) H. Algal Constituent Products (kg/yr) ‘Green Box’ Provides a e.g. Dry algal biomass, protein, oil etc. B. Total Energy Input (kWh/yr) Technical, Economic, I. Indirect Algal Products (kg/yr) C. Total Consumables Input (kg/yr) & Environmental e.g. Ethanol, Isobutyraldehyde, fish etc. Boundary D. Total Required Labor (FTEs) - J. Un-captured Gas Emissions (kg/yr) e.g. CO22 , NOx , H O, Hydrocarbons etc. E. Water Input (Liters/yr) Accounting for an Algae Operation’s K. Liquid Waste Output (Liters/yr) F. Total Nutrient Input (kg/yr) Total Yearly Inputs e.g. Saline or biologic discharge etc. G. Carbon Input (kg/yr) & Outputs L. Solid Waste Output (kg/yr) e.g. Organics, salts, airborne dust etc. p Figure 1: ‘Green Box’ approach to describe distinct operational components via the collective inputs and outputs, forming the basis of the descriptive parameter and metrics discussion in this document.

Algae Biomass Organization | 3 recommends that when large-scale algal A note on “algae” versus “algal”: operations are proposed or analyzed, ABO has adopted the common the sets of descriptive metrics and parlance of using “algae” when methodologies are adopted to uniformly describing the industry as the “algae characterize these operations. By industry” and the ABO organization harmonizing a common set of descriptive as the “Algae Biomass Organization”. metrics, the algal industry will accelerate However, the correct scientific usage its growth by eliminating confusion in the applied elsewhere in this document business and LCA arena of this industry. By and recommended to users for identifying the sources and characteristics technical and scientific discussions of inputs, and the intended fates and is as follows: algae is the plural noun characteristics of outputs, we will allow referring to a multitude of cells, alga for upstream and downstream life cycle is a single cell and algal is the proper environmental and techno-economic 1 adjectival form. analysis (TEA). By knowing the quantity of inputs and outputs we can feed data into techno-economical models to arrive at cost and process productivity parameters. is no harmonized descriptive language set, By knowing the character of the inputs nor have measurement methodologies and outputs we add an understanding of been specifically developed to describe the value flow. By knowing the upstream the diverse technologies being proposed source and downstream fate of inputs and for scaled algal farms. The lack of a suitable outputs we further add an understanding common language and methodology has of the sustainability and enduring footprint created confusion in expressing attributes of an operation. As the size of an algal and represents a barrier to industry facility increases, the importance of a expansion. comprehensive understanding of the inputs and outputs expands. Additional guidance With the distribution of this document, on LCA studies may be found in the ISO the ABO Technical Standards Committee 1404x series of documents that describe proposes a set of descriptive language and goals, scoping, quality, transparency, and measurement methodologies tailored to requirements for data collection. The EPA’s the growing needs of our industry across its Renewable Fuel Standard (RFS) guidance diverse technologies, operation sizes, and also provides background on boundary product types. ABO’s approach manages conditions assumed when performing complexity by measuring and characterizing sustainability calculations within the context process inputs and outputs only at the of an LCA. boundary that might encompass just an algal farm or fermentation facility, or it could The Committee welcomes the voluntary further include the plants’ infrastructure, its adoption of the IAM 8.0 language and water source, or a portion of a biorefinery or measurement methodologies into peer- power plant connected to the farm. In this reviewed research. Likewise, the committee way, the delineated boundary conditions depends upon peer-reviewed research and outlined throughout the document (and its own peer-review processes to form its in the ‘Green Box’ approach) provide a recommendations. We welcome growth descriptive method that can be adapted in academic and industry contribution to to compare algal operations having wholly the Committee. This IAM 8.0 document different inner workings yet having similar is designed to meet the evolving needs inputs and outputs. The essential set of input of the algal industry and its stakeholders. and output variables required to characterize Accordingly, the ABO Technical Standards the economic and environmental footprint Committee invites formal stakeholder is described through the balance of this comments on furthering the scope document (Figure 1). and specifics of this document. Please contact ABO directly to log comments Environmental and economic footprint and contributions. The Committee will accounting should be mostly indifferent to formally review comments and recommend the particular technologies a commercial improvements on a periodic basis: operation might employ during production. [email protected]. Companies who wish to keep their inner processes confidential can nonetheless provide useful information for regulatory 1 Laurens LML, Slaby EF, Clapper GM, Howell S, Scott D. Algal agencies and for site location licensing. Biomass for Biofuels and Bioproducts: Overview of Boundary Con- The ABO’s Technical Standards Committee ditions and Regulatory Landscape to Define Future Algal Biorefiner- ies. Ind Biotechnol 2015; 11: 221–228.

Algae Biomass Organization | 4 Chapter 1: State-of-the-Art algal Product and Operations Measurements

In order to establish long-term cultivation dry weight. Thus, it is not useful to derive cultivation. However, CO2 is dissolved in an and biorefinery operational trials, different a highly sensitive measurement for cell aqueous system and forms a weak acid-base stakeholders need to harmonize their data content, such as cellular protein/cell, when buffer system according to Eqn. 1: * inputs towards a more uniform description that figure will be divided by a parameter where H2CO 3 includes CO2 (aq) and H2CO3. and testing of algal biomass and products. that has lower statistical confidence. The relative amount of the dissolved We encourage an open dialogue on the Sampling frequency and sample size will inorganic carbon (DIC) species in the above adoption of a set of descriptive parameters also influence the ratio. For this reason, an equilibrium depends on the pH of the to help eliminate confusion, and accelerate accurate measurement of sample volume 2 - system. Therefore, bicarbonate (HCO3) is growth of the algal industry. We believe that and representative sampling are essential the dominant inorganic species in the pH standardization across an industry cannot be as well. A standard method that is routinely where most microalgae thrive (i.e., between enforced, rather will have to be encouraged, used throughout the water and wastewater pH 6.5 and 10). However, the pH of an algal and will ultimately happen through industry is the ASTM D5907 method, culture is manipulated by N-assimilation and consensus among a group of stakeholders. which provides a detailed assessment the amount of photosynthesis activity,2-4 of filterable matter (or dissolved solids) and these metabolic events can cause the Possibly two of the most pressing and in a water environment (Table 1.1). The - dissolved CO2 and HCO3 concentrations fundamental areas of standardization are implementation of an existing standard to be displaced far from equilibrium.5 By in the measurement of algal productivity method and adoption throughout the consequence, microalgae have developed and biomass composition. Algal biomass industry could help set the stage for like- carbon concentrating mechanisms (CCMs) productivity is the denominator in for-like comparisons between different to increase the carbon flux to ribulose- any description of algal yield, and the cultivation systems and reports on the 1,5-bisphosphate carboxylase/oxygenase composition provides the respective yield performance of reactors. (RuBisCo), which catalyzes the first step of the constituents that give it market Dry weight can be assessed by “primary in carbon fixation.6 Microalgal CCMs value. The composition of biomass forms measurements”, in which the desired employ a number of carbonic anhydrases a crucial point in any algal bio-production component is separated and weighed and bicarbonate transport proteins that process, and there is an effort to suggest directly, or by “indirect measurements”, such effectively and reversibly shuttle inorganic universally accepted analytical methods - as cell counting or fluorescence, where carbon, in the forms of HCO3 and CO2, across that would allow researchers and industry the measured quantity is calibrated to the the periplasmic membrane, through the members to compare processes and track actual mass and used as an analog. Indirect cytosol, into the chloroplast, and convert individual components, including lipids, measurements are very useful because of it to CO2 in the direct vicinity of RuBisCo in proteins, carbohydrates, and ash. We discuss their typical speed and sensitivity when the pyrenoid. This is an effective strategy to the current challenges for the application calibrated. It is the aim of the Technical maintain high levels of carbon in the cell and of these methods in the algal industry and Standards Committee to provide relatively avoid loss of CO2 by passive diffusion across recommend measurement practices that are easy and inexpensive primary measurement the cell membrane. based on a review of existing methods. methods that are useful across a variety of algae and different types of aquatic biomass. Microalgae will grow at atmospheric Trade and testing organizations will have Although other methods exist, the ones concentrations of inorganic CO2 (~400 to work together to define the required mentioned in this document have been ppm); however, biomass productivity can be biomass, oil, and other product properties, tested and confirmed by many laboratories. improved by supplementing the media with and encourage the use of select test Where possible, the described methods additional inorganic carbon. It is often cited methods for the analysis of algal biomass include their respective advantages and that this additional carbon source could composition. The applicability of test disadvantages. come from industrial waste such as coal- methods currently in use (American Society fired power, cement production, or plant flue for Testing and Materials, ASTM; Association Carbon content measurements of Official Analytical Communities, The carbon content of the biomass is AOAC; American Oil Chemists’ Society, often one of the primary measurements 2 Markou G, Vandamme D, Muylaert K. Microalgal and AOCS; and International Organization for to determine the energetic value of the cyanobacterial cultivation: the supply of nutrients. Water Res 2014; Standardization, ISO) should be evaluated 65: 186–202. biomass and to provide information on using reference material in the context of 3 Eustance E, Gardner RD, Moll KM, Menicucci J, Gerlach R, Peyton the efficiency of carbon conversion in a BM. Growth, nitrogen utilization and biodiesel potential for two comprehensive interlaboratory studies. For cultivation system. Carbon is assimilated chlorophytes grown on ammonium, nitrate or urea. J Appl Phycol example, culture health parameters which 2013; 25: 1663–1677. by either photosynthesis in autotrophic are known to be important in the industry 4 Shiraiwa Y, Goyal A, Tolbert NE. Alkalization of the Medium by algal cultivation systems, or from an organic Unicellular Green Algae during Uptake Dissolved Inorganic Carbon. and for objective assessment of reactor and carbon source such as sugar in heterotrophic Plant Cell Physiol 1993; 34: 649–657. culture performance can be found on the fermentations. 5 Nedbal L, Cervený J, Keren N, Kaplan A. Experimental validation ASTM website.1 of a nonequilibrium model of CO₂ fluxes between gas, liquid medium, and algae in a flat-panel photobioreactor. J Ind Microbiol

Carbon utilization and measurements Biotechnol 2010; 37: 1319–26. Whole cell dry weight, cell 6 Giordano M, Beardall J, Raven JA. CO₂ concentrating mechanisms Inorganic carbon (CO2) is the primary in algae: mechanisms, environmental modulation, and evolution. number, biomass productivity nutrient required for sustainable algal Annu Rev Plant Biol 2005; 56: 99–131. Biomass productivity in cultivation systems depends on both the cell number and the

!!!≈!.! !!!≈!.! !!!≈!".! ∗ ! ! !! ! 1 ASTM. Water Testing Standards. http://www.astm.org/Standards/ ! (!") ! ! ! ! ! water-testing-standards.html (accessed Aug 2015). !" + ! ! ! !" !"# + ! !" + 2!

Algae Biomass Organization | 5 selection for microalgae that can tolerate to discern the total DIC. Some dual chamber high pH and ionic strength.2 However, carbon analyzers consist of both a heated the solubility and application efficiency is chamber and an acidic sparging chamber, much higher when bicarbonate salts are which can be configured to measure total used as supplemental DIC, as compared DIC in only one step.

to gaseous CO2, and contamination from microorganisms is reduced due to the high A CO2 electrode can be used to measure ionic strength of the media. Furthermore, dissolved CO2 in a system. Basically, a CO2 depending on the concentration and timing permeable membrane allows the electrode of the culture amendments, bicarbonate solution to equilibrate with the surrounding supplementing can increase both growth aqueous environment and the resulting and lipid accumulation.13-15 A related source pH is measured. In order to measure the of DIC is anaerobic digestion wastewater, total DIC concentration, the sample must where organic carbon has been converted be acidified to drive the dissolved carbon to methane and CO . The latter will have 2 species to CO2. The disadvantages of using dissolved into the wastewater stream, a CO2 electrode include membrane fouling establishing a bicarbonate-carbonate buffer from algal cultures and potential electrode with elevated DIC concentrations.2 interferences with volatile weak acids (e.g., - - NO2, HSO3 , acetic acid, and formic acid). Inorganic carbon measurements Carbon dioxide and other gases consisting Organic carbon measurement of dissimilar atoms absorb infrared radiation of the biomass 7,8 gas. However, CO2 solubility is dependent at unique and discrete wavelengths. Thus, A variety of C analyzers are available, along on the temperature, total pressure, and the most common technique for measuring with standard procedures (e.g. ASTM D4129) concentration of total dissolved solids, and gaseous CO2 is to use infrared spectroscopy. to measure total organic carbon in aqueous in shallow depths a fraction of supplied CO2 Total inorganic carbon (or DIC) is typically samples. Solid phase CHN analyzers will gas could escape into the atmosphere.9,10 measured by acidification of the sample measure total carbon on a dried filter or a The efficiency of CO2 dissolution into driving the carbonate equilibrium to CO2, dried pellet. However, primary methods aqueous solutions is dependent on the which is then sparged from the solution are needed to calibrate to the cell weight deviation of chemical conditions from using oxygen or inert gas, and trapped of each algal species. While this method is equilibrium, the contact time (e.g., aqueous for quantification. Quantification can be accurate even for small sample sizes if the depth), and the contact surface area (e.g., done using infrared spectroscopy, gas calibration is accurate, its disadvantages are bubble parameters).5,11-13 chromatography, or by coulometry. Current that it is an indirect measurement requiring state-of-the art gaseous CO2 measurements expensive equipment, and additionally, the An alternative approach to using gaseous are done using off-gas sensors employing C/N ratio in algal cells changes with time of CO2 directly in algal cultures is to use infrared technology, which has become day and growth conditions, complicating the solutions with high non-carbonate alkalinity relatively inexpensive. Furthermore, current interpretation of the results. (e.g., high hydroxyl ion concentration state-of-the-art DIC measurements are and high pH) to absorb CO2 and convert performed by filtering a sample (0.45 µm or it to solid phase bicarbonate salts (e.g., smaller) and analyzing it using a total carbon

NaHCO3, KHCO3, and NH4HCO3). The main analyzer. A portion of a filtered sample is concerns with using bicarbonate salts are combusted under high temperature using a higher cost than gaseous CO2 and strain a heavy metal catalyst, thereby converting the total organic and inorganic components

to CO2. The resulting CO2 gas is then moved across an infrared sensor using a carrier gas 7 Collet P, Hélias A, Lardon L, Ras M, Goy R-A, Steyer J-P. Life-cycle assessment of microalgae culture coupled to biogas production. and quantified by comparison to known Bioresour Technol 2011; 102: 207–14. concentrations of organic and inorganic 8 Sheehan J, Dunahay T, Benemann J, Roessler P. A Look Back at standards. A second analysis must be done the U.S. Department of Energy’s Aquatic Species Program: Biodiesel from Algae. Natl. Renew. Energy Lab. Goldon, CO. 1998. http:// on the sample, and the sample must be www.nrel.gov/biomass/pdfs/24190.pdf (accessed Aug 2015). acidified and sparged with oxygen (or 9 Benemann JR. Utilization of carbon dioxide from fossil fuel- another inert gas) to remove all of the DIC, burning power plants with biological systems. Energy Convers Manag 1993; 34: 999–1004. and then reanalyzed by combustion. Thus, 10 Milne JL, Cameron effrey C, Page LE, Benson SM, Pakrasi HB. total carbon content and the dissolved Perspectives on Biofuels: Potential Benefits and Possible Pitfalls. organic carbon (DOC) content are measured, American Chemical Society: Washington, DC, 2012 http://dx.doi. org/10.1021/bk-2012-1116.ch007 (accessed Aug 2015). and the latter is subtracted from the former 11 Danckwerts PV, Kennedy AM. The kinetics of absorption of carbon dioxide into neutral and alkaline solutions. Chem Eng Sci 1958; 8: 201–215. 14 Gardner RD, Cooksey KE, Mus F, Macur R, Moll K, Eustance 12 Putt R, Singh M, Chinnasamy S, Das KC. An efficient system for E et al. Use of sodium bicarbonate to stimulate triacylglycerol carbonation of high-rate algae pond water to enhance CO₂ mass accumulation in the chlorophyte Scenedesmus sp. and the diatom transfer. Bioresour Technol 2011; 102: 3240–5. Phaeodactylum tricornutum. J Appl Phycol 2012; 24: 1311–1320. 13 Lohman EJ, Gardner RD, Pedersen T, Peyton BM, Cooksey KE, 15 White DA, Pagarette A, Rooks P, Ali ST. The effect of sodium Gerlach R. Optimized inorganic carbon regime for enhanced bicarbonate supplementation on growth and biochemical growth and lipid accumulation in Chlorella vulgaris. Biotechnol composition of marine microalgae cultures. J Appl Phycol 2012; Biofuels 2015; 8: 82. 25: 153–165.

Algae Biomass Organization | 6 Carbon accounting It is possible that temporary storage of for biodiesel or green diesel.16-19 Not all In the prior section, the possibility of carbon can have positive impacts, however lipids can be considered equally valuable using carbon dioxide as a feedstock for this is more of a consideration for long term for fuel or even food or feed applications. algae growth opened up the possibility biomass capture, such as reforestation.20 The The lipid composition, with respect to polar for large-scale algae cultivation to be following methods demonstrate examples (phospho- and glycolipids) and non-polar considered a carbon sequestration of carbon accounting methods21: (triglycerides and sterols) lipids and the technology. Carbon accounting attempts respective impurities found in each fraction, • Source Reduction: CO2 is only reported to assess the environmental impact of an when it enters the atmosphere, and is highly dependent on the origin and type industrial process by determining how sequestered carbon is not taken into of biomass. Autotrophically grown algae effective a process is at storing carbon. account. are rich in polar lipids, waxes, sterols, and Though there are different techniques for pigments, whereas heterotrophic cultivation carbon accounting, however they follow • Sink Enhancement: All emissions are will yield triglyceride-rich oil similar to plant- a similar development process. First, the reported, but sequestering options are derived oils, but often with very different boundaries for the process are determined, converted to negative emissions fatty-acid profiles.26,27,58 which involves determining which inputs • Value Chain: Considers all inputs and and outputs to consider, as well as whether outputs in cradle-to-grave greenhouse Traditionally, lipids have been measured 16 or not to consider upstream emissions. gas emissions, as well as energy balances gravimetrically after solvent extraction. The For example, indirect land use change completeness of extraction and composition • Point of Uptake and Release: all (ILUC) causes some carbon dioxide release, depends on the biochemistry of the alga emissions of CO (combustion, decay, etc.) however it is currently difficult to quantify. In 2 and the recent physiological conditions count as positive emissions and all uptake some carbon accounting methodology, such experienced by the organism, as well as the of atmospheric carbon counts as negative as those used in the EU and British Columbia, compatibility of the solvent polarity with the 17 emissions. ILUC are excluded. The boundaries of a lipid molecule polarity and the extraction carbon accounting method have a large conditions used, resulting in inconsistent impact on whether the process can be Carbon accounting is used for comparison lipid yields.22-25 Inevitably, the extractable oil considered carbon neutral, carbon positive, between different technologies and fraction will contain non-fuel components or carbon negative. After the boundaries for established to provide a consistent and (e.g., chlorophyll, other pigments, proteins, the process are determined, an emissions standardized method. For this reason, it and soluble carbohydrates). Thus, it may be inventory is carried out. Usually the is normalized and expressed as a flow of necessary to assess its fuel fraction (i.e., fatty 19 emissions are normalized to a designated carbon per unit area per unit time. acid content) by transesterification followed CO amount on a consistent unit for easy 2 Compositional analysis of algal by quantification of the fatty acid methyl comparison. In addition, different types of esters (FAMEs). Due to the large number emissions have different residence times in biomass, lipids, carbohydrates, of variables, it is difficult to standardize the atmosphere, so it may be appropriate and protein an extraction-based lipid quantification to consider the time dimension of carbon Characterization of algal biomass consists of procedure. There are two extraction systems emission.16 Once an analysis is preformed it the accurate measurement of lipids, proteins, is usually compared to process alternatives and carbohydrates as the major constituents to determine whether the process is of all biomass samples. The degree to which 22 Haas MJ, Wagner K. Simplifying biodiesel production: The direct advantageous compared to existing these are characterized depends primarily or in situ transesterification of algal biomass. Eur J Lipid Sci Technol 2011; 113: 1219–1229. technology.16 on the information required and different 23 Davis RE, Fishman DB, Frank ED, Johnson MC, Jones SB, Kinchin methods provide different information. CM et al. Integrated evaluation of cost, emissions, and resource In the carbon cycle, algal biomass would potential for algal biofuels at the national scale. Environ Sci Technol 2014; 48: 6035–42. take the form of a carbon stock, as carbon Algal lipids vs. extractable oils 24 Knothe G. A technical evaluation of biodiesel from vegetable dioxide is converted to biomass. Accounting vs. fuel fraction oils vs. algae. Will algae-derived biodiesel perform? Green Chem 2011; 13: 3048. for carbon in terms of algal biofuels is The detailed composition and molecular complicated since part of the carbon is only 25 Knothe G. Biodiesel and renewable diesel: A comparison. Prog profile of lipids is required for reporting on Energy Combust Sci 2010; 36: 364–373. temporarily stored. Some of the carbon oil quality and biomass valorization and 26 Li MH, Robinson EH, Tucker CS, Manning BB, Khoo L. Effects of will be released back to the atmosphere as will be highly influential when targeting dried algae Schizochytrium sp., a rich source of docosahexaenoic emissions during e.g. biofuel combustion. acid, on growth, fatty acid composition, and sensory quality particular bioproduct markets, for example of channel catfish Ictalurus punctatus. Aquaculture 2009; 292: However other carbon will be present in the 232–236. residual biomass. This biomass may be used 27 Pyle DJ, Garcia R a, Wen Z. Producing docosahexaenoic acid as a co-product or disposed as waste, which (DHA)-rich algae from biodiesel-derived crude glycerol: effects of impurities on DHA production and algal biomass composition. J 18,19 would have other carbon implications. Agric Food Chem 2008; 56: 3933–9. 28 Guckert JB, Cooksey KE, Jackson LL. Lipid sovent systems are not equivalent for analysis of lipid classes in the microeukaryotic green alga, Chlorella. J Microbiol Methods 1988; 8: 139–149. 16 Caffrey K, Chinn M. Carbon Accounting: Description and 29 Iverson SJ, Lang SL, Cooper MH. Comparison of the Bligh and Methology. 2015 https://content.ces.ncsu.edu/carbon-accounting- Dyer and Folch methods for total lipid determination in a broad description-and-methodology range of marine tissue. Lipids 2001; 36: 1283–7. 17 Plevin RJ, Beckman J, Golub AA, Witcover J, O’Hare M. Carbon 30 Laurens L, Quinn M, Van Wychen S, Templeton D, Wolfrum Accounting and Economic Model Uncertainty of Emissions from EJ. Accurate and reliable quantification of total microalgal fuel Biofuels-Induced Land Use Change. Environ Sci Technol 2015; 49: potential as fatty acid methyl esters by in situ transesterification. 2656–2664. 20 Costa PM. Carbon accounting, trading and the temporary Anal Bioanal Chem 2012; 403: 167–178. 18 Sayre R. Microalgae: The Potential for Carbon Capture. Biosci- nature of carbon storage. 2002 http://pdf.usaid.gov/pdf_docs/ 31 Bigogno C, Khozin-Goldberg I, Boussiba S, Vonshak A, Cohen ence 2010; 60: 722–727. Pnacy491.pdf Z. Lipid and fatty acid composition of the green oleaginous alga 19 Vardon M. Carbon and Ecosystem accounting. 2014 http://doc. 21 Pena N, Frieden D, Bird DN. Accounting for Algae. GCB Bioen- Parietochloris incisa, the richest plant source of arachidonic acid. teebweb.org/wp-content/uploads/2017/01/ANCA-Tech-Guid-5.pdf ergy 2013; 5: 243–256. Phytochemistry 2002; 60: 497–503.

Algae Biomass Organization | 7 texture (impurities, unsaponifiable matter; i.e. non-fatty-acid-containing lipids, such as sterols, pigments, and hydrocarbons), properties that may affect shelf life (moisture, storage stability), and overall purity of the triglyceride oil (i.e., low level of free fatty acids). Some of these oils or fats are marketed as detergents, lubricants, or as other industrial or cosmetic applications. These markets capitalize on the properties that are already present for edible purposes, and typically add or emphasize one or currently in use across algal biomass attributes common to naturally occurring several properties that are important for the analytical laboratories: conventional Soxhlet triglycerides, making up the majority of the particular industrial application. extractor systems or the more recently vegetable-derived oils and fats, or other developed pressurized fluid extraction important characteristics for the use of Inedible oils are commonly used for animal systems (as in the commercially available these oils. The algae industry will need to feed rations, biodiesel, and other industrial Accelerated Solvent Extractor, Thermo provide a list of the necessary properties and applications. These generally require only Scientific, Massachusetts, USA). attributes of algal oils, allowing them to be low-grade oil or fat and there tends to be easily compared to already approved oils less concern with oil color, some impurities, As an alternative to extraction there is a and fats. While it is premature at this time to and free fatty acid levels. On the other growing emphasis on the quantification develop specific standards or trading rules hand, some other properties become more of lipids through a direct (or in situ) of algal oils, those for existing oils and fats important, such as viscosity, energy content transesterification of whole algal biomass. can serve as a useful guide for the attributes (BTU), and low levels of poly-unsaturated The process consists of either a two-step and specific values of algal oils that will be fatty acids to enhance storage stability. alkaline and subsequent acid hydrolysis demanded by customers (Table 1.1). of the biomass,32,33 or a single-step acid High-throughput measurement catalysis,24,34,35 followed by the methylation Traditional oils and fats are often derived of lipids in algae of the fatty acids to FAME and quantification from vegetable oil feedstocks and are In addition to the procedures listed by gas chromatography (GC). These generally characterized by application above, there has been an emphasis to procedures have been demonstrated to be (edible or industrial applications), by their accelerate the quantification of lipids. robust across species and their efficacy is source (plant, animal, algal, etc.), and then Often, researchers need to tailor the less dependent on the parameters listed by various industry terms, which generally analysis to the screening of thousands of above that influence lipid extraction. describe their quality or purity (crude, individual strains for bioprospection or However, if the relative composition of intact refined, or refined and bleached) or their metabolic engineering projects. These lipids is required (e.g., polar versus neutral end use (technical grade, feed grade, etc.). In high-throughput methodologies are based lipid content), an extraction process may particular the end use will often determine on hydrophobic (lipophilic) fluorescent be the only way to isolate intact lipids from the list of properties and quality targets that the rest of the biomass, with the utilization need to be met. These oils consist mostly of advanced instrumentation, such as liquid of triglycerides and have a premium on chromatography for the characterization of properties important to their end use (color, the lipid molecular profile. Several reports cold properties, heat stress properties), in the literature and AOAC (Association of minor compounds that can affect flavor or Official Analytical Communities) official methods suggest in situ transesterification as the lipid quantification procedure of choice for algal biomass (Table 1.1).24,26–29

Quality attributes and considerations for algal oils Trading rules and important quality attributes of existing vegetable-derived oils and fats are based on a combination of key

32 Griffiths MJ, van Hille RP, Harrison STL. Selection of Direct Transesterification as the Preferred Method for Assay of Fatty Acid Content of Microalgae. Lipids 2010; 45: 1053–1060. 33 AOAC. Analysis of Fatty Acids. In: Official Method 991.39. 1995 34 Bigelow NW, Hardin WR, Barker JP, Ryken SA, Macrae AC, Cat- tolico RA. A Comprehensive GC-MS Sub-Microscale Assay for Fatty Acids and its Applications. J Am Oil Chem Soc 2011; 88: 1329–1338. 35 Lohman EJ, Gardner RD, Halverson L, Macur RE, Peyton BM, Gerlach R. An efficient and scalable extraction and quantification method for algal derived biofuel. J Microbiol Methods 2013; 94: 235–244.

Algae Biomass Organization | 8 dyes, such as Nile Red29,36 and BODIPY.37,38 CH OH As these dyes are soluble in a lipid or OH 2 hydrophobic environment, the fluorescence O OH intensity increases proportionally with the Chemicals H H O HO H lipid content and this principle has been O HO O O used extensively in the screening for high OH Enzymes HO OH H OH lipid-producing cells (Figure 1.2). Note that OH n BODIPY staining may not be a substitute for H OH Nile Red in semi-quantitative fluorescence measurements of total lipids, as the dye does not exhibit a Stokes wavelength shift when binding to hydrophobic areas of an algal cell, such as neutral lipids. Furthermore, although fluorescent dyes are a powerful requirement for quantitative prediction of accumulation stage. The FAME-GC method and potentially high-throughput approach a new set of materials is a robust predictive measures all fatty acids, irrespective of for screening lipid-producing cells, caution model of near-IR spectra based on a fully their origin and thus both polar and neutral has to be taken with the quantitative characterized calibration sample set. lipid-derived fatty acids will be measured, interpretation of fluorescence results from An alternative rapid non-destructive while NMR will be specific in only measuring both BODIPY and Nile Red Dyes, due to method for in vivo analysis of oil content in the neutral lipid content. This discrepancy the possibility of inconsistent dye-uptake live algal cultures by 1H Nuclear Magnetic between the two methods and two derived between different algal species. Resonance (1H NMR) has recently been values becomes smaller at high neutral lipid developed.41 The method is specific for concentrations. neutral lipids including free fatty acids and Infrared (mid- and near IR) spectroscopy mono-, di-, and tri-acyl glycerides (MAG, offers an alternative possibility of a rapid and Algal carbohydrate measurements DAG and TAG) stored in cellular lipid bodies. sensitive determination of the composition Carbohydrates can comprise a significant Less than 1 mL of algal culture is required of algae because IR vibrations of organic portion of the algal biomass and thus for analysis, and the measurement takes compounds directly follow Beer’s law their accurate quantification is crucial only minutes on commonly available NMR and can be used for quantitative analysis. to determine the feasibility of using an spectrometers (300 MHz or greater). Virtually Mid- and near-IR wavelengths are able algal species for specific biofuel and co- no sample preparation is required, and to quantitatively determine the amount product pathways. Often carbohydrate drying is unnecessary. The lower limit of of lipids to algal biomass from different determination is reported by calculation, detection of neutral lipids in a culture by species. By combining the measured lipid which means that the sum of ash, protein this in vivo method is approximately 30 µg/ content with the spectra using multivariate and lipids is subtracted from the mass mL. In a typical analysis, a < 1 mL sample statistical approaches, predictive calibration balance, leading to the rest of the biomass of algal culture is placed in a NMR tube, models can be built.39 Near-IR spectra were being carbohydrates. In some food/feed and a coaxial capillary insert containing correlated with increasing concentrations of sources that may approximate the true a calibrated reference solution is inserted lipids, allowing for the distinction between carbohydrate content, however, in single- into the tube. The assembly is then placed neutral and polar lipids. Recently, a similar cell organisms such as algae, this may in the magnet of a NMR spectrometer for approach was taken, where near-IR spectra cause a significant overestimation of the analysis. Since 1H NMR is an inherently and of a set of biomass samples were used true carbohydrates in the biomass. One directly quantitative measurement of all to build quantitative prediction models method for algal biomass carbohydrate the observable protons in the sample, the of the full biochemical composition of determination is based on an analytical NMR signals between 0.25 and 2.85 ppm three microalgal strains.40 This approach hydrolysis step in which polymeric due to TAGs (static proton NMR) can be is capable of taking the full quantitative carbohydrates are released to their integrated and compared to a reference biochemical analysis of algal biomass monosaccharide constituents. There signal.35 Anticipated applications of algal from several days down to a minute, using are historical methods based on a rapid lipid 1H NMR include prospecting or only a fraction of the material needed for phenol sulfuric acid method, which claim screening of oleaginous algal cultures, traditional chemical analyses. The only to hydrolyze and react quantitatively process optimization studies, and process with the carbohydrates in solution.42,43 monitoring and control in large culturing However, the phenol-sulfuric acid method operations. The method is complimentary is notoriously variable and not all sugars 36 Cooksey KE, Guckert JB, Williams SA, Callis PR. Fluorometric to FAME-GC methods, including direct exhibit a similar colorimetric response. determination of the neutral lipid content of microalgal cells using Nile Red. J Microbiol Methods 1987; 6: 333–345. transesterification, and can be used to Thus some carbohydrates could cause an 37 Govender T, Ramanna L, Rawat I, Bux F. BODIPY staining, an distinguish neutral lipid production over- or underestimation if a calibration alternative to the Nile Red fluorescence method for the evaluation from lipids derived from cell membrane is performed based on one neutral of intracellular lipids in microalgae. Bioresour Technol 2012; 114: 507–11. components. Neutral lipid concentrations monosaccharide such as glucose and 38 Cooper MS, Hardin WR, Petersen TW, Cattolico RA. Visualizing have been found to be consistently lower ‘green oil’ in live algal cells. J Biosci Bioeng 2010; 109: 198–201. by 1H NMR than total lipids by FAME-GC for 39 Esbensen KH. Multivariate Data Analysis - in practice: an introduc- the same culture samples throughout the oil tion to mutlivariate data analysis and experimental design. CAMO 42 DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimet- Process AS: Oslo, Norway, 2002 ric Method for Determination of Sugars and Related Substances. Anal Chem 1956; 28: 350–356. 40 Laurens LML, Wolfrum EJ. High-Throughput Quantitative Bio- chemical Characterization of Algal Biomass by NIR Spectroscopy; 41 Davey PT, Hiscox WC, Lucker BF, O’Fallon J V., Chen S, Helms GL. 43 Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S-I, Lee Multiple Linear Regression and Multivariate Linear Regression Rapid triacylglyceride detection and quantification in live micro- YC. Carbohydrate analysis by a phenol-sulfuric acid method in Analysis. J Agric Food Chem 2013; 61: 12307–14. algal cultures via liquid state 1H NMR. Algal Res 2012; 1: 166–175. microplate format. Anal Biochem 2005; 339: 69–72.

Algae Biomass Organization | 9 this method is not recommended for a colorimetric47 and a nitrogen-ratio compounds between different strains and quantitative reporting of biomass content method.42-44 The latter is based on growth conditions of algae will affect the in algae.36,44 Alternative carbohydrate measuring elemental nitrogen and then applicability of the averaged conversion quantification procedures involve sequential applying an algae-specific nitrogen-to- factor. A detailed investigation of the strain hydrolysis of carbohydrate polymers in protein conversion factor to measure the and physiology of algae on the factor algae, and identification and quantification total nitrogen content in the biomass.42 A calculation has recently been published, and of the monomers via liquid (HPLC) or GC (as fluorometric procedure to measure algal concluded that a one-factor-fits-all approach alditol acetates or silylated derivatives).45 protein has been developed that offers may not be applicable to algae. The amino Because of the use of chromatography, the advantages of requiring only a minute acid composition of algae is perhaps the these procedures are likely to be more amount of biomass, excellent specificity, most accurate protein determination and accurate and will also provide a relative compatibility with a wide suite of reagents, official AOAC standard procedures have monosaccharide composition of the and a high throughput potential. The been published for the quantification of acid algae. There are a number of reports in the colorimetric and fluorometric procedures hydrolyzed amino acid determination. A literature but a comprehensive comparison can be susceptible to interferences from validation of nitrogen-to-protein conversion and test of robustness across strains are non-protein cellular components as well as should be carried out by users and algae currently lacking. from extraction buffer constituents, and are producers based on amino acid data for each highly dependent on the protein standard new strain or process that is implemented. Since starch represents a common storage used for calibrating the absorbance/ carbohydrate in algae, the measurement fluorescence values. The measurements Similarly to carbohydrates, the digestibility of this carbohydrate subset is among the are also dependent on the efficacy of cell and nutritional availability of the protein routine compositional analysis methods fractionation (solubilization of cellular fraction of algal biomass and residues is for algae. This method is selective for proteins). A detailed investigation of the important. Currently, those methods are alpha-1,4-linked glucan due to a specific colorimetric data against amino acid and implemented based on what is available enzymatic hydrolysis step, which is known nitrogen conversion data indicates a species- from the food/feed industry. Often, an to be present in some algal strains but and growth condition-dependent variability approximate value can be derived from the not in others. The method of biomass that underpins an up to 3-fold difference amino acid composition using a theoretical preparation and the enzyme assay kit highly between the colorimetric and the amino calculation referred to as the Protein affect the measurement.38 A protocol that acid data.51 Some of the variability that has Digestibility Corrected Amino Acid Score has been demonstrated to give accurate been observed between spectrophotometric (PDCAAS).52 The PDCAAS is representative and precise starch determination on a and alternative methods underscores the of protein quality based on both the amino variety of strains of algae is detailed in need for a careful interpretation of data from acid requirements of people (where the reference.46 Alternative methods exist and spectrophotometric assays. relative value represents completeness of can be considered equally valid after a proteins) and the ability to digest proteins. careful consideration of the accuracy and Calculating protein content using a The US Food and Drug Administration (FDA) precision of the method. Currently, the nitrogen-to-protein conversion factor has has adopted this rating as a standard to standard methods available from ASTM are proven to be a robust representation for determine protein quality. However, it has being evaluated for application to algae. whole biomass protein measurement. to be taken into account that calculating Similarly, there is a need to get more than Measuring elemental nitrogen is based the PDCAAS of a diet solely based on the just compositional information from the on high-temperature combustion and is PDCAAS of the individual constituents is carbohydrate pool of algae. For example, the much less susceptible to interferences. An impossible. Mainly because one food may digestibility of carbohydrates in the context algal biomass-specific conversion factor provide an abundance of an amino acid of nutritional value of biomass or residue is was calculated from the typical amino acid that the other is missing, in which case the an increasingly important area. In order to composition of 12 species of algae grown PDCAAS of the diet is higher than that of any determine this parameter, existing methods under different conditions.42,44 An overall one of the constituents. To arrive at the final for neutral and acid detergent fiber (NDF/ average ratio factor of 4.78 grams of algal result, all individual amino acids would have ADF) determination of feed and terrestrial protein for each gram of elemental nitrogen to be taken into account. feedstocks fall short of providing the detected has been used successfully for necessary information and these methods algal protein quantification. However, Measurement of volatiles and should be assessed and improved in future variation in the non-protein nitrogenous semi-volatiles in algal cultures years. There are several different approaches that are suitable for the determination of volatile 47 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measure- Algal protein measurements ment with the Folin phenol reagent. J Biol Chem 1951; 193: 265–75. and semi-volatile chemicals present in algal Protein content in algal biomass can be 48 Lourenço SO, Barbarino E, Lavín PL, Lanfer Marquez UM, Aidar E. cultures. These analytes may be naturally quantified using two common procedures: Distribution of intracellular nitrogen in marine microalgae: Calcula- occurring compounds or co-products tion of new nitrogen-to-protein conversion factors. Eur J Phycol 2004; 39: 17–32. present as a result of strain development 49 Diniz GS, Barbarino E, Oiano-Neto J, Pacheco S, Lourenço SO. activities. GC with Headspace Sampling and 44 Laurens LML, Dempster TA, Jones HDT, Wolfrum EJ, Van Wychen Gross Chemical Profile and Calculation of Nitrogen-to-Protein Flame Ionization Detection (GC-HS-FID) S, McAllister JSP et al. Algal biomass constituent analysis: method Conversion Factors for Five Tropical Seaweeds. Am J Plant Sci 2011; uncertainties and investigation of the underlying measuring chem- 2: 287–296. comprises an effective method for volatile istries. Anal Chem 2012; 84: 1879–87. 50 Templeton DW, Laurens LML. Nitrogen-to-protein conversion measurement and forms the basis of several 45 Templeton DW, Quinn M, Van Wychen S, Hyman D, Laurens factors revisited for applications of microalgal biomass conversion LML. Separation and quantification of microalgal carbohydrates. J to food, feed and fuel. Algal Res 2015; 11: 359–367. Chromatogr A 2012; 1270: 225–34. 51 Laurens LML, Van Wychen S, McAllister JP, Arrowsmith S, Demp- 46 Megazyme. Total starch assay procedure (amyloglucosidase/ ster T A, McGowen J et al. Strain, biochemistry, and cultivation- 52 Boutrif E. Recent developments in protein quality evaluation. alpha-amylase method). 2011. https://secure.megazyme.com/files/ dependent measurement variability of algal biomass composition. 1991 http://www.fao.org/docrep/U5900t/u5900t07.htm (accessed Booklet/K-TSTA_DATA.pdf (accessed Aug 2015). Anal Biochem 2014; 452: 86–95. Aug 2015).

Algae Biomass Organization | 10 of the standard methods for volatile analysis the gas headspace. However, in most cases, and semi-volatile molecules including (Table 1.1). It is the preferred method for the addition of heat and/or salts can lower ethanol, isopropanol, acetone, acetaldehyde rapid and high-throughput analysis of algal the partition coefficient (K) by reducing gas methanol, acetonitrile, ethyl acetate, methyl culture volatiles, since algal samples can solubility. The partition coefficient (K) =s C / ethyl ketone, and others. be directly placed in a vial with little to no Cg, where Cs is the concentration of analyte preparation. in sample phase and Cg is the concentration Considerations for using a of analyte in gas phase. A slightly modified standard reference Volatile components from complex sample form of the Blood Alcohol Content (BAC) biomass material mixtures are isolated from non-volatile method53 can be used to quantify low levels Reference Materials (RMs) are ‘controls’ or sample components in the headspace of a of volatiles. This process requires heating standards used to check the quality and sample vial. Headspace GC is most suited to volatilize the compounds from the metrological traceability of products, to for the analysis of small molecular weight matrix, and therefore is not concentration validate analytical measurement methods, volatiles in samples as they are efficiently dependent. This method can detect volatile or for the calibration of instruments. A partitioned into the headspace gas volume standard RM is prepared and used for from the liquid or solid matrix sample. three main purposes: (1) to help develop Higher boiling point volatiles and semi- 53 Firor R, Meng C, Bergna M. Static Headspace Blood Alcohol accurate methods of analysis; (2) to calibrate volatiles may not be detectable with this Analysis with the G1888 Network Headspace Sampler. Agil. Appl. Note 5989-0959. 2004. http://www.chem.agilent.com/Library/ measurement systems used to facilitate technique due to their low partitioning into applications/5989-0959EN.pdf (accessed Aug 2015). exchange of goods, institute quality control, determine performance characteristics, Cultivation Characteristics Agency and method reference or measure a property at the state-of-the- Total suspended solids ASTM D5907 art limit; and (3) to ensure the long-term Total dissolved organic carbon ASTM D4129, adequacy and integrity of measurement Total dissolved nitrogen ASTM D3590 quality assurance programs.54 Unlike other Volatile and semi-volatile organics ASTM D2908 well-established food and oil commodities, Volatile alcohols ASTM D3695 the lack of a universal standard RM in the Biomass characteristics algae industry inhibits the direct comparison Moisture* AOAC 930.15; AOAC 934.06 of methods and measurements used to Fiber AOAC 991.43 compare processes and products from algae. Ash* AOAC 942.05; AOAC 923.03 The universal adoption of a RM provides Protein AOAC 990.03; AOAC 984.13 commercial sites and laboratories with a Carbohydrates AOAC 986.25 common platform to compare, for example, Fat (total lipids)* AOAC 954.02; AOAC 920.39 the fatty acid composition of different algal Fatty acids* ISO15304M strains grown under various environmental Chlorophyll* AOAC 942.04; AOCS Cc13i-96 conditions, and subjected to different oil Total phosphorus ASTM D5185 recovery processes. One example is to Total nitrogen ASTM D4629 generate a laboratory-produced natural Sodium AOAC 985.01 matrix standard, which has two distinct Zinc AOAC 990.08 advantages: (1) as a reproducibly generated Oil characteristics standard, it can supplant conventional Fatty acids not part of the AOCS Ca 5a-40 reference products that vary markedly triglyceride* among production batches; (2) such a material might help in the identification Color or clarity AOCS Cc 13a-43; AOCS Cc 13e-92; AOCS Td 1a-64 and elimination of errors in lipid extraction, Water and low boiling AOCS Ca 2f-93; AOCS Ca 2e-84 derivatization and analytical techniques, compounds by being able to provide a reference value General impurities AOCS Ca 3a-46 for measurements allowing for historical Sterols, alcohols, hydrocarbons AOCS Ca 6b-53 data tracking and outlier identification. One Storage stability AOCS Cd 12b-92; AOCS Cd 1b-90; AOCS Cd 18-90; candidate standard RM for fatty acid analysis AOCS Cd 22-91 is the newly identified haptophyte strain, Chain length of triglyceride fatty AOCS Ce 1i-07; AOCS Ce 1b-89 Chrysochromulina tobin (Haptophyceae).55 acids This optimized alga is amenable to this Freezing or cloud point AOCS Cc 6-25; AOCS Cc 12-59; AOCS Cc 1-25 purpose because: (1) as a soft bodied Thermal stability, frying suitability AOCS Cc 9a-48 organism, it is readily susceptible to all Metals AOCS Ca 17-01; AOCS Cs 20-99 conventional disruption and fatty acid Special acids (either highly AOCS Ce 1i-07 extraction techniques, (2) it has a high desired, or not desired for stability fatty acid content (~40% dry weight), (3) its purposes) Fuel properties Gasoline ASTM D4814 54 NIST. Standard Reference Materials. http://www.nist.gov/srm/ Jet fuel ASTM D1655 (accessed Aug 2015). Diesel ASTM D975 55 Bigelow N, Barker J, Ryken S, Patterson J, Hardin W, Barlow S et al. Chrysochromulina sp.: A proposed lipid standard for the algal biofuel industry and its application to diverse taxa for screening lipid content. Algal Res 2013; 2: 385–393.

Algae Biomass Organization | 11 methods. If a novel method describes the selected for improvement57: measurement of a raw material (e.g., oil or • Moisture: A method needs to be whole biomass), generally AOAC or AOCS established for thermally unstable should be contacted. However, for a method for a fuel or fuel parameter, it would be samples. This may be based on NREL TP- better to contact the method development 5100-60956 or AOAC 934-06 division of ASTM. If the new method aligns • Ash: The EN-ISO 18122:2015 is generally with an established subcommittee, it will be satisfactory, however improvements presented for approval and comments. If this could be made for marine algae samples. is a new area for the standards development In addition, clarification could be made organization, a new committee may be between the two temperature options. created. For example, AOCS has an Algal Products Expert Panel that is convening a • Fat (Total Lipids): A definition should be collaborative study on several analyses. Once created for the term “algae lipids” and there is consensus that a method should be a new standard based on Ryckebosch58 studied, there are set procedures to follow method could be developed for the for a collaborative study with the aim of extraction. determining the precision and accuracy of • Fatty acids: There is no standard for the method. testing by direct trans-esterification, and one should be created. Various trade groups already publish trading standards, guidelines, or quality targets for • Chlorophyll: The different methods oils and fats. Those listed and used by the for chlorophyll detection are difficult to AOCS, AOAC, ASTM, and ISO are listed in compare, and definite standard should be Table 1.1. When comparing the standard developed growth response and lipid profiles are highly test methods currently in use at commercial • Fatty acids not part of the triglyceride reproducible, and (4) unlike many algae analytical laboratories, for example for lipid (Unsaponifiable matter): There are that have limited fatty acid distributions, quantification, it is clear that there are a different methods of extraction and each the cells of this organism contain a broad handful of different extraction procedures should be tested specifically for algae. representation of both saturated and available that are incompatible; for unsaturated fatty acids ranging from 14 to example AOAC 920.39, a traditional Soxhlet 22 carbons long (C14 to C22). extraction with diethylether, and AOAC 954.02, which includes an acid hydrolysis Alternatively, a standard material can step prior to extraction. Both methods do be generated that is representative extract fat, however, the yields and chemical of cultivation trials and represents a composition of the resulting oils are very model organism approach. One such different, which can lead to very different example is Nannochloropsis, an organism conclusions. The former method will extract that is commonly used for commercial intact lipids from the cell’s interior and developments and in government- requires diffusion of the lipids through the sponsored research projects. To generate cell wall, which, as has been demonstrated a standard RM of Nannochloropsis, a large before, is a function of the cell’s properties Different end users or customers may amount of one cultivation batch would have and the strain of algae. The latter method want additional information; i.e., more to be made available to the community employs an acid to hydrolyze cell walls and specific fatty acid profiles for high value and stored and distributed in a manner that liberate the entrained lipids. A comparison nutraceuticals, or more information on the protects the material against degradation. of lipid quantification procedures in algae acid number and inorganic constituents for There is initial work underway with a RM highlighting the strain, physiological status, a diesel-type fuel application, and thus the algal biomass that is used by members of and cell wall discrepancies was recently attributes and values currently covered in a national consortium of algae growers published and can be translated to some of the list of existing methods provide some and testbed sites.56 The data obtained in 30,51 the standard test methods employed. good targets for the types of information that work could set the stage for further that will need to be provided in order for development and implementation of a The differences between test methods has the algal oil industry to continue to grow. As standard RM available to the algae industry. recently prompted the European Committee the algal oil industry develops, the different

for Standardization (CEN) to conduct a stakeholders should carefully consider these Standard methods available review of relevant algae biofuels guidelines existing markets and their requirements and for constituent and whole in order to determine which test methods should illustrate how algal oils may provide biomass analysis need standardization and are currently advantages compared to existing oils and not represented in the suite of methods AOAC, AOCS, and ASTM are standard fats. available by CEN or ISO, or represent development organizations that offer information needs where the available paths for requesting and developing new methods do not provide an adequate 57 European Committee for Standardisation. CEN/BT/WG 218 Final answer. The following measurements were Report - Year 1. 2016 58 Ryckebosch E, Muylaert K, Foubert I. Optimization of an Analyti- 56 ATP3, Algae Testbed Public Private Partnership, Algae Testbed. cal Procedure for Extraction of Lipids from Microalgae. J Am Oil http://atp3.org/ (accessed Aug 2015). Chem Soc 2012; 89: 189–198.

Algae Biomass Organization | 12 Chapter 2: Life Cycle and Techno-Economic Analysis for the Uniform Definition of Algal Operations

Life cycle assessment (LCA) and techno- quantified, including the release of CO2 from a highly flexible framework in the form economic analysis (TEA) are used to assess combusting fossil fuel for energy, methane, of robust engineering models is needed the total environmental, energy, and and other greenhouse gas (GHG) emissions that anticipates both existing and future financial footprint of a manufacturing from energy and material production. pathways for the algae-based production process. Commonly analyzed processes An overall LCA will clearly define which of food, fuel, and high-value chemicals. This include the powering and provisioning of processes are within its boundary or scope framework should be set up by describing algal production facilities, the conversion and often uses the 100-year global warming in a comprehensive manner the many or use of algae for products like fuel and potential for emissions of CH4, N2O and CO2. inputs and outputs that occur in algal co-products compatible with a biorefinery Alternative bioenergy-focused product engineering operations and by identifying concept or installation, the delivery of pathways are being investigated with the the methodologies required to measure those products to the market, the use aim of assessing the environmental impact these data. While the algae industry of the product, and the displacement of of the respective operations.5 continues to grow, harmonization between equivalent products such as fossil fuels or LCA and TEA methods is necessary, as the other co-product substitutes. The entire Similarly, a TEA approach is used for current evaluation of industry processes process and value chain is divided and modeling the conversion of biomass to depends on extrapolation of laboratory life cycle emissions are allocated to each fuels and includes a cultivation modeling data, and is affected by differences in fraction, most commonly on a mass-ratio approach. These process models compute production pathways and inconsistencies basis, underscoring the need for highly thermodynamically rigorous material and in the definition of the system boundaries. accurate quantification of the biomass energy balances for each unit operation. Similarly, the legislative landscape in and bioproduct composition, where The material and energy balance data different countries can impact the rate of bioproduct LCA credits are incorporated from such simulations are used to aid with adoption of renewable fuels. For example, on a displacement basis.1-4 For each of determining the number and size of capital the EU requires certain sustainability the steps, all net inputs and outputs are equipment items. As process conditions and criteria to be met and liquid fuels to have flows change, baseline equipment costs a sustainability certification prior to their are automatically adjusted using a scaling implementation into existing infrastructure.

1 Wang M, Huo H, Arora S. Methods of dealing with co-products of factor. These baseline cost estimates come biofuels in life-cycle analysis and consequent results within the U.S. from vendor quotes or from historical cost LCA and TEA tend to be complex, not only context. Energy Policy 2011; 39: 5726–5736. databases (for secondary equipment such as because of the many inputs, outputs, and 2 Sheehan J, Camobreco V, Duffield J, Graboski M, Shapouri H. Life Cycle Inventory of Biodiesel and Petroleum Diesel for Use in an tanks, pumps, and heat exchangers). inter-relationships that are involved, but Urban Bus. Golden, CO, 1998 http://www.nrel.gov/docs/legosti/ To generate input data for LCA and TEA, also because algal product manufacturing fy98/24089.pdf (accessed Aug 2015). processes vary widely and continue to be 3 Pradhan A, Shrestha DS, Gerpen J Van, Duffield J. The Energy Bal- ance of Soybean Oil Biodiesel Production: A Review of Past Studies. developed. There is currently no consistent Trans ASABE 2008; 51: 185–194. 5 Bradley T, Maga D, Antón S. Unified approach to Life Cycle Assess- or standardized reporting on LCA or TEA 4 Delucchi MA. Emissions of greenhouse gases from the use of ment between three unique algae biofuel facilities. Appl Energy approaches.6 Since LCA and TEA are crucial transportation fuels and electricity. Argonne, IL, 1993 2015. for the development of the algae industry, the need for their standardization is glaring.

Life cycle analysis A LCA process would minimally involve determining the energy and mass inputs, the emission of carbon and other GHGs, and the water balance associated with the production of one unit of product such as a gallon or liter of fuel. For example, the amount of energy embodied in an algae-based fuel is compared to the fossil or alternative energy required for its manufacturing. This concept is referred to as the net energy ratio (NER) or energy return on investment (EROI) of a given fuel product.3 LCA also factors in the waste products, air emissions, and raw materials. General guidelines for quantifying carbon footprint and GHG emissions are given in ISO/TS 14067 and ISO 13065 respectively, while LCA analysis guidance is given in the ISO standards 14040 and 14044.

6 Quinn JC, Davis R. The potentials and challenges of algae based biofuels: A review of the techno-economic, life cycle, and resource assessment modeling. Bioresour Technol 2015; 184: 444–52.

Algae Biomass Organization | 13 Often, the inclusion of co-products from defines four categories of renewable fuel assessment of algal fuels.15–17 It has been biofuels production can complicate the with minimum GHG reduction thresholds suggested that water is an important way LCA boundary conditions are set (as as a key requirement for each category. resource to be considered by LCA. However, defined in ISO 14040/44) and can impact EISA requires 20% GHG reduction for any the consistent framing of the use of water the LCA outcome for each scenario.1,5 The renewable fuel facility constructed after remains a complex challenge. Water itself is impact of each individual co-product and 2007, 50% reduction for advanced biofuel, a renewable resource, but the ways in which process scenario should be considered 50% reduction for biomass-based diesel, it is used for different energy strategies separately and reported in a consistent and 60% reduction for cellulosic biofuel. are not directly comparable. For instance, manner. The boundaries of a LCA should All of these are measured against the underground injection of water for hydraulic be clearly defined before it is performed. 2005 average petroleum baseline. Having fracturing and emulsification with fracking A LCA focusing on the main product and achieved significant success through 2014, fluids has a much different environmental system is known as an attributional LCA, the majority of growth remaining in the implication than the use of water to produce while one including broader environmental program is to be fulfilled by advanced electricity via hydroelectric power or cooling impacts is a consequential LCA.7 A LCA biofuels, which include biomass-based water for thermoelectric generation. These may assist in determining eligibility diesel and cellulosic biofuel. Implementation applications are all very different from the for government incentive programs, of the RFS requires the EPA to estimate the transpiration of water by organisms during evaluating the environmental impact of life cycle GHG emissions for renewable fuels biomass production or its recycling during farm operations, projecting economic to determine eligibility in the prescribed biomass processing. The impact of water performance, or performing a resource categories. EISA defines life cycle GHG use also varies dramatically by region, and assessment (RA). The latter is used to emissions as “the aggregate quantity of GHG therefore, a universal or even national calculate the total amount of fuel or other emissions (including direct emissions and framework is rarely appropriate. There is no product that can be manufactured using a significant indirect emissions such as those compliance threshold or inclusion of water specific process given the amount of input from land use changes), related to the full in LCAs required by EISA. Instead, following resources available within a specific area, or fuel life cycle, including all stages of fuel and EISA enactment, the EPA and the National alternatively, to inform the LCA of the need feedstock production and distribution, from Academies of Sciences with the National to bring resources to the cultivation facility feedstock generation or extraction through Research Council (NRC) have completed from remote locations.8,9 In all cases, the the distribution and delivery and use of qualitative assessment of water impact of importance of uniform approaches to these the finished fuel to the ultimate consumer, renewable fuels complying with the RFS. The analyses is increasing as the algae industry where the mass values for all GHGs are goal of this work was to assess and avoid seeks to rapidly develop, finance, and build adjusted to account for their relative global potential negative environmental impacts of out its operations. The standardized GREET warming potential.”14 the RFS.18 Again, as commercial algal facilities LCA tool developed by Argonne National are being developed, this may need follow- Laboratories has been adapted to over 100 LCA can include other resource inputs up to ensure that previous assumptions still feedstock-to-fuel pathways, yet it continues and impacts besides energy and GHGs. In hold true. to require adaptation to accommodate the particular, LCA can be used to compare diversity of algae-based fuels.9-11 environmental and human health impacts Techno-economic Analysis between renewable and conventional TEA focuses on assessing the economic In the US, the Energy Independence products. These impact categories can feasibility and commercial viability for a and Security Act of 2007 (EISA) included include potential effects with regard to given process pathway. The analysis can the national Renewable Fuel Standard acidification, eutrophication, air pollution, be represented by Equation 2-1 and allows (RFS)13 with the purpose of diversifying ozone depletion, smog formation, performance of a process to be quantified fuel alternatives and increasing the ecotoxicity, human toxicity, and fossil contribution from renewable fuels. EISA fuel depletion. Table 2.1 presents a list of suggested indicators employable in the 15 Efroymson RA, Dale VH. Environmental indicators for sustainable production of algal biofuels. Ecol Indic 2015; 49: 7 McManus MC, Taylor CM. The changing nature of life cycle assess- 1–13. ment. Biomass and Bioenergy 2015; 82: 13–26. 16 Bradley T, Maga D, Antón S. Unified approach to Life Cycle 8 Venteris ER, Skaggs RL, Coleman AM, Wigmosta MS. A GIS cost Assessment between three unique algae biofuel facilities. Appl model to assess the availability of freshwater, seawater, and saline Energy 2015. doi:10.1016/j.apenergy.2014.12.087 groundwater for algal biofuel production in the United States. 17 McBride AC, Dale VH, Baskaran LM, Downing ME, Eaton LM, Environ Sci Technol 2013; 47: 4840–9. Efroymson RA et al. Indicators to support environmental sustain- 9 Venteris ER, McBride RC, Coleman AM, Skaggs RL, Wigmosta ability of bioenergy systems. Ecol Indic 2011; 11: 1277–1289. MS. Siting algae cultivation facilities for biofuel production in the 14 U.S. Environmental Protection Agency. EPA Lifecycle 18 National Research Council (NRC). Sustainable Development of United States: trade-offs between growth rate, site constructability, Analysis of Greenhouse Gas Emissions from Renewable Fuels. EPA- Algal Biofuels. The National Academies Press: Washington D.C, water availability, and infrastructure. Environ Sci Technol 2014; 48: 420-F-10-006. 2010; : 3. 2012 3559–66. 10 Woertz IC, Benemann JR, Du N, Unnasch S, Mendola D, Mitchell BG et al. Life Cycle GHG Emissions from Microalgal Biodiesel - A CA- GREET Model. Environ Sci Technol 2014; 48: 6060–8. 11 Davis RE, Fishman DB, Frank ED, Johnson MC, Jones SB, Kinchin CM et al. Integrated evaluation of cost, emissions, and resource potential for algal biofuels at the national scale. Environ Sci Technol 2014; 48: 6035–42. 12 Frank ED, Han J, Palou-Rivera I, Elgowainy A, Wang MQ. User Manual for Algae Life-Cycle Analysis with GREET: Version 0.0. 2011 p Equation 2-1: General and simplistic principle of cost calculations for TEA, where Cproduction = Total cost of production https://greet.es.anl.gov/files/algae-life-cycle-manual (accessed Aug per unit product, Ccapital,i = Capital cost (installed), including direct and indirect capital costs, of sub-category i. Coperating,j = 2015). Operating cost of sub-category j. Vbio-products,k = Value of k co-product, capital and operating costs are time dependent and

13 USEPA. Renewable Fuel Standard (RFS). http://www.epa.gov/ assumed to be over the lifetime of the facility (Ccapital) or, for Coperating and Vbio-products on an annual basis and dependent on otaq/fuels/renewablefuels/index.htm (accessed Aug 2015). the yield of the products.

Algae Biomass Organization | 14 Category Indicator Unit Potential environmental effects Ref. Soil quality Bulk density g cm-3 Water holding capacity, infiltration, 15 crop nutrient availability

Terrestrial acidification kg SO2 equivalent to air 16 Terrestrial eco-toxicity kg 1,4 dichlorobenzene to industrial soil 16 Water quality Peak storm flow L s-1 Erosion, sediment loading, infiltration 17 Minimum base flow L s-1 Habitat degradation, lack 17 of dissolved oxygen Consumptive water use (incorporates feedstock production: m3 ha-1 day-1; Availability of water for other uses 15 base flow) biorefinery: m3 day-1 Water quality in Nitrate concentration in streams concentration (mg L-1, ppm); export kg-1 Eutrophication, hypoxia, 15 consumption and (and export) ha-1 yr-1 potability discharge water Total phosphorus (P) concentration in concentration (mg L-1, ppm); export kg ha-1 Eutrophication, hypoxia 15 streams (and export) yr-1 Salinity Conductivity (no unit) water composition change 15 Fresh/Marine water eutrofication kg P and N equivalent 16 Energy Fossil energy consumption MJ kg ha-1 yr-1 Fossil resource depletion 20

-1 Greenhouse gases CO2 equivalent emissions (CO2 and kg C eq GJ Climate change, plant growth 15

N2O)

Land use Agricultural/Urban land occupation m2 x year of land 16

Natural land transformation m2 x year of natural land 16

Resource depletion Mineral resource depletion kg Fe equivalent 16

Fossil resource depletion kg oil eq 16

Biodiversity Presence of taxa of special concern presence increased or decreased biodiversity 15 Habitat of taxa of special concern ha increased or decreased biodiversity 15 Abundance of released algae Number L-1 increased or decreased biodiversity 15 Air quality Tropospheric ozone ppb human and plant health 15 carbon monoxide ppm human health 15 Total particulate matter less than 2.5 µg m-3 visibility and human health 15 um diameter (PM2.5) Total particulate matter less than 10 µg m-3 visability and human health 15,16 um diameter (PM10) Ozone depletion kg CFC-11 equivalent 16 Human air toxicity kg 1,4 dichlorobenzene to urban air 16

Photochemical oxidant formation kg NMVOC compound equivalent to air 16

Productivity Primary productivity or yield gC L-1 year-1 or based on chlorophyll a Climate change, soil fertility, cycling 15 of carbon and other nutrients

Algae Biomass Organization | 15 as cost per unit product.19 This equation were selected as an initial focus (1) a lipid lipid co-products.24-26 One example of an delivers a break-even cost of production, extraction, (2) a hydrothermal conversion ALU approach is based on a biochemical and should be modified to include return on of whole algal biomass, and (3) a volatile processing strategy to selectively recover capital investment and income taxes in order biofuel product (e.g., ethanol) pathway. and convert certain algal biomass to represent profits. These pathways have been adopted by the components to fuels, namely carbohydrates US Department of Energy as baseline cases to ethanol and lipids to a renewable Much like LCA, the TEA should have for technology and process optimization diesel blendstock (RDB) product.27,28 The well defined boundaries. The final cost towards future design cases that improve overarching process design converts estimates are highly dependent on the cost basis for production of fuels as algal biomass, delivered from upstream location, as biological parameters vary with shown through full TEA and LCA and with cultivation and dewatering, to ethanol, RDB, meteorological and geographical conditions. comparative sustainability assessment and minor co-products, using dilute-acid Furthermore, local policies and economics reports available in the peer-reviewed pretreatment, fermentation, lipid extraction, play into construction and operation literature. We selected these pathways here and hydrotreatment. Additional areas, e.g., costs. Once algae biomass production or as examples only, of well-documented anaerobic digestion of spent algal residues, conversion TEAs are completed, they can be reports covering both the engineering and combined heat and power generation, and integrated into an overall process TEA. thermodynamic modeling assumptions. utilities are also included in the design, and so are detailed material and energy balances Sustainability considerations It is worth mentioning that the TEA and capital and operating costs for this for algal cultivation referenced here are based on “nth-plant” baseline process. This case study techno- During algal production and processing economics. The key assumption implied economic model provides a production th operations, gaseous, liquid, and solid by n -plant economics is that our analysis cost for the fuel products that can be used emissions can include indirect and direct does not describe a pioneer plant; instead, to gauge the technology potential and to GHG emissions associated with the it assumes several plants using the same quantify critical cost drivers. In brief, the production of input energy and materials as technology have already been built and process can be described as follows: whole well as their consumption during operations. are operating. In other words, it reflects a algal biomass, grown autotrophically in open This includes water evaporation, effluent mature future in which a successful industry pond systems, is utilized at a dewatered waters with entrained organic and inorganic of n plants has been established. Because paste concentration of 20% directly into a materials not otherwise suitable for recycled the techno-economic model is primarily a biomass-pretreatment process, followed use within the operation, solid biomass tool for studying new process technologies by aqueous-phase fermentation of sugars residue fractions not included in algal or integration schemes in order to comment liberated after pretreatment to ethanol, constituent products or indirect products, on their comparative economic impact, and finally hexane solvent extraction to th and solid inorganic, organic, and biological n -plant analysis avoids artificial inflation of separate the neutral lipid-rich oil from the particulates that can become airborne project costs associated with risk financing, biomass. The solvent is separated from the emissions or liquid effluent suspensions.15 longer start-ups, equipment overdesign, and oil and recycled. The lipid-extracted residual other costs associated with first-of-a-kind or biomass is sent to anaerobic digestion. The pioneer plants, lest these overshadow the anaerobic digestion of the spent biomass Techno-economic and life cycle real economic impact of research advances provides methane for process heat, and analysis for example pathways in conversion or process integration. It recycles CO2, nitrogen, and phosphorous to To assist in realizing the goals of increasing should be emphasized, however, that a large the algal cultivation ponds. The digestate bioenergy production from algae, a number number of the assumptions included in the (‘sludge’ solids) product from anaerobic of techno-economic evaluations have economic assessments carry a degree of digestion is sold as a fertilizer co-product. been developed for both biological and uncertainty and are subject to refinement. The raw oil is upgraded to finished fuels thermochemical pathways for converting (diesel-range hydrocarbons with a small algal biomass to fuels. These conceptual Algal lipid extraction and upgrading naphtha co-product) via hydrotreatment. evaluations of example processes, termed Conversion pathways focused around The TEA study calculated an overall cost “design cases”, provide a detailed basis for the extraction and upgrading of algal potential of $4.35/gallon gasoline equivalent understanding the potential of various lipids, are referred to as lipid extraction (gge) representing a plausible future conversion technologies and help identify pathways.21-23 They are often less destructive technical barriers where research and than comparative thermochemical development could potentially lead to avenues and thus allow for the utilization significant cost improvements. Consistent 24 Davis R, Kinchin CM, Markham J, Tan ECD, Laurens LML, Sexton and development of additional non- D et al. Process Design and Economics for the Conversion of Algal assumptions for items such as plant Biomass to Biofuels: Algal Biomass Fractionation to Lipid- and lifetimes, rates of return, and other factors Carbohydrate-Derived Fuel Products. 2014 http://www.nrel.gov/ docs/fy14osti/62368.pdf are used in all design cases so the various 20 International Energy Agency Bioenergy Task 39. State of 25 Laurens LML, Nagle NJ, Davis R, Sweeney N, Van Wychen S, Low- conversion pathways may be assessed on a Technology Review – Algae Bioenergy. 2017 http://www. ell A et al. Acid-catalyzed algal biomass pretreatment for integrated comparative basis. ieabioenergy.com/wp-content/uploads/2017/01/IEA-Bioenergy- lipid and carbohydrate-based biofuels production. Green Chem Algae-report-update-20170114.pdf 2015. doi:10.1039/c4gc01612b

21 Davis R, Fishman D, Frank ED, Wigmosta MS. Renewable Diesel 26 Jones S, Zhu Y, Anderson D, Hallen RT, Elliott DC. Process Design To highlight examples of pathways for from Algal Lipids: An Integrated Baseline for Cost, Emissions, and and Economics for the Conversion of Algal Biomass to Hydrocar- production of fuels from algae, the following Resource Potential from a Harmonized Model. Golden, CO, 2012 bons : Whole Algae Hydrothermal Liquefaction and Upgrading. 22 Lohman EJ, Gardner RD, Halverson L, Macur RE, Peyton BM, Richland, WA, 2014 Gerlach R. An efficient and scalable extraction and quantification 27 Approval for Algenol Fuel Pathway Determination under the method for algal derived biofuel. J Microbiol Methods 2013; 94: RFS program. 235–244. 19 IEA Bioenergy. State of Technology Review - Algae Bioenergy 28 Luo D, Hu Z, Choi DG, Thomas VM, Realff MJ, Chance RR. Life (ISBN: 978-1-910154-30-4). 2017 http://www.ieabioenergy.com/ 23 Halim R, Danquah MK, Webley P a. Extraction of oil from cycle energy and greenhouse gas emissions for an ethanol produc- wp-content/uploads/2017/01/IEA-Bioenergy-Algae-report- microalgae for biodiesel production: A review. Biotechnol Adv tion process based on blue-green algae. Environ Sci Technol 2010; update-20170114.pdf 2012; 30: 709–32. 44: 8670–8677.

Algae Biomass Organization | 16 target to be achieved by 2022, based on Metric Unit Notes 24 processing high-lipid biomass. The study 1. Cultivation: Continuous data - weather also identified a number of technology gaps Precipitation cm/day Precipitation data (as available from weather events) and uncertainties that would require further research and development in order for the Air temperature °C Minimum hourly basis pathway to achieve fuel costs at a minimum Dew point temperature °C Hourly basis of $3/gallon of gasoline equivalent. The W/m2 Solar radiation/insolation/photosynthetically or Hourly basis overall feedstock cost was identified as active radiation (PAR) one major determinant (at the time of µmol/m2 sec publication of that report the projected Wind speed m/s Hourly basis feedstock cost target was assumed to be Air pressure mm Hg Hourly basis $430/ton). 2. Cultivation: Continuous data - culture

Water salinity mg/L The whole algal biomass hydrothermal Water pH pH liquefaction (HTL) conversion pathway Water temperature °C A similar assessment was carried out Dissolved oxygen mg/L to perform a TEA for a whole biomass Oxidation reductive potential mV HTL process. The whole algal biomass thermochemical conversion pathway has 3. Cultivation: Installation/logistics been summarized in a Pacific Northwest Land use/cost Upon installation 29 National Laboratory report. In this process, Scale of production (pond/cultivation size) Ha whole algal biomass at a dewatered paste Days of operation Steady state/dynamic/culture crash ratio concentration of 20% is directly transferred 4. Cultivation: Discrete data - culture into a hydrothermal liquefaction (HTL) process reactor to generate a bio-oil Pond depth cm Daily basis destined for catalytic upgrading to RDB or Make-up water (evaporation) L Volume of make-up water added to the pond (if applicable) other fuel products. Thus, the attractiveness Make-up water (after harvest) L Volume of water added back after harvest (if applicable) of the pathway lies in the processing of Nutrients - nitrogen mg N/L Daily basis, measured as ppm N whole biomass with high biocrude yields. Nutrients - phosphorus mg P/L Daily basis, measured as ppm P

The HTL process operates at high-pressure CO2 source (flue gas/purified CO2) Wt % and temperature (typically 14-20 kPa and Water supply Fresh/saline/brackish water, stating source 300-350°C) to produce crude bio-oil along Biomass concentration (ash free dry weight) g/L Measured according to standard procedure of total suspended solids with a water-soluble organic phase. The count bio-oil is then catalytically hydrotreated Contamination count (type)/mL to end-product fuels. The water-soluble 5. Cultivation/productivity and other calculated metrics organic phase is treated through a catalytic hydrothermal gasification (CHG) process Total productivity (ash free dry weight) g/L !"#$! !"#$% ! − !"#$! !"!#!$% (!) that produces methane fuel; water and represents total biomass produced during an experiment or batch nutrients in the aqueous effluent are !"#$ !"#$%& ! Average (and peak and low) biomass areal recycled to the algae ponds. The projected g/(m2 x day) productivity !"#$!"!#$ (!) ! target (2022) case stipulates 77% recovery !"#$ !"#! ! × !"!#$ !"#$ of the algal carbon into the bio-oil fraction 2 g/(m x day) for final conversion to fuels, 13% carbon Daily Biomass areal or volumetric productivity or !"#$! ! ! ! − !"#$! (!) where n = number! of days between measurements, allowing for n > 1, typical off-gas for hydrogen production, and 9% as g/(L x day) ! × ! sampling plans are AFDW every other day and calculated on a m2 or L basis dissolved CO2 in the water fraction for algal

production. TEA of the process targeted Average biomass volumetric productivity g/(L x day) !"#$!"!#$ (!) for 2022 goals (extrapolated from current experimental data) shows that an overall !"#$ !"#$%& ! × !"!#$ !"#$

cost of $4.49/gge is possible, if the algal Nitrogen depletion rate mg/(L x day) !"#$%&!#' !! !" − !"#$%&!#' !!!! (!") 25 where n = number of days between measurements and nutrient N > 0 feedstock is obtained at $430/ton. ! × !

Phosphorus depletion rate mg/(L x day) Direct-to-ethanol pathway !"#$%&!#' !! !" − !"#$%&!#' !!!! (!") where n = number of days between measurements and nutrient P > 0 A third example pathway involves the ! × ! production of ethanol using Cyanobacteria. 6. Cultivation/strain specific parameters for productivity Ethanol can be collected from closed Light absorption coefficient Needed for physics-based modeling of strain productivity photobioreactors, where it is produced via theLight bioreacter extinction coefficient requires a large amount of Neededaround for physics the-based ethanol modeling separation of strain productivity process, the intracellular photosynthesis. The purification energy.7. Cultivation/other Unlike in LCA/TEA other metrics biofuel pathways, net life cycle energy consumption, excluding of fuel grade ethanol from the dilute there is little waste biomass available to photosynthesis, ranges from 0.55 MJ/MJ ethanol-in-water solution collected from Water evaporation rate cm/day provide process heat and electricity to offset !"#$EtOH !"#$ℎ !down ! ! (!" )to− 0.20 MJ/!"#$ !"#$ℎ !MJ (!" EtOH,) and the where n = number of days between measurements those energy requirements. In a scenario net GHG emissions! range from 29.8 g CO2e/ % downtime due to unplanned events, crashes, contamination, emergency basedReactor downtimeon a natural-gas-fueled (unplanned) combined% of month MJ EtOH down to 12.3 g CO2e/MJ EtOH maintenance 29 Batan L, Quinn J, Willson B, Bradley T. Net energy and heat and power system to provide process for initial ethanol concentrations from 0.5 kWh/day/m3 greenhouse gas emission evaluation of biodiesel derived from energyReactor mixing and energyconservative assumptions to 5 wt %. EPA recently certified Algenol’s microalgae. Environ Sci Technol 2010; 44: 7975–7980. volume 8. Cultivation: Biomass component analysis Algae Biomass Organization | 17 Moisture/Ash % DW Based on harvested, centrifuged representative material Total lipids % DW Based on harvested, centrifuged representative material Total protein % DW Based on harvested, centrifuged representative material

Total carbohydrates % DW Based on harvested, centrifuged representative material C:N:P molar ratio Based on harvested, centrifuged representative material

Biomass elemental composition (C, H, N, S, O, P) Wt % Based on harvested, centrifuged representative material

9. Harvesting and conversion Dewatered algal biomass concentration g/L Harvesting efficiency % Specify at each stage of harvesting process

As As much detailed information on conversion process, heat supply and Processing applicable efficiency of conversion or extraction as possible

As As much detailed information on processing of residual biomass as possible, Spent biomass usage applicable including recycling nutrient and energy credits

Metric Unit Notes 1. Cultivation: Continuous data - weather

Precipitation cm/day Precipitation data (as available from weather events) Air temperature °C Minimum hourly basis

Dew point temperature °C Hourly basis

W/m2 Solar radiation/insolation/photosynthetically or Hourly basis active radiation (PAR) µmol/m2 sec

Wind speed m/s Hourly basis

Air pressure mm Hg Hourly basis 2. Cultivation: Continuous data - culture

Water salinity mg/L

Water pH pH

Water temperature °C

Dissolved oxygen mg/L

Oxidation reductive potential mV

3. Cultivation: Installation/logistics

Land use/cost Upon installation

Scale of production (pond/cultivation size) Ha

Days of operation Steady state/dynamic/culture crash ratio

4. Cultivation: Discrete data - culture Pond depth cm Daily basis

Make-up water (evaporation) L Volume of make-up water added to the pond (if applicable)

Make-up water (after harvest) L Volume of water added back after harvest (if applicable) Nutrients - nitrogen mg N/L Daily basis, measured as ppm N Nutrients - phosphorus mg P/L Daily basis, measured as ppm P

CO2 source (flue gas/purified CO2) Wt % Water supply Fresh/saline/brackish water, stating source Biomass concentration (ash free dry weight) g/L Measured according to standard procedure of total suspended solids

count Contamination count (type)/mL 5. Cultivation/productivity and other calculated metrics

Total productivity (ash free dry weight) g/L !"#$! !"#$% ! − !"#$! !"!#!$% (!) represents total biomass produced during an experiment or batch !"#$ !"#$%& ! Average (and peak and low) biomass areal g/(m2 x day) productivity !"#$!"!#$ (!) ! !"#$ !"#! ! × !"!#$ !"#$

g/(m2 x day) Daily Biomass areal or volumetric productivity or !"#$! ! ! ! − !"#$! (!) where n = number! of days between measurements, allowing for n > 1, typical g/(L x day) ! × ! sampling plans are AFDW every other day and calculated on a m2 or L basis

Average biomass volumetric productivity g/(L x day) !"#$!"!#$ (!)

!"#$ !"#$%& ! × !"!#$ !"#$

Nitrogen depletion rate mg/(L x day) !"#$%&!#' !! !" − !"#$%&!#' !!!! (!") where n = number of days between measurements and nutrient N > 0 ! × !

Phosphorus depletion rate mg/(L x day) !"#$%&!#' !! !" − !"#$%&!#' !!!! (!") where n = number of days between measurements and nutrient P > 0 ! × ! 6. Cultivation/strain specific parameters for productivity that could offer performance targets for

Light absorption coefficient Needed for physics-based modeling of strain productivity an integrated design case. This ensures that consistent inputs for each type of Light extinction coefficient Needed for physics-based modeling of strain productivity model result in an integrated methodology 7. Cultivation/other LCA/TEA metrics to develop cost, emission, and resource potential baselines. Water evaporation rate cm/day !"#$ !"#$ℎ! ! ! (!") − !"#$ !"#$ℎ! (!") where n = number of days between measurements ! % downtime due to unplanned events, crashes, contamination, emergency Alternative methodologies exist that allow Reactor downtime (unplanned) % of month maintenance for a direct comparison between processes,

kWh/day/m3 e.g., the calculation of Energy Return on Reactor mixing energy 28-30 volume Investment (EROI). The EROI provides 8. Cultivation: Biomass component analysis a direct comparison not only between the

Moisture/Ash % DW Based on harvested, centrifuged representative material energy inputs and outputs of each case, but also among other energy production Total lipids % DW Based on harvested, centrifuged representative material technologies. In one example that was Total protein % DW Based on harvested, centrifuged representative material recently published,28 the EROI is calculated Total carbohydrates % DW Based on harvested, centrifuged representative material as the ratio of (total) energy outputs (Eout) C:N:P molar ratio Based on harvested, centrifuged representative material to (total) energy inputs (E ), where E is in BC Biomass elemental composition (C, H, N, S, O, P) Wt % Based on harvested, centrifuged representative material the energy output from biocrude material

9. Harvesting and conversion (53.0 MJ/kg), EEtOH is the energy output Dewatered algal biomass concentration g/L from ethanol (41.9 MJ/kg), EAF is the energy Harvesting efficiency % Specify at each stage of harvesting process credit from animal feed (25.1 MJ/kg), EE is the energy input from electricity (3.8 MJ/ As As much detailed information on conversion process, heat supply and Processing applicable efficiency of conversion or extraction as possible MJ for the Texas grid, 3.9 MJ/MJ for the Hawaii grid, and 1.13 MJ/MJ for wind power), As As much detailed information on processing of residual biomass as possible, Spent biomass usage applicable including recycling nutrient and energy credits EH is the energy input from heat (1.2 MJ/ MJ), and ∑EMATL is the sum of all embedded energy inputs from operating materials, typically derived from established inventory Direct-to-ethanol fuel as an advanced to provide an example of how crucial databases.30 When onsite heat or electricity biofuel with a 69% reduction in life cycle measurements and their standardization are (produced from combined heat and power, GHG emission compared to gasoline.30 in serving as inputs for modeling. In 2011, CHP) is used, the associated amount is Energy consumption and GHG emissions it was concluded that without consistent subtracted from the inputs. If non-renewable can be further reduced via employment of approaches towards input metrics, energy impacts are used rather than the higher efficiency heat exchangers in ethanol independent TEA, LCA, and RA models total energy impacts, the EROI results purification and/or by use of solar thermal would not provide systematic comparisons change significantly, especially for the cases energy for some of the process heat. The life amongst different biomass feedstock with wind power and large oil yields. cycle energy and GHG emissions for three and fuel production systems.21 Argonne different system scenarios for this Direct-to- National Laboratory (ANL), the National Carbon Capture and Utilization ethanol pathway, using process simulations Renewable Energy Laboratory (NREL), and Algae consume carbon, especially in the and thermodynamic calculations, were the Pacific Northwest National Laboratory form of CO2. Every ton of algae can consume recently published.31 (PNNL) convened a workshop to assist with up to 1.8 tons of CO2 (though this heavily harmonizing the analyses of algal-oil-based depends on the strain and cultivation 21 Harmonization of inputs and fuel pathways. This effort was supported conditions), which means algae utilize crucial measurements by the Department of Energy, and brought carbon more efficiently than any other In order to compare the inputs and outputs together leaders in the field who agreed organism. As algae assimilate the waste of a process or even compare the economic on a common set of assumptions to aid carbon, the process gives off oxygen, or life cycle impact, there is a need to with the modeling of process benchmarks, creating a clean technology, referred to harmonize the inputs to computational cost and sustainability quantification as as carbon capture and utilization (CCU). models. Metrics and crucial measurements well as metrics and boundary conditions. Over 150 companies are working to have been proposed to characterize the This harmonization effort can be used as an commercialize cleantech advances that example and could aid with future voluntary inputs into a cultivation system (Table 2.2) convert concentrated sources of CO2 to and a more detailed overview of reactor industry consensus harmonization, through renewable fuels, food, feed, fertilizer, green and cultivation performance comparative support from the ABO and future directions chemicals, and plastics. Other companies metrics is given in Chapter 7. The purpose of the Technical Standards Committee. The are creating high-value products such of the data and metrics listed here is workshop team established a common understanding of TEA, LCA, and RA modeling systems, as separate disciplines that (1) offer perspectives to improve specific parameters, 32 Frischknecht R, Jungbluth N, Althaus H-J, Doka G, Dones R, Heck 30 Beal CM, Gerber LN, Sills DL, Huntley ME, Machesky SC, Walsh T et al. The ecoinvent Database: Overview and Methodological MJ et al. Algal biofuel production for fuels and feed in a 100-ha process assumptions, and systems Framework. Int J Life Cycle Assess 2004; 10: 3–9. facility: A comprehensive techno-economic analysis and life cycle integration; (2) identify areas in need of 33 USEPA. Clean Power Plan for Existing Power Plants. assessment. Algal Res 2015; 10: 266–279. harmonization; and (3) propose process 34 Executive Order 13783: Promoting Energy Independence 31 Beal CM, Smith CH, Webber ME, Ruoff RS, Hebner RE. A and Economic Growth. https://www.whitehouse.gov/the-press- Framework to Report the Production of Renewable Diesel from improvements and emerging technologies office/2017/03/28/presidential-executive-order-promoting-energy- Algae. BioEnergy Res 2010; 4: 36–60. independence-and-economi-1

Algae Biomass Organization | 18 as Omega-3 nutraceuticals, powerful carbon emissions can be found in in from CCU. However, several studies show antioxidants, cosmetics, pharmaceuticals, California. The Global Waming Solutions that algae offer promising pathways for CO2 and medicines. Act of 2006, Assembly Bill AB32, prompted reduction or sequestration.27,36 Many algal the creation of the Low Carbon Fuel CCU platforms under development will The EPA’s Clean Power Plan implementation Standard (LCFS) in 2009. The LCFS mandates permanently sequester captured carbon in to reduce CO2 emissions from existing power petroleum refiners to reduce the carbon- enduring products such as plastics or other plants, positions algae to play a major role intensity (CI) of their fuels by 10% by 2020.35 industrial chemicals. The production of algal 34 in curbing CO2 and other GHG emissions. Under this program, each organization’s CI biofuels does not sequester the harvested

The Clean Power Plan sets federal guidelines must meet the target for the year. A fuel CO2 when the biofuel is burned. However, for states to follow in order to cut carbon that has a CI below the target is rewarded the algal biofuel produced displaces emissions by 32% before 2030. As a result CI credits, which represent a metric ton of petroleum-derived fuel, avoiding the CO2 of successful advocacy by the ABO, the CO2 avoided. If the target is not met, the emissions associated with extraction, CPP specifically endorses CCU and names institution may no longer be in compliance. refining, and combustion of the displaced algae as a qualifying clean technology. The An intuition can remain in compliance by petroleum. CPP specifies that “state plans may allow purchasing CI credits from another business affected Electricity Generating Units (EGUs) under compliance.35 The price of the credits In the CPP, EPA establishes a protocol for to use qualifying CCU (carbon capture and is highly volatile, however have remained obligated parties to certify CO2 reductions utilization) technologies to reduce CO2 around $90 per credit during the first half of from CCU projects, and commits to work emissions that are subject to an emission 2017. collaboratively with stakeholders to develop standard, or those that are counted when appropriate monitoring, reporting and demonstrating achievement of the CO2 Algae producers offer a method to reduce accounting protocols for CCU platforms. emission performance rates or a state rate- emissions from electricity production and The consideration of how emerging based or mass-based CO2 emission.” The fuel production. Carbon utilization will algae alternatives could be used to meet

CPP gives new opportunities to companies reduce the cost of emission reduction for CO2 emission goals requires a better commercializing algae-based technologies utilities and create new revenue streams. understanding of the ultimate fate of the that convert CO2 generated at power plants Algal CCU technologies will accelerate captured CO2 and the degree to which the into valuable bioproducts. Several peer- the development of job-creating clean method permanently isolates the captured reviewed LCAs of algal production systems technologies and support the CPP and LCFS. CO2 or displaces other CO2 emissions show that utilization of carbon by algae from the atmosphere. The ABO Technical reduces CO2 emissions to the atmosphere CCU accounting standards Standards Committee aims to reach a target substantially.14,23,27 The CPP acknowledges for Currently no universally accepted audience that will help develop a common the first time the value of carbon utilization. monitoring and reporting mechanisms approach for the set up of metrics and tools Currently, the CPP implementation is under have been adopted for the quantification to assess carbon utilization using algal review by the EPA, and parts of it may be and verification of CO2 emission reductions technologies in future developments. rescinded or revised based on findings in the future. 35 Promotum. California’s Low Carbon Fuel Standard: Evaluation of the Potential to Meet and Exceed the Standards. Cambridge, MA, 36 Air Resources Board. Weekly LCFS Credit Transfer Another example of legislation to reduce 2015 http://www.ucsusa.org/sites/default/files/attach/2015/02/ Activity Reports. https://www.arb.ca.gov/fuels/lcfs/credit/ California-LCFS-Study.pdf lrtweeklycreditreports.htm

Algae Biomass Organization | 19 Chapter 3: Regulations and Policy on Algal Production Operations

Sustainable algal production is governed deployment of genetically engineered programs for technical and financial by an entanglement of regulations focused organisms in the algal industry.1,2 assistance. Some of the water quality on measures of conservation and air, water, parameters that are monitored as part of and soil quality. Algae producers need to Water quality and the NPDES permitting process are shown in comply with the requirements of the nation discharge regulation Table 3.1. or jurisdiction in which their facilities are The Clean Water Act authorizes the National sited. In the US, this requires understanding Pollutant Discharge Elimination System Air quality and gaseous emission the intricacies of conservation and quality (NPDES) permit program that controls water To protect public health and welfare metrics defined by the USDA and EPA. pollution by regulating point sources that nationwide, the Clean Air Act requires EPA discharge pollutants into waters. Point to establish national ambient air quality In the US, environmental laws and sources are discrete conveyances such as standards for certain common pollutants regulations to which algal production pipes or man-made ditches. Industrial, based on the latest scientific findings. EPA operations are subject typically regulate municipal, and other facilities must obtain has set air quality standards for six common (1) water pollution or discharges to water, permits if their discharges go directly to criteria pollutants: particulate matter, ozone, (2) gaseous emission or air pollutants, surface waters. sulfur dioxide, nitrogen dioxide, carbon (3) the handling and disposal of solid monoxide, and lead. State enforceable and hazardous waste, (4) facility siting The National Pollution Discharge Elimination plans must control emissions and air quality and permitting, and (5) handling of toxic System (NPDES)3 requires point sources standards that drift across state lines and substances. Some state and local regulatory (PS) to comply with technology-based harm air quality in downwind states. Other authorities have requirements that relate effluent limits. Concentrated Animal Feeding key provisions are designed to minimize to the production, importation, and Operations (CAFOs) that discharge directly pollution increases from new or expanded genetic engineering of algae and other to surface waters are treated as point industrial plants such as algal production microorganisms, including their processing sources and must obtain NPDES permits. sites. The law calls for new stationary sources for R&D and commercial activities, and their Algal production is governed under NPDES (e.g., power plants and factories) to use release to the environment. Algae producers and requires a federal discharge permit. the best available technology. Relevant air use a broad array of process designs. Non-Point Source (NPS) water pollution quality metrics for algal producers are listed The reagents used (e.g., microorganisms, reaches receiving waters through diffuse in Table 3.2. enzymes, chemicals), determine the and complex pathways. The Clean Water Act quantity and nature of the waste produced. allocates authority for PS and NPS control Soil quality and biodiversity Various biological processes amplify to both federal and state authorities. With The avoidance of soil pollution follows the natural microbial populations (including some exceptions, the states have generally avoidance of water and air pollution that metabolically or genetically engineered opted for voluntary compliance strategies needs to form the basis of algae operations. varieties), algal toxins (potentially inducing for agricultural NPS control, supported dermatitis, neurological disruption, and to a varying degree by state and federal hepatotoxicity), as well as enzymes that may 4 Rice EW, Baird RB, Eaton AD, Ciesceri LS. Standard Methods for be potentially hazardous to the environment the Examination of Water and Wastewater. In: American Public and individuals. Each process may contain Health Association. American Public Health Association, 2012, p 1 Henley WJ, Litaker RW, Novoveská L, Duke CS, Quemada HD, 724 constituents that are potentially pathogenic, Sayre RT. Initial risk assessment of genetically modified (GM) 5 USEPA. Determination of trace elements in waters and wastes microalgae for commodity-scale biofuel cultivation. Algal Res 2013; toxic, infectious, or allergenic and that are by inductively couples plasma-mass spectrometry. EPA Method 2: 66–77. of concern for affecting native microbial 200.8 Revis. 5.4. 1994. http://water.epa.gov/scitech/methods/cwa/ 2 Glass DJ. Pathways to Obtain Regulatory Approvals for the Use bioindicators/upload/2007_07_10_methods_method_200_8.pdf populations and, consequently, ecosystem of Genetically Modified Algae in Biofuel or Biobased Chemical (accessed Aug 2015). Production. Ind Biotechnol 2015; 11: 71–83. balance. The USDA and EPA have created 6 Pate R, Klise G, Wu B. Resource demand implications for guidelines to protect the environment and 3 NPDES. State Program Status. http://water.epa.gov/polwaste/np- US algae biofuels production scale-up. Appl Energy 2011; 88: the public from harmful environmental des/basics/NPDES-State-Program-Status.cfm (accessed Aug 2015). 3377–3388. pollution. Algal producers need to monitor, and in some cases report, metrics for Parameter Unit Notes potentially harmful pollutants that enter Water waters, air, or soil. Initial risk assessments Feedstock production in an open consumption for m3/ha/day are underway to accommodate the future raceway cultivation Water Feedstock production in a closed or consumption for m3/day semi-closed PBR biorefinery Quality: Nitrogen, Concentration: mg/L Nutrient utilization for cultivation Phosphorus Export and loss: kg/ha/yr subtracting recycling credits

Salinity µSiemens/m Conductivity 6 Herbicides, metals, toxins, agricultural Chemicals Concentration, mg/L chemicals, flocculants Pathogen density Cells or particles/L For desired species or indicator species

Algae Biomass Organization | 20 Parameter Unit Notes by governmental agencies. Aflatoxins, microcystins, and β-Methylamino-L-alanine Tropospheric ozone ppb Combination of sources and methods (BMAA) are common toxins associated 9 necessary. EPA Multiscale Air Quality model with cyanobacterial growth. In particular, Greenhouse gases ppb CO2 equivalent emissions (CO2 and N2O) microcystins and BMAA are cyanobacteria

Carbon monoxide ppb CO2 equivalent emissions toxins. Table 3.4 presents the summarized Particulates g/m3 > 10 mm diameter; < 2.5 mm diameter hazards associated with the toxins, regulation limiting them, and detections Volatile organic compounds g/m3 Concentration methods.

Aflatoxins are a group of fungal toxins that Parameter Unit Notes are commonly found in maize and other Total organic carbon (N) mg/ha If digested algae are mixed with soil. crops during production, harvest, storage or processing. Inadequate control of moisture Extractable phosphorus mg/ha If digested algae are mixed with soil. and hygienic processes during production, (P) harvest, storage and processing often results Abundance of released cells/L Initially calculated from known biomass in culture in contamination by these fungi. Crops algae and estimated release rate or estimated using in commerce are routinely screened for genetic markers. these toxins following standard methods. Food supplies that test over the regulatory limit are considered unsafe for human consumption and destroyed. The FDA Defect Most algae producers do not have additional Safer Chemical Ingredient List.10 Action Level (DAL) for aflatoxins is 20 ppb. requirements for soil quality reporting. Algae In general, there are no specific methods producers that are generating products used Siting, permitting, strain for aflatoxins in algae but there are several as biofertilizers or growth stimulants need deployment AOAC method for different types of foods to monitor and report soil carbon, nitrogen, Siting requirements and operating permits particularly nuts and corn. These methods phosphorus, and possibly the abundance are controlled by local jurisdictions. Water are AOAC 975.36, 991.31 and 998.03 among of algal cells in the soil (Table 3.3). Similarly, access and use in most jurisdictions are others. to track the release of chemicals and toxins tightly controlled and monitored. Algal from algal cultures in the environment, the operations are highly dependent on water. The main algal toxins of concern among chemical distribution in the soils has to Therefore, permits and water monitoring are consumers of algae are the microcystins be monitored. To improve chemical safety critical. The typical steps in the siting and (primarily cyanobacteria-derived). These and provide more streamlined access to permitting process are shown in Figure 3.2. are hepatotoxins and are implicated in information on chemicals, EPA has built and liver cancer as well. The WHO standard continues to populate a new database. This Algae Toxins for drinking water is 1µg/L. The Oregon new database, named ChemView, greatly Department of Health has set a limit of 1 Preventing the presence of toxins in improves access to health and safety data µg/g for Aphanizomenon products from an algae products is beneficial for on chemicals regulated under the Toxic Klamath Lake. This is also currently the consumer health and often required Substances Control Act (TSCA). It contains industry standard for other algae and information EPA receives and develops algal biomass. The most common method about chemicals including those on EPA’s used is a competitive enzyme-linked 10 USEPA. Safer choice. 2015. http://www2.epa.gov/ immunosorbent assay (ELISA) method, saferchoice#about (accessed Aug 2015). which is a plate-based assay technique 7 Gaffney JS, Marley NA. The impacts of combustion emissions on 11 Bremner J, Mulvaney C. Nitrogen-total. In: Page AL, Miller air quality and climate – From coal to biofuels and beyond. Atmos RH, Keeney DR (eds). Methods of Soil Analysis, Part 2, Chemical designed for detecting and quantifying e.g. Environ 2009; 43: 23–36. and Microbiological Properties. Madison, WI: American Society of proteins, peptides, antibodies. Agronomy, 1982, pp 595–624 8 USEPA. Methods TO14A and TO15. Compendium of Methods for the Determination of Toxic Compounds in Air Second Edition. 12 Doran JW, Jones AJ. Methods for assessing soil quality. Soil A third toxin is β-Methylamino-L-alanine 1999 Science Society of America Inc., 1996 or BMAA, which is also a cyanobacterial 9 Appel KW, Gilliland AB, Sarwar G, Gilliam RC. Evaluation of the 13 Nelson WL, Mehlich A, Winters E. The development, evaluation, Community Multiscale Air Quality (CMAQ) model version 4.5: and use of soil tests for phosphorus availability. In: Pierre WH, neurotoxin has attracted attention recently Sensitivities impacting model performance. Atmos Environ 2007; Norman AG (eds). Soils and Fertilizer Phosphorus in Crop Nutrition. because it is implicated as a cause for 41: 9603–9615. Academic Press, New York, 1953, pp 153–188

p Figure 3.2: Overview of the main steps of a permitting process for an algal farm or production operation

Algae Biomass Organization | 21 Toxin Hazard Regulation Detection Title V facilities.

Aflatoxins Fungal toxins believed FDA defect action level: AOAC 975.36, 991.31, Major Source – Larger emitting facilities to be linked to cancer 20 ppb 998.03 with complex permitting requirements Mycrocystins Cyanobacterial toxin WHO: 1µg/L drinking ELISA method (e.g., medium to large operations, linked to liver cancer water utilities, refineries, or forging operations) Oregon Department of need to adhere to different regulations. Health: 1 µg/g algae Voluntary restrictions on the quantity of BMAA Cyanobacterial None reference 26 air contaminants can be placed on some toxin linked to operations at these types of sources in neurodegenerative order to avoid certain rule requirements, diseases (amyotrophic i.e., synthetic minor restrictions. If the lateral sclerosis, facility potentially emits substances that Parkinson’s) trigger at least one major source permitting requirement and/or Title V threshold, the facility is referred to as a “Major Source” or “Title V facility.” Algal Strains & Farm Cultivation & Production Algae-based Products Inputs/Outputs Processes The type of permit issued to one of these sources depends on when a given operation at the facility was installed or modified. R&D Specific Regulation Commercial Specific Product Specific Generally speaking, new installations and Regulation Regulation • NIH - rDNA guidelines modifications to operations at these major • HHS screening; DS rDNA • EPA TSCA - MCAN • Chemicals - EPA - TSCA sources are required to apply for and be issued • EPA TSCA - TERA, R&D • EPA FIFRA - pest management PMN a permit-to-install. Then, they must apply for a contained-use exemption • FDA for FDA-regulated • Fuel - EPA /DOD/DOT/FAA Title V permit-to-operate or a permit revision • USDA - genetically engineered products fuel certification if an effective Title V permit-to-operate has organism importation, • TTB - for ethanol production • Fuel - EPA - RFS already been issued for the facility. interstate movement, and • NEPA, ESA with federal compliance open-system research with funding • Ethanol - TTB known plant pests Algal strain selection R&D and Commercial • Food - FDA CFSAN R&D and Commercial • Feed - FDA CVM / AAFCO Algal strain selection is essential in • USDA - plant pests • Items listed as R&D order to identify and maintain suitable • State biotechnology and/ or • OSHA - general duty clause specific promising algal strains for cultivation and aquaculture regulations • State biotechnology and/or development. The isolation of new algal • Federal, state, and/or local aquaculture regulations permits (RCRA, OSHA, building strains from a wide variety of environments and fire codes, etc.) • Federal, state, or local permits will enable metabolic versatility. The • RCRA, OSHA, building and fire • EPA TSCA - TERA, R&D isolation of algae can be done from a codes etc., as applicable contained use exemption large variety of natural aqueous habitats including freshwater, brackish water, marine,

soil, and hypersaline environments.17,18 Additionally, large-scale sampling should amyotrophic lateral sclerosis, Parkinson’s biomass).14-16 be coordinated to ensure a broad coverage disease, dementia and potentially of environments. The specific location can other neurodegenerative diseases. The Minor Source – Smaller emitting facilities be determined by advanced site-selection cyanobacteria normally exist as symbionts have less complicated permitting criteria through the combined use of with other plants like cycads. Consumers requirements (e.g., small industrial dynamic maps, geographical information do show concern from time to time but it is operations or gas stations), generally system (GIS) data, and analysis tools for not in the mainstream as in the case of the referred to as “Non-Title V.” These permits are selection. In order to maximize the genetic microcystins. There is no regulatory limit set often voluntary restrictions and may specify diversity, the ecosystems to be sampled at this moment. There is no official method the quantity of air contaminants included in may include aquatic environments such as such but there is a single method that has issued permits that prevent the source from as oceans, lakes, rivers, streams, ponds, or been published in the AOAC journal.26 becoming subject to the Title V operating geothermal springs covering hyper-saline, permit program. A permit-to-install and fresh, brackish, acidic, and alkaline waters, Siting process operate (PTIO) is issued for these types of and terrestrial environments in a variety of Algae farms can be split into small and sources. Permits last for 10 years for Non- large facilities, which are mainly separate in their permitting requirements based on the type of biomass processing or the 14 Pienkos PT, Darzins A. The promise and challenges of microalgal-derived biofuels. Biofuels, Bioprod Biorefining 2009; 3: extraction scenarios anticipated to be 431–440. 17 Sieracki ME, Gobler CJ, Cucci TL, Thier EC, Gilg IC, Keller MD. used at the newly built facility (e.g., VOCs 15 Zuo Z, Zhu Y, Bai Y, Wang Y. Acetic acid-induced programmed Pico- and nanoplankton dynamics during bloom initiation of produced during extraction or drying of the cell death and release of volatile organic compounds in Aureococcus in a Long Island, NY bay. Harmful Algae 2004; 3: Chlamydomonas reinhardtii. Plant Physiol Biochem 2012; 51: 459–470. 175–84. 18 Elliott LG, Feehan C, Laurens LML, Pienkos PT, Darzins A, 16 NRC. Sustainable Development of Algal Biofuels. The National Posewitz MC. Establishment of a bioenergy-focused microalgal Academies Press: Washington D.C, 2012 culture collection. Algal Res 2012; 1: 102–113.

Algae Biomass Organization | 22 geographical locations.19,20 Moreover, algae Toxic Substances Control Act (TSCA) that regulations primarily cover only those are typically found in planktonic and benthic governs the use of new microorganisms modified organisms containing nucleic environments within an aqueous habitat. for certain industrial uses. Briefly, if a acids from plant pest species or genera, In suspended mass cultures, planktonic modified algal strain contains coding so that modified algae would only be algae may be used, whereas biofilm-based nucleic acids from more than one genus, it covered if they contained such sequences.2 production facilities may use benthic is considered a “new microorganism” under Use of engineered algae strains in other algae for attached growth and cultivation. these regulations. Although many R&D countries would, in most cases, be subject to Traditional cultivation techniques such uses of new microorganisms are exempt regulatory requirements similar to those in as enrichment cultures may be used for from EPA oversight, R&D in open ponds the US, under applicable national laws. the isolation of new strains from natural would require EPA’s advance review and habitats.21 Because of morphological approval of an application called a TSCA The regulatory distinction of monitoring, similarities when comparing many algal Environmental Release Application (TERA), treating, and ultimately controlling species, actual strain identification should and there has been at least one field trial biotechnology fall under the purview of the be based on molecular methods like rRNA of modified algae that has been conducted EPA’s NPDES and the respective permitting sequence comparison, or in the case of under an approved TERA. Commercial use process is unclear.24 Beyond nutrient load, closely related strains, other gene markers.22 of a new microorganism, whether in an genetically engineered organisms contained open pond or a contained reactor, would in NPDES discharges have the potential to Biotechnology require prior EPA review of a Microbial impact drinking water supplies as well as The use of genetically engineered algae Commercial Activity Notice (MCAN) the surrounding environment. The EPA list strains in the US for industrial purposes describing the strain and the proposed use, of US environmental laws can be found (other than food or feed production) and there are several examples of MCANs online.25 The primary federal regulations for might be subject to regulation by the successfully filed for commercial uses of protection of the environment are in the Title Environmental Protection Agency or the algae and cyanobacteria.2 It is possible 40 Code of Federal Regulations (CFR). State US Department of Agriculture. Uses of that USDA’s biotechnology regulations and local regulatory requirements must be genetically engineered algae for production might apply to modified algae. The USDA considered. A more detailed discussion of of fuels or chemicals may fall under regulates genetically engineered algae from wastewater pollutants and their potential regulations maintained by EPA under the the standpoint of preventing the spread implications in algae cultivation systems is of pests, weeds, and diseases under the discussed further in Chapter 4. Federal Plant Pest Act (FPPA).23 The USDA 19 Venteris ER, Skaggs RL, Coleman AM, Wigmosta MS. A GIS cost also regulates the spread of new varieties of model to assess the availability of freshwater, seawater, and saline feedstock whether they are developed by groundwater for algal biofuel production in the United States. 24 USEPA. NPDES. http://water.epa.gov/polwaste/npdes/ (accessed Environ Sci Technol 2013; 47: 4840–9. selection or hybridization, or are genetically Aug 2015). 20 Quinn JC, Catton K, Wagner N, Bradley TH. Current Large- engineered. However, USDA’s biotechnology 25 USEPA. www.epa.gov/lawsregs/laws (accessed Aug 2015). Scale US Biofuel Potential from Microalgae Cultivated in Photobioreactors. BioEnergy Res 2011; 5: 49–60. 26 Glover W, Baker TC, Murch SJ, Brown P (2015) Determination of β-N-methylamino-L-alanine, N-(2-aminoethyl) glycine, and 2, 21 Andersen R, Kawachi M. Traditional Microalgae Isolation 4-diaminobutyric acid in food products containing cyanobacteria Techniques. In: Algal culturing techniques. 2005 23 Plant Protection Act. https://www.aphis.usda.gov/plant_health/ by ultra-performance liquid chromatography and tandem mass 22 Fishman DB, Majundar R, Morello J, Pate R, Yang J. National Algal plant_pest_info/weeds/downloads/PPAText.pdf (accessed Aug spectrometry: single-laboratory validation. J AOAC Int 98(6):1559– Biofuel Technology Roadmap. Washington D.C, 2010 2015). 1565

Algae Biomass Organization | 23 Chapter 4: Use of Wastewater in Algal Cultivation

By integrating algal production and phosphorus.5,6 Nevertheless, the application Wastewater pollutants wastewater treatment (WWT), both of municipal wastewater for algal production The pollutants to be removed from processes might be accomplished with holds most promise to be economically wastewaters during treatment fall into improved economic and environmental feasible even in the short term. several major types: sustainability. This chapter covers the major issues to be considered in algal facility Some wastewaters contain inhibitors of Gross pollutants are mainly dissolved planning and the metrics to use in the algal growth, for example, high ammonia and particulate organic matter, typically evaluation of combined algal wastewater concentrations in animal waste and toxic characterized and regulated in two ways: and biofuels production projects. The two compounds in industrial wastewaters. (1) biochemical oxygen demand (BOD) main approaches to this integration are (1) Such wastes are often also highly turbid, determined over 5 days of incubation or WWT using algae and (2) consumption of reducing light availability to the algae. When as chemical oxygen demand (COD), and wastewater to produce algal biomass. In algal growth media is recycled, inhibitory (2) total suspended solids (TSS), based on the former, the algal production per unit organic compounds, including allelopathic dry weight particulates captured on an wastewater volume is low, and treated agents excreted by algae themselves, can analytical filter. Another measure, volatile wastewater is discharged or reused offsite. In accumulate in the media and potentially suspended solids (VSS), is equivalent to the latter, algal production per wastewater inhibit growth of competing algae.7 In the ash-free dry weight (AFDW) used in volume is maximized, and the wastewater cultivation systems that extensively recycle algal biomass analysis. The initial removal is consumed through evaporation and water, salts can build up to high enough of particulate organic matter by settling is blowdown during cultivation.1 concentrations to become inhibitory, but called “primary treatment,” while removal of for low salinity waters, such as municipal biodegradable organics is called “secondary Recycling of wastewater in algal wastewater, organic inhibitors are more treatment.” Further removal of organic biofuel feedstock production likely to be the limiting factor for water matter is termed “advanced” or “tertiary” recycling. In such cases, the concentration of treatment and usually involves filtration. The two main areas of intersection for algal inhibitory compounds can be controlled by cultivation for biofuels and wastewater are disposing of a portion of the water in each Pathogens (bacteria, viruses, protozoa, in (1) WWT with discharge or offsite reuse cycle, i.e., blowdown disposal. etc.) removal is an even more important of the treated effluent (the wastewater is goal in WWT, and in the US, it is generally only used once for algal production), and (2) accomplished by chemical (e.g., chlorination) use of treated or untreated wastewater as a Background on wastewater or UV light treatment following secondary culture medium that is recycled repeatedly treatment treatment and suspended solids removal. for production of algal biofuel feedstock. An understanding of wastewater Pathogens can also be removed to a major In the WWT application, the main products characteristics and the standard steps in extent through natural die-off in a series would be reclaimed water, algae-based treatment are essential for the evaluation of of ponds with long hydraulic residence fertilizer, and algal biofuels. However, algae wastewater schemes. times. This process can be accelerated by biofuels and fertilizers would not be major withholding CO supply to algal systems, economic drivers at current prices. Instead, Wastewater sources and types 2 thereby causing the pH to rise to levels WWT fees and reclaimed water sales would Many types of wastewaters can be treated deleterious to bacteria and viruses. provide most of the revenue. The dedicated with algal technologies and could be suited biofuels application has thus far only been for supporting algal biomass production. Nutrients, such as N and P, are required carried-out experimentally or at a small pre- Each wastewater type (e.g., municipal, to be removed to relatively low levels. pilot plant scale.2-4 agricultural, or industrial, and sub-categories within these) would require optimization Potassium (K) is generally not regulated. Nutrient removal is generally termed Algae have been grown on a wide variety of of algal technologies to fit their specific “tertiary treatment;” though this term is also wastewaters, most prominently municipal, requirements. Wastewater types with sometimes used to refer to filtration and but also agricultural (animal barn flush water large flows would be required to justify other advanced treatments. and field drainage) and industrial (food the expense of such development efforts. processing, aquaculture, etc.) wastewaters. At 60-100 gallons per person per day of Salts, measured as total dissolved solids For municipal wastewaters, the limiting domestic wastewater production, treatment or conductivity, degrade groundwater nutrients for algal growth are typically (in of municipal wastewaters is a large market, quality and can be detrimental in irrigation sequence of limitation) inorganic carbon, which is tied to the potential revenue stream reuse. The main salt present in municipal nitrogen, possibly some trace metals, and of wastewater fees. Similarly, industrial WWT might provide revenue for algae growers wastewater is sodium chloride. However, including wastewaters from food processing, algae are not known to accumulate sodium fossil fuel development, mining, etc. chloride and so are not useful for salt 1 Lundquist TJ, Woertz IC, Quinn NWT, Benemann JR. A Realistic removal. Technology and Engineering Assessment of Algae Biofuel Produc- tion. 2010 http://www.energybiosciencesinstitute.org/media/ AlgaeReportFINAL.pdf (accessed Aug 2015). Toxic metal ions (e.g., lead, chromium, 5 Fulton LM. Nutrient Removal by Algae Grown in CO₂-Enriched 2 Mehrabadi A, Craggs R, Farid MM. Wastewater treatment high Wastewater over a Range of Nitrogen-to-Phosphorus Ratios. copper, mercury, uranium) must often rate algal ponds (WWT HRAP) for low-cost biofuel production. 2009. http://digitalcommons.calpoly.edu/cgi/viewcontent. be removed from wastewaters. In the Bioresour Technol 2015; 184: 202–214. cgi?article=1206&context=theses (accessed Aug 2015). US, metal concentrations in municipal 3 Rogalla F, Banks CJ, Heaven S, Lara Corona ES. Algae Biofuel: 6 Woertz IC, Feffer A, Lundquist TJ, Nelson Y. Algae Grown on Dairy Symbiosis between Nutrient Removal and Water ReuseReuse - and Municipal Wastewater for Simultaneous Nutrient Removal and wastewater are generally low compared the EU FP7 All-Gas project. In: IWA Reuse conference proceedings. Lipid Production for Biofuel Feedstock. J Environ Eng 2009; 135: to the discharge limits and also lower 2011 1115–1122. than the concentrations toxic to algae 4 Lundquist TJ, Rodrigues M, Ripley E. Nutrient removal perfor- 7 Bacellar Mendes LB, Vermelho AB. Allelopathy as a potential mance of a new algal high rate pond pilot plant. In: WEFTEC confer- strategy to improve microalgae cultivation. Biotechnol Biofuels or bacteria. Conventional wastewater ence, Water Environment Federation. 2012 2013; 6: 152.

Algae Biomass Organization | 24 secondary treatment plants incidentally be recycled with the blowdown ratio can be divided into two major groups: (1) remove substantial fractions of many metals, controlling water quality in the cultivation electro-mechanical technologies such as which partition to the produced sludges. units. Blowdown or other discharges from activated sludge and trickling filters, which Industrial wastewaters are more challenging, such facilities using municipal wastewater use heterotrophic microbes, mostly bacteria; and sometimes require removal of not are likely to be regulated by wastewater and (2) photosynthetic technologies such as only toxic but even radioactive elements. authorities. However, when algal cultivation algal ponds, floating aquatic plant systems, Microalgae are known to accumulate such uses wastewater from aquaculture, and wetlands, which use algae or higher metals and radionuclides from very low agriculture, or mining and fossil fuel plants, in addition to bacteria. Only the concentrations.8,9 extraction industries, discharges may be major treatment technologies currently used regulated by sector-specific agencies. in each category—activated sludge and Trace organic compounds include algal ponds—are discussed here. components of pesticides, herbicides, Wastewater reuse and the treatment leading WWT plants of all types use a series of household chemicals, personal care up to it are regulated at the state or local standard treatment steps (unit operations). products such as lotions, plastics residuals, level. In California, for example, allowed First, large objects and stringy matter pharmaceuticals, etc. These are typically treatment prior to reuse can range from are removed from the wastewater in a removed only partially in conventional minimal (e.g., secondary treatment only preliminary treatment, followed by grit or and algal WWT through degradation or for pasture irrigation) to intensive (i.e., sand removal by sedimentation. During partitioning to sludge via adsorption.10 secondary treatment plus coagulation, primary treatment or clarification, the However, even low levels of some of these filtration, and disinfection for landscape organic matter is settled, yielding primary compounds can be of concern. For example, and golf course watering) treatment as sludge. Next, oxidation of organic matter endocrinedisrupting compounds (EDCs, regulated by the State Water Resources occurs during secondary treatment, as well hormones or hormone mimics present in Control Board.12 Thus, a regulatory issue will as conversion of soluble organic matter into urine and personal care products) affect be whether algal biofuel production using microbial cells, and biological flocculation aquatic wildlife even at extremely low municipal wastewater will be considered of colloidal matter. In order to achieve concentrations (nanograms per liter). a WWT activity (able to accept raw oxidation, it is necessary to increase the EDC concentrations are now being widely wastewater) or a reuse activity (restricted to dissolved oxygen content of wastewater monitored in municipal wastewater accepting treated wastewater). Regulations (e.g., through mechanical aeration or algal effluents, but so far, regulations targeting and permitting for algae operations are photosynthetic oxygenation). In a secondary EDC discharges are rare or absent in the US. discussed in more detail in Chapter 3. clarification, the bioflocculated microbial

solids formed during secondary treatment Regulations and permitting Wastewater treatment and are removed, usually by sedimentation. In the US, wastewater discharge permitting recycling technologies In bacterial technologies, the resulting authority stems from the EPA. The EPA WWT is accomplished through a series sludge is called secondary sludge or delegates specific permitting authority of generic steps or “unit operations,” for aeration solids. Disinfection of the clarified to the states, which each have their own example, sedimentation and oxidation. secondary effluent would complete basic water boards or environmental quality At a treatment facility, unit operations are treatment. As mentioned earlier, disinfection departments that analyze potential impacts combined to achieve the targeted levels of is commonly achieved with one of a variety from wastewater discharges and promulgate water purification and solids processing. of chlorine compounds or with UV light. policies, regulations, and permits to protect The unit operations used in conventional Ozone, bromine, and even pasteurization the environment, while maintaining the mechanical and algal treatment processes are also occasionally used for wastewater minimum national requirements determined have similar objectives, though they disinfection. The wastewater sludge is by the EPA. Enforcement of discharge differ in design. In algal systems, primary thickened to 2-6% solids content and then permits falls to the local water agencies. sedimentation is usually done in deep ponds anaerobically or aerobically digested to For secondary treatment, the EPA has set rather than tanks, and dissolved oxygen is covert some of the organic matter to CH national minimum standards for discharge 4 provided by algal photosynthesis instead and/or CO . Next the residual sludge is dosed of treated wastewater to waters of the US. 2 of the electrically-powered aeration used in with chemical flocculants and dewatered to The 30-day mean concentrations of BOD conventional “activated sludge” processes. In up to 20% solids content. At this point, the and total suspended solids (TSS) are both 30 algal systems, several unit operations often sludge is usually transported to agricultural mg/L. Details and exceptions are described take place in a single pond, though these are 11 by EPA’s NPDES. Nutrient limits are set by typically operated in series. In conventional state agencies, and the EPA does not have a treatment, unit operations are segregated national minimum discharge standard. in different reactors (e.g., primary settling tanks, aeration tanks, and secondary settling In algal biofuel production, water would tanks).

Conventional and algal wastewater 8 De la Noüe J, Laliberté G, Proulx D. Algae and waste water. J Appl Phycol 1992; 4: 247–254. treatment processes 9 Wong Y-S, Tam NFY (eds.). Wastewater Treatment with Algae. Conventional municipal WWT technologies Springer Berlin Heidelberg: Berlin, Heidelberg, 1998 10 Jasper JT, Sedlak DL. Phototransformation of wastewater- derived trace organic contaminants in open-water unit process treatment wetlands. Environ Sci Technol 2013; 47: 10781–90. 12 California Code of Regulations, Titles 17 and 22. Calif. Dep. Pub- 11 NPDES. Technology-Based Effluent Limitations. http://water.epa. lic Heal. http://www.waterboards.ca.gov/drinking_water/certlic/ gov/polwaste/npdes/basics/upload/pwm_chapt_05.pdf (accessed drinkingwater/documents/lawbook/RWregulations_20140618.pdf Aug 2015). (accessed Aug 2015).

Algae Biomass Organization | 25 fields for application as fertilizer, to landfills intense investigation. Biogas (anaerobic used in most commercial algal production for disposal, or to compost facilities for digestion) and liquid fuels (oil extraction, systems and in most projections of algal conversion to soil amendment. Treatment hydrothermal liquefaction) technologies biofuel production, was originally developed 14 plants with nutrient discharge limits will are advancing as well. Supplying CO2 could for WWT with algae. Algal turf scrubbers use additional unit operations, including improve biomass production and nutrient were originally designed for the removal + aerobic nitrification of ammonium (NH4 ) to removal, but must be demonstrated at scale. of nutrients from recirculating commercial - nitrate (NO3 ) and denitrification of nitrate With regard to N and P removal, ammonia aquaria and aquaculture systems. Both to nitrogen gas (N2). N and P can both be outgassing and nitrification/denitrification types of processes have been implemented assimilated by bacteria or algae, which are in ponds could aid in N removal, while P in a small number of actual WWT and subsequently removed by clarification. removal can also result from precipitation nutrient removal operations, respectively.15,16 Some wastewater bacteria are capable in the ponds, and might be increased with However, wider use of these technologies of enhanced phosphorus assimilation. selected algal cultures. has been lagging. The other major type Alternatively, phosphorus can also be of algal production reactor, the enclosed removed by precipitation. The original algal WWT technology is an photobioreactor (Chapter 7), has not unmixed pond or lagoon, generically called advanced beyond the research stage for The core process in the above is the a waste stabilization pond. Such engineered WWT. provision of dissolved O2 that allows the ponds have been used in the US for over natural microbial populations (mainly a century. They are built in earthwork Raceway ponds bacteria) to grow and convert the and harbor a changing variety of algae So-called ‘Oswald’-, raceway- or ‘high- biodegradable organics into biomass including green algae, cyanobacteria, and rate’ ponds achieve high algal biomass and CO2. The two basic processes used to diatoms, which provide photosynthetic productivity, and thus O2 production, provide O2 are mechanical aeration and oxygenation, often supplemented with organics destruction, nutrient removal and, algal photosynthesis. As already noted, minor mechanical surface aeration. Bacteria therefore, more rapid WWT than stabilization mechanical aeration requires electricity to and other microorganisms can be a major ponds. High rate ponds are typically 30-60 run the blower or aerators that transfer O2 component of the suspended biomass. cm deep, and are operated at a hydraulic from air into the wastewater, while in algal These pond system designs are not residence time of 3-6 days and channel 17 processes solar energy supports algal O2 standardized, but typically involve a series velocities of 15 cm/s. production. Further, while both bacteria of ponds, with an initial primary deep (> 2 and algae assimilate dissolved N and P into m) “anaerobic” or “facultative” pond, with Algae-based WWT has depended on cellular compounds, algal processes fix CO2 an aerobic algal culture on the surface. This native poly-cultures of algae. In raceways, allowing more nutrient assimilation but also type of pond is often followed by a series of the dominant taxa are often Chlorella, producing more biomass. several shallower (1 m) “oxidation” ponds. In Scenedesmus, Micractinium, Pediastrum, contrast, heavily aerated and mixed “aerated Actinastrum, etc. and sometimes diatoms. Algal technologies lagoons” are dominated by bacteria rather Some control of algal taxa has been Compared to conventional treatment than algae and are not included in this demonstrated outdoors by recycling processes, algal processes have several discussion.13 Algal productivity can be highly of settled biomass, leading to a culture pros and cons. Algal WWT requires much variable, from about -1 to +10 g AFDW/m2 dominated by large easily settled Pediastrum more land as it is, of course, a solar energy per day, resulting in intermittent discharges cells.18 process. Additionally, settling the algal from a few tenths to a hundred kilograms of biomass is currently not as reliable as that of AFDW of algal-bacterial biomass per hectare Recent advances in the use of raceways the bacterial biomass produced in activated per day. However, most stabilization pond for WWT are based on more consistent 19,20 sludge processes. While the large amount of systems do not employ algal harvesting. bioflocculation for harvesting, CO2 algal biomass has good potential for biogas, Instead, long hydraulic residence times allow biofuels, and recapturing of nutrients, it a majority of the algae to settle in the ponds, could also represent a disposal issue. In leaving a more or less clarified effluent. To some situations, CO2 supplementation meet discharge limitations on suspended is required to maximize productivity. N solids, some pond facilities use chemical 14 Oswald WJ, Gotaas HB. Photosynthesis in sewage treatment. removal by assimilation at ~10% N in coagulation to facilitate settling, often Transcr Am Soc Civ Eng 1957; 122: 73–105. the dry weight algal biomass requires including dissolved air flotation clarification. 15 Adey WH, Goertemiller T. Coral reef algal turfs: master producers in nutrient poor seas. Phycologia 1987; 26: 374–386. maximizing productivity and additional Almost all of these facilities dispose of the 16 Zivojnovich MJ. Nitrogen Areal Removal Rates for Algal Turf land over that needed for secondary resulting biomass sludge by returning it to Scrubber® Systems in Nonpoint Source Applications. 2006 http:// treatment. Additionally, P assimilation at the floor of the treatment ponds, where it hydromentia.com/Products-Services/Algal-Turf-Scrubber/Product- Documentation/Assets/2006_Zivojnovich-Nitrogen-Areal-Removal- ~1% of algal dry weight is much lower decomposes over the course of years. Rates-for-ATS.pdf (accessed Aug 2015). than the > 10% assimilation which can be 17 Oswald WJ. Large-scale algal culture systems (engineering achieved in advanced activated sludge aspects). In: Borowitzka MA, Borowitzka LJ (eds). Micro-algal Raceway ponds and algal turf scrubbers Biotechnology. Cambridge University Press, Cambridge, 1988, pp biomass. Nonetheless, algal processes are are the two algae-based technologies that 357–395 of lower cost than conventional treatment, have made inroads as alternative WWT 18 Park JBK, Craggs RJ, Shilton AN. Recycling algae to improve depending on land availability and cost. species control and harvest efficiency from a high rate algal pond. technologies, and both have potential Water Res 2011; 45: 6637–49. for large-scale production of algal biofuel 19 Gutzeit G, Lorch D, Weber A, Engels M, Neis U. Bioflocculent Additional R&D can help to overcome some feedstock. The raceway pond technology, algal-bacterial biomass improves low-cost wastewater treatment. of these limitations of microalgal processes. Water Sci Technol 2005; 52: 9–18. Currently, low cost harvesting through more 20 Lundquist TJ, Kraetsch M, Chang A, Hill E, Fresco M, Hutton R et al. Recycling of Nutrients and Water in Algal Biofuels Production. reliable settling of the algal biomass is under 13 Llorens M, Sáez J, Soler A. Primary productivity in a deep sew- 2015 http://www.energy.gov/sites/prod/files/2015/04/f21/algae_ age stabilization Lagoon. Water Res 1993; 27: 1779–1785. lundquist_132201.pdf (accessed Aug 2015).

Algae Biomass Organization | 26 addition for enhanced nutrient removal,4,21,22 HydroMentia) and vertical plastic media plant to irrigation sites. Urban land-use strain control,18 and a better understanding (Grower Harvester™, BioProcess Algae). planning should reserve land for sustainable of the hydraulic design of raceways.23,24 Another innovative approach using rope wastewater reclamation, which will also Raceway ponds are a generic technology, media has been tested at small pilot scale provide additional benefits such as open although various designs have been built. (Utah State University).28 space and aquatic wildlife habitat near Many full-service consulting engineering urban areas. firms should be capable of generating Harvesting raceway designs, but standard and custom Separating microscopic algal cells from Evaluation metrics for wastewater raceway and facility designs are also growth media, including wastewater, is a treatment and recycling provided by smaller, specialized engineering major cost challenge in algal biotechnology. Several criteria and metrics can be used in firms (e.g., RNEW® by MicroBio Engineering). In bacterial-based WWT, low-cost separation evaluating the feasibility of algal wastewater of bacterial cells (e.g., to < 30 mg/L projects for either treatment and/or biofuel Photobioreactors suspended solids concentration) has been production. Due to the waste-origin of such Photobioreactors (PBRs) have been accomplished for over a century by growing algae, fertilizer and biofuel are probably the considered for WWT.25 However, their bacteria in settleable flocs (activated sludge) only outlets for the biomass. The order in high cost, small module size, fouling, and or biofilms (trickling filters). For more which the criteria are discussed here reflects complexity have prevented the scale-up of complete suspended solids separation, their likely impact on project feasibility: PBRs for WWT (see Chapter 7 for a dedicated chemical coagulation and sedimentation discussion of PBRs). One interesting or filtration are used.29 Oxidation pond Footprint: Treatment facility footprint (land area) is usually the first decision factor for approach, which avoids the need to support systems have generally depended on slow project siting. If flat, low-cost land is not water-filled tubes or bags, is the floating sedimentation of individual algal cells available, then solar technologies such as PBR, first patented in 197626 with several or small algal-bacterial flocs. Oxidation algae are unlikely to be competitive with pond systems with low suspended solids companies recently promoting this idea. conventional mechanical technologies. Most prominent was the OMEGA project, discharge limits have traditionally used funded by US NASA, in which bags filled chemical coagulation and dissolved air 30 Scalability: Scalability is an issue for all with sewage would be floated on reservoirs flotation. For both bacterial and algal technologies to move from bench to 27 and other protected waters. technologies, common chemical coagulants pilot to full-scale. To be competitive with are alum (aluminum sulfate), ferric chloride, conventional technologies, a recommended Attached growth technologies and a wide variety of synthetic organic minimum size for an individual treatment Growing algae in biofilms attached to polymers (poly-electrolytes). However, the module is 100 m³/day-reactor (26,000 physical media has the advantage that the salts associated with inorganic coagulants gallons/day). Raceways and turf scrubbers biomass can be harvested with scrapers can be problematic for algal facilities that are examples of algal technologies fitting (sometimes rakes) at a relatively high recycle media and wastewater facilities this criterion. solid concentration (0.5-4% solids). A that discharge to salt-sensitive receiving disadvantage at large scale is the need for waters. Membrane filters have been used Treatment capabilities and reliability: Local the scraper to move over the medium or the for harvesting in a few algae production regulatory requirements will control the medium to move under the scraper, whereas systems,31 and bioflocculation of algae level and reliability of treatment. Due to the in suspended growth reactors, the biomass without use of chemicals is often observed possibility of monetary fines or the need for is pumped in the media to the harvesting in raceway ponds. Practices to better control retrofits, treatment plant engineers design unit. Moving large quantities of water, of the process are being developed (see for discharge concentrations substantially course, also has its costs. Raceway pond section above). lower than the proscribed regulatory values. For algal systems, the seasonality of treatment performance is a major issue. Several algal biofilm reactors have been Facility siting tested at large-scale—horizontal plastic Due to the high cost of land near cities, Cost: To overcome the natural reluctance geomembrane media (Algal Turf Scrubbers™, treatment of wastewater with algal systems would be reasonable only for rural of project owners to employ new communities or where long pipelines carried technologies, it is recommended that the wastewater into areas with affordable land. total cost (combined capital and operational 21 Park JBK, Craggs RJ. Nutrient removal in wastewater treatment Such pipelines are found in many cities expenditure) of algal technologies is at least high rate algal ponds with carbon dioxide addition. Water Sci one third less than competing technologies. Technol 2011; 63: 1758–1764. either for transport of wastewater to the However, individual owners may have a 22 Woertz IC, Fulton L, Lundquist TJ. Nutrient Removal & Green- treatment plant or from the treatment house Gas Abatement with CO₂ Supplemented Algal High Rate preference for lower capital or operational Ponds. In: Proceedings of the 2009 WEFTEC Annual Conference: expenses, rather than considering only Orlando, FL. 2009 http://works.bepress.com/tlundqui/4/ (accessed Aug 2015). total cost. For example, municipalities with 28 Christenson LB, Sims RC. Rotating algal biofilm reactor and 23 Hadiyanto H, Elmore S, Van Gerven T, Stankiewicz A. Hydrody- access to low-interest loans may be more spool harvester for wastewater treatment with biofuels by- namic evaluations in high rate algae pond (HRAP) design. Chem products. Biotechnol Bioeng 2012; 109: 1674–84. concerned with operational expenses. Eng J 2013; 217: 231–239. 29 Metcalf & Eddy Inc., Tchobanoglous G, Burton F, Stensel HD, 24 Hreiz R, Sialve B, Morchain J, Escudié R, Steyer J-P, Guiraud Tsuchihashi R, Abu-Orf M et al. Wastewater Engineering: Treatment Sustainability factors: The wastewater P. Experimental and numerical investigation of hydrodynamics and Reuse. 5th ed. McGraw-Hill, 2014 in raceway reactors used for algaculture. Chem Eng J 2014; 250: treatment industry generally focuses on 230–239. 30 Colic M, Morse D, Morse W, Miller JD. New developments net energy consumption and recycled in mixing, flocculation and flotation for industrial wastewater 25 Burlew JS. Current status of the large-scale culture of algae. In: pretreatment and municipal wastewater treatment. In: WEFTEC, water production as sustainability metrics. Algal culture: From laboratory to pilot plant. 1953 Washington DC. 2005 However, the use of recovery wastewater 26 Gudin. US patent 3955317: Method of growing plant cells. 31 Bhave R, Kuritz T, Powell L, Adcock D. Membrane-based energy 1975. nutrients as fertilizer has been the topic of efficient dewatering of microalgae in biofuels production and 27 Trent J, Wiley P, Tozzi S, McKuin B, Reinsch S. The future of biofu- recovery of value added co-products. Environ Sci Technol 2012; 46: els: is it in the bag? Biofuels 2012; 3: 521–524. 5599–606.

Algae Biomass Organization | 27 Chapter 5: Regulatory and Process Considerations for Marketing Algal-Based Food, Feed, and Supplements growing interest to the industry over the Cultivation and processing of algae for the and storage.2 The recent passage of the Food past few years (e.g., dedicated conferences food and supplement market has been Safety Modernization Act (FSMA) introduced organized by the Water Environment heralded as an attractive market opportunity many new requirements for the food and feed Federation). The fertilizers are in the form that can provide high economic margins industry and FDA guidance has not yet been of either treated sludge (“biosolids”) or even at modest plant size. Algal biomass published for many of these requirements. In concentrated nutrients (such as N- and contains edible oils, proteins, carbohydrates, April 2012, the FDA published new guidelines P-rich mineral struvite). In addition, other pigments, antioxidants, and other useful on safety assessment of food nano-materials. environmental quality characteristics might dietary ingredients or additives. Algal-based Some microalgal cell and cell fragment sizes be considered as sustainability factors, for food and supplements, now mostly found in may qualify as nano-scale materials.3 Food example, effluent water quality, air pollutant health food stores, are expected to become or feed companies may also require other emissions, truck traffic, noise, etc. Land-use increasingly common in the mainstream food certifications, such as ISO 9000, which may be changes typically are not considered (for a market. necessary for international sales.4 more detailed sustainability discussion, see

Chapter 2). The challenge confronting algae in the Economic support for algaculture Sustainability may be the biggest advantage context of food production is compliance Though algae is subject to regulatory of algal treatment technologies over with well-established but potentially complex framework similar to most crops, it is not conventional facilities, particularly in regulations. However, a significant number nationally defined as agriculture, making terms of decreased electricity use and of producers have successfully navigated it difficult to obtain the same type of improved recovery of nutrients in the form these regulations including companies governmental economic support.5 For of algal biomass. However, algal treatment such as Qualitas, Heliae, DSM, Solazyme, example, the Biomass Crop Assistance facilities built to assimilate nutrients are and Earthrise. The regulations covering this Program excludes algae from matching expected to produce more biomass than market vary from country to country and payments to help with harvest, storage, and conventional technologies, and thus have only US regulations are considered in this transportation.5 Several states have attempted higher GHG emissions associated with document release. to remove this disadvantage by passing bills handling and transport of this additional in their own senates. In Florida, SB 1830 gave biomass. Alternatively, more concentrated Regulatory framework aquaculture taxation exemptions similar to nutrient streams would be generated for food in the US those of agriculture.6 Arizona passed two (lowering transport costs) if the biomass The Environmental Protection Agency bills allowing algaculture to take place on were subjected to hydrothermal liquefaction (EPA), Food and Drug Administration (FDA), State Trust Lands and be given the same instead of directly used as fertilizer. As United States Department of Agriculture taxation exemptions as agriculture.7,8 HB 276 with the recommended cost advantage in Ohio reclassified algaculture as a form of criterion, algal technologies might need an (USDA), Federal Trade Commission (FTC), Association of Animal Feed Control Officials agriculture.9 In Iowa, land used for algaculture advantage of at least a one-third decrease 10 in GHG emissions to attract the attention of (AAFCO), and the International Organization is considered to be agricultural. communities seeking to lower the carbon for Standardization (ISO) all have regulatory footprint of their WWT facility. authority over various aspects of food Though algae crops do not have all the production, distribution, and marketing. The financial opportunities applied to most Biofuel metrics FDA regulates the safety of all foods, except agricultural crops, there are federal programs When wastewater is used in algal biofuel most meat and poultry products, which fall that may apply. The 2008 Farm Bill established feedstock production, the evaluation under the purview of the USDA. The FDA at tax credit for producers of cellulosic metrics would mostly focus on the cost and authority covers foods, dietary supplements, ethanol.11 The Noninsured Crop Disaster sustainability of the biofuel, as discussed in food additives, color additives, medical Assistance Program provides assistance to Chapter 2. Any revenue or savings derived foods, and infant formulas. EPA regulates business producing noninsurable crops low from the WWT service could be used to pesticides, including residues on food, and lower the cost of the biofuels. The amount antimicrobials. of revenue could be expected to scale with 2 FDA. Current Good Manufacturing Practices (CGMPs). http://www. In the US, food is defined as “articles fda.gov/Food/GuidanceRegulation/CGMP/default.htm (accessed the cost of alternative WWT and disposal Aug 2015). used for food or drink for man or other options at the specific location. The ability of 3 IFT. Assessing the Safety of Nanomaterials in Food. http://www.ift. an algal biofuel enterprise to capture WWT animals, chewing gum, and articles used org/food-technology/past-issues/2011/august/features/assessing- for components of any such article.”1 Algal the-safety-of-nanomaterials-in-food.aspx?page=viewall (accessed revenue is not certain. Communities may Aug 2015). biomass and other products will be either instead require algae growers to pay for use 4 ISO. 9000. www.iso.org/iso/iso_9000 (accessed Aug 2015). a food for human consumption, a dietary of the water and nutrient resources in the 5 Trentacoste EM, Martinez AM, Zenk T. The place of algae in wastewater. supplement that is a subset of food, or agriculture: policies for algal biomass production. Photosynth Res feed for animal consumption. The FDA 2015; 123: 305–315. also requires that food, feed, and dietary 6 The Florida Senate. Ad Valorem Taxation. 2013https://www. flsenate.gov/Session/Bill/2013/1830 supplement production follow current Good 7 State of Arizona. HB 2225. 2012 http://www.azleg.gov/ Manufacturing Practices (GMP), which is an legtext/50leg/2r/bills/hb2225p.pdf extensive checklist covering food production 8 State of Arizona. HB 2226. 2010 http://www.azleg.gov/ legtext/49leg/2r/bills/hb2226p.pdf 9 Ohio General Assembly. Substitute House Bill Number 276. 2012 http://archives.legislature.state.oh.us/bills.cfm?ID=129_HB_276 10 The Iowa Legislature. House File 632. 2013 https://www.legis. iowa.gov/legislation/BillBook?ga=85&ba=HF 632 11 Schnepf R. Renewable Energy Programs and the Farm Bill: Status 1 USP. Food Ingredient Standards. http://www.usp.org/food-ingre- and Issues. 2013 http://nationalaglawcenter.org/wp-content/up- dients (accessed Aug 2015). loads/assets/crs/R41985.pdf

Algae Biomass Organization | 28 foods.15 A GRAS substance can meet current Proposed regulatory requirements through preparation Ingredient of either a GRAS self-determination (no notice to FDA required) or filing a GRAS notification with the FDA. An FDA review of a GRAS notification (GRN) requires approximately 180 days and the submission becomes publicly NDIN to FDA NDI or GRAS GRAS Determination accessible. GRNs can be accessed on the FDA website,16 and currently, the Schizochytrium,17 Dunaliella,18 Spirulina,19 Chlorella,20 Prototheca,21 and Haematococcus22 algae have products which are GRAS. Table 5.1 Self-determined GRN to FDA displays some of the information found in the FDA non-response (75 days) GRAS documents.

FDA response A GRAS safety dossier contains the product’s (180 days) biological and/or chemical identity and characterization, product specifications and batch data, the method of production, the intended use (including food types), the Dietary Supplement Market Food ingredient Market estimated dietary intake of the product, and a review of the publicly available scientific literature and supporting studies. Once a substance is determined to be GRAS, it is only so for the intended use and amounts Algae Species Production Method Product Ref. specified in the GRAS determination. A dietary supplement is composed of 17 Schizochytrium fermenter docosahexaenoic acid one or more dietary ingredients. Dietary Dunaliella bardawil raceway dried powder 18 supplements are governed by their own set of regulations as specified in the Dietary Spirulina platensis raceway ingredient in foods 19 Supplement Health and Education Act of Chlorella vulgaris raceway ingredient in foods 20 1994 (DSHEA).23 New dietary ingredients require a New Dietary Ingredient Notification Prototheca moriformis fermenter algal structuring fat 21 (NDIN) to be delivered to the FDA. The term Haematococcus pluvialis photo-bioreactor astaxanthin esters 22 “new dietary ingredient” means a dietary ingredient that was not marketed in the US in a dietary supplement before October 15, 1994. Draft guidance for the preparation of yield occurs. This program covers crops (3) generally recognized as safe (GRAS) a NDIN is provided on the FDA website.24 grown for food and livestock production and ingredients with food additive exemption, A substance that is GRAS may be used in a includes feedstocks such as algae.12,13 The or (4) dietary supplements. Note that a color dietary supplement without a NDIN. USDA provides rural development grants additive also requires a different regulatory for renewable energy systems, including process with substantially more testing than biomass, under the Rural Energy for America other food ingredients, as well as FDA review Program (REAP).14 Many federal agencies, and approval. The common marketing terms such as the DOE, USDA, and DOD supply “nutraceutical,”“functional food,” and “animal 15 FDA. Generally Recognized as Safe (GRAS). http://www.fda.gov/ Food/IngredientsPackagingLabeling/GRAS/ (accessed Aug 2015). grants for the production of algae biofuels.5 dietary supplement” do not have regulatory 16 FDA. GRAS Notices Inventory. recognition in the US. 17 U.S. Food and Drug Adminstration. GRAS Notification for DHA Food for human consumption Algal Oil Derived from Schizochytrium sp. 2003. http://www.fda. Food ingredients are used in a variety gov/ucm/groups/fdagov-public/@fdagov-foods-gen/documents/ Foods can be made using algae as the main document/ucm264032.pdf of products with regulatory definitions, constituent (e.g., algal flour), as an additive 18 U.S. Food and Drug Adminstration. GRAS Notification Dunaliella including conventional foods, foods for (e.g., algae in energy drinks), or as the source Bardawil Food Usage Conditions for General Recognition of Safety. special dietary use (medical foods), and infant 2008. for a supplement (e.g., capsules containing formulas. The first step in ensuring regulatory 19 U.S. Food and Drug Adminstration. Spirulina platensis GRAS self omega-3 fatty acids). In the US, an algal affirmation 7-1-2011. 2011; : 43. compliance of a proposed food additive is to product intended for human consumption 20 U.S. Food and Drug Adminstration. Generally Recognised As determine whether the additive should be falls into one of four regulatory categories: Safe (GRAS) Notice 000396: Chlorella vulgaris. 2011. a food additive or a GRAS substance. A food (1) food additives, (2) color additives, 21 U.S. Food and Drug Adminstration. Algal Structuring Fat GRAS additive requires that a petition be submitted Notification. to the FDA for review and approval, followed 22 U.S. Food and Drug Adminstration. GRAS Notice 000356: natural astaxanthin complex, a Haematococcus pluvialis extract. 2010. 12 U.S. Department of Agriculture. Noninsured Crop Disaster As- by a public comment period, and then 23 FDA. Dietary Supplements. http://www.fda.gov/food/Dietary- sistance Program (NAP) for 2011 and Subsequent Years. 2011; : 1–2. published in the Federal Register. This can be Supplements/default.htm (accessed Aug 2015). 13 U.S. Department of Agriculture. Noninsured Crop Disaster As- a protracted process that is avoided when 24 FDA. Draft Guidance for Industry: Dietary Supplements: New sistance Program; Interim Rule. 2013 possible. GRAS is a food additive exemption Dietary Ingredient Notifications and Related Issues. http://www.fda. 14 U.S. Department of Agriculture. Part 4280 – LOANS AND gov/food/guidanceregulation/guidancedocumentsregulatoryinfor- GRANTS. 2016 and is a more common approach for new mation/dietarysupplements/ucm257563.htm (accessed Aug 2015).

Algae Biomass Organization | 29 Total All Livestock Poultry Pig Ruminant Aquaculture countries where flue gas is considered a form of waste, it cannot be used for the production Production (106 Tonnes) 980 939 439 256 196 41 of food.36Use of microalgae to treat wastewater or flue gas can lead to the uptake Percentage 100% 96% 45% 27% 20% 4% of heavy metals.37,38 The presence of excess China (106 Tonnes) 183 158.2 65 85 8.2 18 heavy metals is a concern in general, as it can reduce the growth and lipid production of USA (106 Tonnes) 173 146 82 24 40 11 microalgae. In the context of food and feed, the uptake in heavy metals has potential to make the de-oiled biomass unsafe for animal Foods and feeds for lead to reduced digestibility and higher or human consumption.39 animal consumption feed intake.31,32 The cellulosic cell wall Algal products intended for animal food or prevents access to protein and other cell Other considerations include setting product 33 feed require a GRAS notification to the FDA or components. The use of lipid extracted specifications based on knowledge of the an approval from the Association of American algae may not have these problems, however, algal biomass, constituent substances, and Feed Control Officials (AAFCO), which is a as the cell wall is weakened by extraction manufacturing process to assure safety. collection of state officials. States impose of lipids. Studies on algae digestibility and Food grade specifications can be found in regulations, inspections, and license fees on organic matter digestibility for ruminants the current Food Chemicals Codex (FCC), pet, specialty pet, and animal foods, which indicated that under certain processing which includes specifications for many major are summarized by AAFCO.25 Algae-derived conditions digestibility increases with chemical constituents such as ash, moisture, 34 1 pet and animal feed additives require AAFCO the addition of algae. Table 5.2 gives a and heavy metal content. Also, the US certification, often referred to as an AAFCO breakdown of the global feed industry, Pharmacopeial Convention (USP) is a scientific monograph. A monograph is typically used indicating a large potential market for algae nonprofit organization that sets standards to describe a standard format by which a products (adapted from reference 44). for the identity, strength, quality, and purity food product may be characterized and gain of medicines, food ingredients, and dietary approval or recognition for use. Buyers must Processing considerations: supplements manufactured, distributed, and comply with AAFCO regulations, which are growth and harvesting of consumed worldwide. FSMA introduced new requirements for food facilities including different than the FDA regulations. The use algae for food preparation of Hazard Analysis and Risk- of algae in feed is often considered a color Algal growth operations that produce based Preventive Controls (HARPC). HARPC additive, due to changes in animal meat material intended for the food market requires food facilities to evaluate chemical, appearance.26 must adhere to a number of regulatory biological, physical, and radiological hazards, requirements that are designed to assure natural toxins, pesticides, etc. that may In 2004, the feed industry consumes 30% consumer safety. There are three key potentially contaminate the product. FDA has of all microalgae production.27 Specifically, regulatory considerations for marketing yet to develop guidance for the preparation in the aquaculture feed industry, it is the an algal food product. First and foremost, a of a HARPC analysis. A more or less similar natural feed for many aquatic animals.28 food facility that manufactures, produces, requirement, hazard analysis and critical Microalgae could also offer a supplement to packages, or holds food for consumption control points (HACCP)40 is mandatory for existing products for livestock consumption in the US must be registered with the FDA meat, poultry, seafood, and cut vegetables, and comprise anywhere from 7-20% of feed regardless of whether it is located in the US or even though some producers of other foods composition depending on the species.27 not.35 Second, the facility, whether foreign or and dietary supplement companies obtain Small amounts of algae could be used for domestic, must follow GMP regulations. And, HACCP certifications for added safety. the delivery of different elements, such as Se third, the product must have either approval or Cu, essential to livestock nutrition.29,30 In as a food additive or a determination of the Safety information includes pertinent this case, the algae are grown in conditions GRAS status of the ingredient. Any changes scientific information, including publicly where they will absorb the element, and to the manufacturing process will require available scientific articles on the safety and only small amounts of (g/kg) of algae are additional review. used. For animals, high levels of algae can toxicity associated with human consumption of the algae or extract, and closely related The need to approve manufacturing materials. Documentation should include processes may eliminate production 25 AAFCO. State Regulatory Requirement Summary. 2015. http:// credible information on known adverse www.aafco.org/Portals/0/SiteContent/Regulatory/State_Regula- opportunities, such as waste water treatment tory_Requirement_Summary_20150330.pdf (accessed Aug 2015). or CO2 sequestration directly from flue gas. In 26 Orr AI, Burkholder WJ, Benz SA. Regulatory Processes for Using Algae Biomass or Algae Products in Animal Food. In: Algae Biomass 36 International Energy Agency Bioenergy Task 39. State of Tech- Summit. 2010 http://algaebiomass.org/wp-content/gallery/2012- nology Review – Algae Bioenergy. 2017 http://www.ieabioenergy. algae-biomass-summit/2010/06/Orr_Adam.pdf 31 Stokes RS. Evaluation of algae meal as a novel feedstuff for com/wp-content/uploads/2017/01/IEA-Bioenergy-Algae-report- ruminants by. 2015; : 1–126. update-20170114.pdf 27 Lum KK, Kim J, Lei XG. Dual potential of microalgae as a sustainable biofuel feedstock and animal feed. J Anim Sci Biotechnol 32 Peiretti PG, Meineri G. Effects of diets with increasing levels of 37 Abdel-Raouf N, Al-Homaidan AA, Ibraheem IBM. Microalgae and 2013; 4: 53. Spirulina platensis on the performance and apparent digestibility wastewater treatment. Saudi J Biol Sci 2012; 19: 257–275. in growing rabbits. Livest Sci 2008; 118: 173–177. 28 Borowitzka MA. Microalgae for aquaculture: Opportunities and 38 Hess DE. Impact of Heavy Metal Contamination From Coal Flue constraints. J Appl Phycol 1997; 9: 393–401. 33 Becker EW. Micro-algae as a source of protein. Biotechnol Adv Gas on Microalgae Biofuel and Biogas Production rough Multiple 2007; 25: 207–10. Conversation Pathways. 2016. 29 Skřivan M, Skřivanová V, Dlouhá G, Brányiková I, Zachleder V, Vítová M. The use of selenium-enriched alga Scenedesmus 34 Lodge-Ivey SL, Tracey LN, Salazar A. Ruminant Nutrition Sympo- 39 Napan K, Teng L, Quinn JC, Wood BD. Impact of heavy metals quadricauda in a chicken diet. Czech J Anim Sci 2010; 55: sium: The utility of lipid extracted algae as a protein source in for- from flue gas integration with microalgae production. Algal Res 565–571. age or starch-based ruminant diets. J Anim Sci 2014; 92: 1331–42. 2015; 8: 83–88. 30 Saeid A, Chojnacka K, Opaliński S, Korczyński M. Biomass of 35 FDA. Registration of Food Facilities. http://www.fda.gov/food/ 40 FDA. Hazard Analysis & Critical Control Points (HACCP). http:// Spirulina maxima enriched by biosorption process as a new feed guidanceregulation/foodfacilityregistration/default.htm (accessed www.fda.gov/food/guidanceregulation/HACCP (accessed Aug supplement for laying hens. Algal Res 2016; 19: 342–347. Aug 2015). 2015).

Algae Biomass Organization | 30 Chapter 6: Regulatory Considerations and Standards for Algal Biofuels effects associated with ingestion of algae or The production of marketable bio-based approval process in less than a year). ABO extract, or closely related materials, and any fuels from algae is an important and exciting and other expert biofuel groups have cited available independent safety or toxicology aspect of the algae industry. New refinery this long time to pathway approval as a assessments. Algal species new to the human technologies are being developed to major obstacle to attracting private capital food supply chain must undergo additional synthesize these fuels from algal biomass, for first-of-a-kind commercial biorefinery toxicology tests and, in some cases, dietary extracted oils, and volatiles like alcohols construction.3 tests with animals, before approval as a food that are generated by algae. However, new additive or determination of the GRAS status fuels must meet current commercial fuel ABO and other organizations report can be made. specifications, such as those for gasoline, that ongoing legislative and regulatory Other regulatory considerations include diesel fuel, biodiesel or ethanol, or require uncertainty around the RFS is further worker safety at the facilities and the development of a new fuel specification. inhibiting advanced biofuel development. marketing standards. The Occupational Additionally, they must meet a complex set Restrictive requirements for co-location of Safety and Health Administration (OSHA) of regulatory and commercial requirements biocrude processing, legislative proposals regulates worker safety, which includes before they can be marketed. These include to weaken or repeal the RFS, and substantial facilities, training, clothing, and accident environmental regulations, safety and reductions in proposed advanced biofuel documentation. Compliance with organic infrastructure compatibility, and engine volume requirements in EPA’s 2014 proposed standards is the responsibility of the USDA compatibility. rule have all contributed to a slowdown and their National Organic Standards Board in advanced biofuels investment.4 EPA’s (NOSB) and claims such as “organic” require revised proposed rule for 2014-2016,5 issued an additional set of constraints provided by earlier this year, would significantly increase the NOSB.41 Environmental regulations for advanced biofuel volume requirements algal food and feeds are similar to those for relative to the original 2014 rule, providing biofuel production. some optimism for renewed investment in the sector. Testing and labeling Food safety and security regulations Fuel certification require constant culture monitoring and and other regulations documentation for the possible presence In the US, the Clean Air Act prohibits the of toxins, bacteria, heavy metals, chemical sale of gasoline or diesel fuel that is not residuals, and other contaminants. GMP “substantially similar” to conventional requires records from each batch of food fuel, and defines this as not causing or quality control tests (21 CFR Part 110). The contributing to the degradation of a FDA’s Food Labeling Guide and a series of vehicle’s emission control system. In general, FDA guidance documents detail food labeling substantially similar fuels are hydrocarbons regulations, including nutritional facts and meeting their respective ASTM standards, ingredient labeling on packaged products.42 however, EPA has ruled that aliphatic Labels that make health claims require that alcohols (except methanol) and ethers additional documentation is provided to FDA can be blended in gasoline at up to 2.7 for proof. Dietary supplement labeling must wt % oxygen and meet this requirement. comply with specific FDA regulations (21 CFR Renewable Fuel Standard Aliphatic alcohols can also be blended into Part 111).43 In its 2012 final rule implementing the RFS gasoline at 3.7 wt % oxygen under the program,1 the EPA certified that commercial Octamix waiver, that requires the inclusion In summary, food regulations vary depending production of biodiesel and renewable of specific corrosion inhibitor additives. For on the intended use and market as well diesel from algal oils (discussed in Chapter other materials it must be demonstrated as country. In the US, several federal and 2) that comply with the 50% threshold that they will not cause or contribute state agencies may have authority over the will qualify as advanced biofuels. EPA also to the degradation of vehicle emission production and marketing of foods, feeds, recently certified Algenol’s Direct-to-ethanol control systems and producers can apply and dietary supplements. Algae producers fuel as an advanced biofuel with a life cycle for a waiver of the “substantially similar” should review the pathways to market and GHG reduction of 69% versus gasoline.2 requirement. There are no corresponding be aware of the requirements. Poor planning Future algae-based fuel pathways that do substantially similar rulings for diesel fuel, could result in lost time and money, and not qualify as biodiesel or renewable diesel but the same concepts apply. A second potentially, legal action. will require full pathway approval by EPA, EPA requirement is fuel registration under a process which currently requires nearly 2 years on average (EPA recently pledged to reduce pathway approval times significantly, 3 ABO. Examining the EPA’s Management of the Renewable Fuel 41 USDA. National Organic Standards Board. http://www.ams.usda. and Algenol reports completing its pathway Standard Program. 2014. http://www.algaebiomass.org/wp- gov/rules-regulations/organic/nosb (accessed Aug 2015). content/gallery/2012-algae-biomass-summit/2014/12/ABO_RFS_ 42 FDA. Labeling & Nutrition. http://www.fda.gov/food/ingre- STATEMENT_DEC10_2014.pdf (accessed Aug 2015). dientspackaginglabeling/labelingnutrition/ucm2006860.htm 4 BIO. Comments on USEPA Proposed Rule on the 2014 Standards (accessed Aug 2015). 1 USEPA. Regulation of Fuels and Fuel Additives: 2012 Renewable for the RFS Program. 2014. https://www.bio.org/sites/default/files/ 43 FDA. Current GMP in Manufacturing, Packaging, Labeling, or Fuel Standards Final Rule. 2012. http://www.gpo.gov/fdsys/pkg/FR- BIO Comments-EPA PR 2014 RFS RVOs-Docket ID No EPA-HQ- Holding Operations for Dietary Supplements. http://www.access- 2012-01-09/pdf/2011-33451.pdf (accessed Aug 2015). OAR-2013-0479.pdf (accessed Aug 2015). data.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?cfrpart=111 2 Approval for Algenol Fuel Pathway Determination under the RFS 5 USEPA. Renewable Fuel Standard Program: Standards for 2014, (accessed Aug 2015). program. http://www.epa.gov/otaq/fuels/renewablefuels/new- 2015, and 2016 and Biomass-Based Diesel Volume for 2017; Pro- 44 http://www.alltech.com/sites/default/files/global-feed- pathways/documents/algenol-determination-ltr-2014-12-4.pdf posed Rule. 2015. http://www.gpo.gov/fdsys/pkg/FR-2015-06-10/ survey-2015.pdf (accessed Aug 2015). pdf/2015-13956.pdf (accessed Aug 2015).

Algae Biomass Organization | 31 Section 211(b) of the 1990 Clean Air Act Property Gasoline Jet Fuel Diesel Amendment, which for biomass-derived materials regardless of composition will ASTM Standard D4814 D1655 D975 require a health effects literature search and detailed engine emissions speciation study. Boiling point Approximately 60- 150-300°C 150-338°C To date, this has been completed for ethanol 185°C (in gasoline) and biodiesel. Vapor pressure or Approximately 40 kPa < 38°C > 37.8°C winter Flashpoint or higher at 37.8°C EPA also regulates underground storage > 52°C Summer tanks to protect ground water and requires that these tanks must be compatible with Freezing point << -30°C or soluble in < -40°C or soluble in < -30°C or soluble in the materials stored in them. Compatibility hydrocarbon hydrocarbon hydrocarbon can be demonstrated by third party testing Composition -- < 25 vol% aromatics -- (such as Underwriters Laboratories, UL) or by the manufacturer of the tank, providing Combustion Research octane 25 mm minimum Centane number > warranty coverage for use with the new number > 90 smoke point (D1322) 40 fuel. Above ground equipment such as fuel dispensers, hoses, and nozzles are required Water Solubility Low Low Low to have a third party listing as being Stability D525 D3241 D6468 compatible with the fuel being handled 3 by the Occupational, Safety, and Health Density/Heats of -- 775-840 kg/m -- Administration (OSHA) and typically also Combustion by local fire marshals. Third party testing > 42.8 MJ/kg normally requires that the fuel has an ASTM standard to serve as the basis for UL to develop a test fluid, and that the manufacturers will be willing to submit their oils.7 The more recent emphasis on creating majority of states use ASTM standards for equipment for testing and potential listing fungible fuels, completely compatible this purpose. Producers of an algal biomass- by UL. with existing infrastructure, has lead to based ethanol, isobutanol, biodiesel (fatty an increased need for information on the acid methyl ester), or hydrocarbon biofuel Fuel properties for gasoline, jet, presence of contaminants in the oils, on the may be able to demonstrate that it meets and diesel applications catalytic hydrodeoxygenation process, and existing ASTM standards. In some cases, New refinery technologies are being on how they influence the characteristics a blendstock standard may be required, developed to produce fuels from algal of the resulting fuels, as well as on whether such as D4806 for denatured fuel ethanol biomass, extracted oils, and volatile this impacts the performance and outcome or D6751 for B100 biodiesel intended for compounds, such as ethyl alcohol, that are of standard test methods for fuel quality blending at up to 20 vol %. Algal-based 8,9 generated by algae. However, new fuels monitoring. fuels that fall outside any of these existing must meet a complex set of regulatory and Because the fuel market is a commodity specifications will need to go through the commercial requirements before they can market, products from different ASTM process to develop the proper fuel be marketed. These include environmental manufacturers are fungible and quality parameters needed for successful regulations, safety and infrastructure interchangeable as long as they meet a operation in the application for which they compatibility, and engine compatibility. common ASTM standard. ASTM standards are intended. The fatty acid structure with respect to are developed by consensus of ASTM chain length and respective degree of members, including fuel producers and In addition to the standards listed in Table unsaturation of algal oils is thought to be the distributors, engine and carmakers, state 6.1, additional regulations and standards main determinant of the quality, in particular fuel regulators, and other interested limit the content of metal contaminants for the cloud point and oxidative stability, parties. ASTM standards are typically gasoline, ethanol, diesel fuel, and jet fuel. 6 of the resulting fuel. In early work, the focused on ensuring safety in distribution The presence of metals in resulting biofuels conversion of oils to biodiesel often involved and handling, as well as fuel-engine from processes that use for example flue gas transesterification for the formation of fatty compatibility (Table 6.1). ASTM standards in the production cycle is a concern and thus acid methyl esters (FAMEs), which make may also be used to describe fuels for the there might be higher scrutiny around the up the biodiesel. In this process the yields purpose of meeting EPA fuel registration propagation of heavy metals throughout and potential contribution of contaminants requirements. Individual states are the process. The EPA has limited Pb levels are dependent on the composition of responsible for regulating fuel quality as in gasoline to its detection point, 0.013 g/L the originating lipids and most successes part of consumer protection laws, and a while Mn is limited to 0.25 mg/L by ASTM and deployment scenarios have been D4814. ASTM D4806 limits Cu content in demonstrated on triglyceride-rich vegetable ethanol to 0.1 mg/kg and 0.0125 mg/kg in 7 Knothe G. Biodiesel: Current trends and properties. Top Catal E10. ASTM D6751 limits Na/K to 5 ppm and 2010; 53: 714–720. Ca/Mg to 5 ppm for B100 and diesel fuel 8 Lapuerta M, Armas O, Rodríguez-Fernández J. Effect of biodiesel fuels on diesel engine emissions. Prog Energy Combust Sci 2008; respectively. Ash cannot comprise more 34: 198–223. 6 Knothe G. A technical evaluation of biodiesel from vegetable oils than 0.01 w% of diesel fuel according to vs. algae. Will algae-derived biodiesel perform? Green Chem 2011; 9 Knothe G. Biodiesel and renewable diesel: A comparison. Prog ASTM D975. ASTM D7566 limits the content 13: 3048. Energy Combust Sci 2010; 36: 364–373.

Algae Biomass Organization | 32 Chapter 7: Open and Closed Algal Cultivation Systems

Phototrophic cultivation of microalgae material, weeds, and even animals to or cyanobacteria in suspension, at its the culture. Careful culture maintenance most basic, requires making nutrients is required for successful growth in the and light available to the algae, which presence of these challenges.5,6 utilize the nutrients and light to power cellular metabolism, producing metabolic Measurements important in products and biomass. Numerous systems establishing and maintaining an open for suspension phase cultivation have been pond algal culture developed, for the most part falling into For a culture to continue producing more two categories: (1) closed photobioreactor product or biomass, biomass concentration systems in which the culture is held within (“culture density“) must be maintained a closed physical container, and (2) open within acceptable boundaries, and the ponds, in which the culture is contained in water chemistry of the culture must remain a pond but exposed to the environment.1,2 compatible with the target organism’s Similar sets of technical measurements are requirements. used in the operation of the two systems, As with any form of farming, pests, weeds, along with some unique measurements. and abiotic stresses can negatively impact culture health. Rapid detection, diagnosis, Open algal cultivation systems and treatment are critical to return a Open pond systems have often been used culture to production and preventing pond for the (relatively) low cost production of crashes. Tools such as microscopy are useful algal biomass. Examples include Arthrospira for diagnostics, however, limitations on and Dunaliella production.3,4 Open pond observable volumes (a 1 μL microscope systems have been scaled to over 40 sample represents only 1/1012 of a large hectares in a single system. Cooling of raceway) mean that pests are often only the culture in sunny environments is observed once a biotic challenge is far accomplished by evaporation of the culture progressed. Tools such as RT-PCR can detect media, which increases water consumption genetic traces of pests or weeds at much but removes the need for physical cooling lower levels, and modern next-generation of the culture. Exposure to the environment sequencing can detect almost all organisms brings a host of environmental challenges, present in a pond using metagenomics. The including introduction of dust, dirt, foreign algae being grown will normally be present at dramatically higher levels than other organisms, so the use of peptide nucleic acid 1 Borowitzka MA. Algal biotechnology products and processes - matching science and economics. J Appl Phycol 1992; 4: 267–279. 2 Borowitzka MA. Microalgae for aquaculture: Opportunities and 5 Shurin JB, Abbott RL, Deal MS, Kwan GT, Litchman E, Mcbride RC constraints. J Appl Phycol 1997; 9: 393–401. et al. Industrial-strength ecology: Trade-offs and opportunities in of Al, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, 3 Belay A. Mass culture of Spirulina outdoors—the Earthrise Farms algal biofuel production. Ecol Lett 2013; 16: 1393–1404. Ni, P, Pb, Pd, Pt, Sn, Sr, Ti, V, Zn to less than 0.1 experience. In: Spirulina Platensis Arthrospira: Physiology, Cell- 6 White RL, Ryan RA. Long-Term Cultivation of Algae in Open- mg/kg in jet fuels. Biology And Biotechnology. 1997 Raceway Ponds: Lessons from the Field. Ind Biotechnol 2015; 11: 4 Borowitzka LJ, Borowitzka MA. Commercial Production of 213–220. β-Carotene by Dunaliella Salina in Open Ponds. Bull Mar Sci -Miami- Fuels from algae can also be derived 1990; 47: 244–252. from non-lipid pathways including the collection of ethanol, hydrogen, ethylene, isobutyraldehyde, and other chemicals exuded by algae in situ. An example of this is Direct-to-ethanol production from cyanobacteria in closed the PBR operations. These can be extracted from algal fluids or PBR headspace and then converted into fuel and other high-value industrial commodities.10 Recently, ethanol produced through the Algenol pathway qualified under the Clean Air Act as an advanced biofuel, assuming the generated fuel meets the previously defined criteria.2

10 Luo D, Hu Z, Choi DG, Thomas VM, Realff MJ, Chance RR. Life cycle energy and greenhouse gas emissions for an ethanol production process based on blue-green algae. Environ Sci Technol 2010; 44: 8670–8677.

Algae Biomass Organization | 33 Output product quality measurements Depending on the product of interest, product quality measures can include protein or oil content, or concentration of relevant biochemicals such as carotenoids or protein pigments. Inorganic salts (ash) will be present at some level, both internal to the algae and from residual media in the product (e.g., wastewater constituents, see Chapter 4). In addition, non-target algae or other microorganisms may negatively impact product quality. Lastly, material destined for food or feed applications will have strict quality requirements around toxic metals, foreign material, etc. (as discussed in Chapter 5).

Closed algal cultivation systems Closed algal growth systems, known as bioreactors, can be classified as photobioreactors (PBRs) or fermentors. PBRs are closed (or almost closed) vessels for phototrophic algal cultivation where light is supplied either directly by the sun or via artificial sources such as LEDs. Fermentors, on the other hand, are closed bioreactors for the heterotrophic production of algae where the energy for growth originates from organic sources such as sugar. While a typical open pond system is open to the environment on at least the top surface, closed systems carefully control liquid, gas, biologics, dust, and solids input and output from the system. Typically, closed systems carefully direct the circulation of the algal culture to distribute the culture’s exposure to natural or artificial light. Since liquid and gas streams have to be brought in and out of the bioreactor via pumps or bubbling- clamps is useful to prevent amplification of Measurements important during induced flow, the energy requirements of host DNA and its subsequent dominance in harvest and water recycle this type of culture are higher. However, metagenomic data.7 Microalgae in culture are extremely dilute. At a PBR typically produces a denser culture normal operation conditions of ~0.5 g/L, the requiring less energy for extracting solids, Culture health can also be negatively biomass is very dispersed and dewatering and environmental contamination is also impacted by either a trace metal deficiency is challenging. Both chemical and physical minimized. Maintaining and optimizing (e.g., iron or manganese), or high methods are used for dewatering algae. water chemistry is easier in a closed system concentrations of metals (e.g., zinc). Analysis Physical methods include centrifugation, that is not diluted or pH-shifted by rain or of metal levels using ICP-OES, flame AA, or settling (clarification), and filtration. concentrated by evaporation. x-ray fluorescence allows for rapid detection Chemical (and electrochemical) methods of these conditions.5,8 are based on flocculants and/or coagulants The geometric configuration of closed PBRs commonly used in waste water treatment is often designed for efficient utilization of to aggregate the algae (as discussed in natural light. Through a variety of methods, Chapter 4), which can then be more easily light is more evenly distributed through settled, or can be floated using Dissolved the growth media in PBRs than in open Air Flotation (DAF). Often, dewatering is pond systems. Daily volumetric harvest conducted in stages, with a primary step rates on the order of 40% and dry biomass achieving 4-6% solids, and a secondary concentrations up to 5 g/L are feasible in dewatering such as decanting centrifugation tubular PBRs. High algal concentrations used to achieve 20-30% solids. After 7 Von Wintzingerode F, Landt O, Ehrlich A, Göbel UB. Peptide lead to increased harvest yields and faster nucleic acid-mediated PCR clamping as a useful supplement in the dewatering, the paste may be dried to downstream processing. determination of microbial diversity. Appl Environ Microbiol 2000; stabilize the material and to allow further 66: 549–57. 8 Mcbride RC, Lopez S, Meenach C, Burnett M, Lee PA, Nohilly F et processing. al. Contamination Management in Low Cost Open Algae Ponds for Biofuels Production. 2014; 10: 221–228.

Algae Biomass Organization | 34 Advantages: Disadvantages: • Accommodates the growth needs for a • Larger costs per infrastructure area broader selection of algal types (although this can be offset by higher • Avoids open pond evaporation losses, product values and production rates) except when closed system evaporative • The need to actively cool and heat above- cooling is required ground systems that often host thermally • Stable water chemistry less affected by vulnerable algal species evaporation and precipitation • Biofilm growth on the culture-container • Isolation from atmospheric pollutants like interface can reduce light transmission airborne dust, biologics and chemicals • Photosynthetically produced oxygen must • More tightly controlled cultivation be actively removed by engineered gas parameters lead to enhanced product transfer systems densities Types of closed photobioreactor systems • Better protection from environmental Closed systems vary widely in size, material, threats, ranging from microbes to rotifers, shape, and technical principles of operation, birds, and animals but they all attempt to prevent undesired • Relatively pure algal inoculums to seed organism intrusion into an otherwise larger reactors or ponds curated algal culture, while at the same time preventing the escape of crop organisms and growth media that could produce environmental damage. Commonly, closed PBRs require induced turbulence of the shorter mean light paths and provide a algal suspension to avoid gradients in the balance between the two aforementioned cultivation medium and to compensate options (Figure 7.3). for cell-on-cell light shading. A detailed overview of several PBR types tested in • Bubble column photobioreactor: concert with an open pond technique has This type of reactor uses a vertical previously been published.9 structure of transparent material containing the algal suspension. Gas is • Tubular fence photobioreactor: introduced at the bottom of the column, Tubular fence bioreactors channel causing a turbulent upwelling stream microalgal suspensions through tubes that provides both suspension mixing made from transparent glass or plastics. of the algae and gas exchange across A horizontal arrangement of tubes in the bubble envelope surface. Due to banks has currently established itself as the simplicity of suspension and oxygen the most standard geometry in industrial removal, this is the most common and production. In these systems, removal of straightforward way to build a PBR. It is photosynthetically generated oxygen is commonly found in lab environments usually accomplished using degassing and can be implemented in any flask by collection vessels every 50 m of closed simply submerging an airstone bubbler. tubing. Biofilms that form on the inside Precision examples are found at ASU’s of the tubes are removed with small Algal Research Laboratory, AzCATI, Mesa, suspended pellets, elastic plug pigs, or AZ (Figure 7.4). chemical off-line cleaning. Tube cooling is often realized by evaporation of water • Plastic film photobioreactor: that drips onto the tubes. However, when Many PBR designs use transparent film hard or saline water is used to cool the (typically polyethylene) to contain algal tubes, external evaporative water deposits cultures. The physical configurations can also diminish light transmission over are varied and in general are designed time. In general, smaller diameter tubes to maximize light distribution evenness increase the pressure needed to circulate through the culture, while minimizing the the liquid, yet expose the algae to a higher cost of replacing or renewing systems. intensity of light, increasing algal density. Companies such as Solix Biosystems Large diameters (> 0.1 m) lead to lower combine bubbling with a submerged algal densities and higher harvesting algae-filled blade system that distributes costs. Oval tube cross sections provide light evenly across the large surface area of the side of the blade (Figure 7.5).

9 Bosma R, de Vree JH, Slegers PM, Janssen M, Wijffels RH, Barbosa MJ. Design and construction of the microalgal pilot facility AlgaePARC. Algal Res 2014; 6: 160–169.

Algae Biomass Organization | 35 • Volatiles harvesting photobioreactor: substrate in a periodic process, where metals, nitrogen, yeast extract, and Ethanol and other volatiles may be the substrate goes on to inoculate and vitamins. Heterotrophic fermentation secreted by some engineered species of propagate the next crop. Bioprocess systems often are fed a nitrogen source cyanobacteria. From the growth media, Algae’s technology is an example of such as ammonia gas allowing for the volatiles transpire into the PBR headspace, growing algae on a synthetic strippable production of extremely high biomass where they are collected as the primary fabric substrate (Figure 7.7). densities on the order of 100-250 g/L in algal product. In these PBRs, the short time frames. The most common microorganisms are stirred, gas managed, • Heterotrophic fermenter: heterotrophic fermentions are usually and nourished with light. However, they In heterotrophic fermentation systems, conducted in a two-phase, fed-batch are not directly harvested and may last algae are fed sugars and oxygen instead mode for production of lipids. The first many months before replacement with of light and carbon dioxide. The algae phase, in which the batched and fed fresh organisms and media. Algenol is are often grown in complex media with nutrients are mainly supporting cell prominent for its ethanol producing a wide variety of batched nutrients to growth, may happen in 24-96 hours. organisms and flat hanging bag support growth and product production. The second phase, in which product production system Examples of batched nutrients are salts, accumulates, is induced by nitrogen (Figure 7.6).

• Floating panel film reactor: System parameters Unit Notes This type of film reactor floats on the surface of water, which serves as Class of bioreactor Descriptive Tubular, at-panel, plastic-lm, open pond structural support and for equalization parameters raceway, etc. of temperature, and selective chemical exchange occurs across engineered Water movement Descriptive Mechanical pump, airlift, jetted raceway, plastic membranes used in containment. mechanism parameters paddlewheel raceway, wind mixed Organizations developing this technology 3 3 include NASA Ames’ Offshore Membrane Operating reactor L or m PBRs typically have volumes of < 1-100 m , 3 Enclosures for Growing Algae (OMEGA). volume Open ponds < 10-1000 m Water usage per kg L/kg and specic Process and evaporative water required to • Internally illuminated well reactor: This reactor type usually consists of a weight of product species grow and harvest 1 kg of product from a chamber filled with algal growth media specic species and organisms where light is introduced Photosynthetic m2 The amount of solar energy intercepted to the organisms via submerged LEDs or 2 LED-illuminated light guides. Examples of footprint of growth may vary from < 1 m to ha companies experimenting with this type operation of reactor include Varicon Aqua and Origin Photosynthetic % Can be up to 90% for closely spaced open Oil. footprint % of total photosynthetically pond and hanging PBR systems • Flat panel reactor: infrastructure area active Flat panel reactors are typically Location of the test Location and Important in scaling production using NREL constructed from thick parallel glass or installations from climate Solar Radiation Database16 plastic sheets, between which a thin (2 to 6 cm) layer of media is circulated and which growth often aerated using air lift mechanisms. system External heat exchangers may need to be speci cations are employed to maintain a healthy culture derived temperature. High growth rates and culture densities can result from short Light source Type, spectrum, Natural sunlight, articial, hybrid light path and minimized self-shading and duty cycle of cells between the transparent sheets. Typical examples are found at ASU’s Algal System operational- % up-time Up-time production, specifying seasonal Research Laboratory (Figure 7.4). time duty cycle closures and maintenance

PBR light % transmission Transmission eciency of PBR materials • Artificial growth substrate transmission and light distribution systems for photobioreactor: photosynthetic active radiation (PAR) light Not all PBRs grow algae in liquid suspension. If they are sufficiently wetted wavelengths with growth media and exposed to PBR transmission % Dust, UV, encrustation, or biolm light carbon dioxide and light, some algae over time loss/year/incident transmission degradation of PBR can grow strongly as biofilms on artificial substrates such as yarns or plastic and encapsulation materials fabric sheets. In these cases, harvesting involves scraping the algae of the artificial

Algae Biomass Organization | 36 limitation, which halts cell growth but biomass and washed biomass (measured polysaccharides like beta-glucan from promotes lipid storage from the continued as total fatty acid content via in situ Euglena (Emsland Stärke GmbH), or sugar feed. During this phase (72-160+ transesterification); non-lipid biomass astaxanthin from a mixotrophic process hours in length), algae can then produce content, after extraction (lean mass); using H. pluvialis (Dainippon Ink & Chemicals large quantities of lipids at 50-85% of carbon conversion efficiency of sugar to Inc.). biomass weight. Since the dark-adapted lipid product; product titer (total oil or process and culture is encased in sterile subcomponent, e.g. DHA, in the oil); oil Proposed standardization of system and stainless-steel fermenters, the growth specific productivity (amount of oil per culture performance related metrics environment can be precisely controlled. unit of time per unit of lean mass. To aid with the standardization of reporting This allows for both growing cultures and parameters for cultivation systems and Most often heterotrophic fermentation controlling conditions over a wide range to allow for comparisons to be drawn of algae is used to produce oil as done by of temperatures, pH, sugar concentration, between different commercial and academic companies like TerraVia (formerly known and dissolved oxygen and carbon dioxide growth systems, we provide an overview as Solazyme) and DSM (formerly known levels. This precise control is important in of characteristic parameters and figures of as Martek). However, some companies fast growing and environment sensitive merit for both PBRs and open systems. For are researching algae production of processes. Though the production rate both of these systems, the CEN recommends is high, the feedstock costs of sugars, production surface area should be taken as oxygen, and other supplied nutrients can pure production area, or space occupied by be inhibitive if the product is of low value. 10 Perez-Garcia O, Escalante FME, de-Bashan LE, Bashan Y. Hetero- production technology. This would exclude To achieve a stable cultivation, the right trophic cultures of microalgae: Metabolism and potential products. indirect production area pertaining to Water Res 2011; 45: 11–36. balance of nutrients and oxygen should inoculation, harvesting, and drying, as well 11 Pignolet O, Jubeau S, Vaca-Garcia C, Michaud P. Highly valuable 15 be provided. The respiration rate, oxygen microalgae: biochemical and topological aspects. J Ind Microbiol as any management buildings on campus. supply per carbon supply, is optimized at Biotechnol 2013; 40: 781–96. The goal of the IAM 8.0 is to standardize the 20-30% of the growth rate.10-14 Other ratios 12 Bellou S, Baeshen MN, Elazzazy AM, Aggeli D, Sayegh F, Aggelis system descriptions regarding the growth G. Microalgal lipids biochemistry and biotechnological perspec- such as carbon, nitrogen, metals and tives. Biotechnol Adv 2014; 32: 1476–1493. system itself and the process details that vitamins to each other are also important 13 Lowrey J, Armenta RE, Brooks MS. Nutrient and media recycling affect its performance metrics and product to consider and can vary widely between in heterotrophic microalgae cultures. Appl Microbiol Biotechnol quality. 2016; 100: 1061–1075. different algae species and even between 14 Minhas AK, Hodgson P, Barrow CJ, Adholeya A. A Review on the strains. Typical metrics captured during Assessment of Stress Conditions for Simultaneous Production of fermentation are lipid content of the Microalgal Lipids and Carotenoids. Front Microbiol 2016; 7: 1–19 15 European Committee for Standardisation. CEN/BT/WG 218 Final (article 546). Report - Year 1. 2016 16 NREL. National Solar Radiation Data Base, https://nsrdb.nrel.gov

Algae Biomass Organization | 37 Culture Parameters Typical Unit Notes: Growth systems are typically optimized for a combination of specific climates, water types, nutrient sources, and product types

Algae and cohorts used Algal species, cohorts, Algal species and origin, or poly-culture origin information in test data and origin

Algae species Alga or poly-culture What types of algae or poly-cultures can be grown in the specific growth system compatibility compatibility chart

Culture density g/L Sustainable culture density of specified test species

Operating volume per L Capacity of each discrete, isolated PBR or pond isolated reactor pH range pH Culture pH for test species and growth system operating limits

Volumetric productivity Harvestable dry weight per liter-day g Areal productivity L × d Harvestable dry weight produced per horizontal illuminated area per day for a specified species g ! m × d Specific energy J/kg Energy cost per kg dry weight product produced. Energy inputs include water consumption movement, harvesting, artificial light, drying, etc.

Nitrogen, phosphorous g/L Steady state levels of major macronutrients

Fluorescence trace Examples: RFUs, or ratios Pulse Amplitude Modulation (PAM) can provide photo system health analysis of metals ppm metal buildup

Microscopy fluorescence Visual RFUs, or ratios Inspection of cell health, detection of pests can be measured using standard fluorescence systems

Real time PCR Cycle threshold (Ct) and Detection of pest or contaminant DNA at low levels; inspection of cell health, microscopy Visual detection of pests

Reflectance spectroscopy Cycle threshold (Ct) Emerging methodology for non-invasive detection of biomass concentration and real time PCR changes in phenotype; detection of pest or contaminant DNA at low levels.

Harvesting aid addition ppm Concentration of harvesting aids (if any) added to system (coagulants, flocculants, metals added by sacrificial electrodes, etc.)

Return water TOC ppm Concentration of organic carbon returning to culture after passing through the harvest system

Product moisture content % For shipped product

Product quality Variable Depending on the product, this may include ash, oil content, protein content, or concentration of specific biochemicals of value

Product purity Variable Many applications will require analysis for hazardous materials including Pb, As, Cd, Hg, etc or organics such as PCBs, plasticizers and antibiotics

Algae Biomass Organization | 38 Members | 2017

Platinum

Gold

Silver

Corporate Applied Chemical Technology FedEx Express OpenAlgae ATP 3 Gas Technology Institute Parker Hannifin Renewable Resources Aurora Algae, Inc. General Atomics PRANA S.r.l. NCMA - Bigelow Laboratory for Ocean Sciences Georg Fischer LLC Qualitas Health CleanTECH San Diego GICON Grossman Ingenieur Consult GmbH Renewable Algal Energy Colorado Lining International Global Algae Innovations SABIC Commercial Algae Management HY-TEK Bio, LLC SCHOTT North America, Inc. Diversified Technologies, Inc. JAIIC Synthetic Genomics Inc DSM Kimberly-Clark T2eEnergy, LLC Duke Energy Corporation Linde AG TASA (Tecnológica de Alimentos S.A.) Earthrise Nutritionals LLC MicroBio Engineering, Inc Texas A&M AgriLife Research Elnusa Daya Kreatif Muradel Pty Ltd University of Kentucky - Center for Applied Evodos B.V. Neste Energy Research

Supporting Organizations American Oil Chemists’ Society European Algae Biomass Association Phycological Society of America Biotechnology Industry Organization National Biodiesel Board

National Laboratories Los Alamos National Laboratory Oak Ridge National Laboratoryw Sandia National Laboratory National Renewable Energy Laboratory Pacific Northwest National Laboratory