Algal Research 2 (2013) 66–77

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Algal Research

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Initial risk assessment of genetically modified (GM) microalgae for commodity-scalebiofuel cultivation☆

William J. Henley a,⁎, R. Wayne Litaker b, Lucie Novoveská a, Clifford S. Duke c,Hector D. Quemada d, Richard T. Sayre e,f

a Department of Botany, 301 Physical Sciences, Oklahoma State University, Stillwater, OK 74078, USAb National Oceanic and Atmospheric Administration, 101 Pivers Island Rd., Beaufort, NC 28516, USAc Ecological Society of America, 1990 M Street, NW, Suite 700, Washington, DC 20036, USAd Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USAe Los Alamos National Laboratory and New Mexico Consortium, 202B Research Center, 4200 W Jemez Road, Los Alamos, NM 87544, USAf Phycal Inc., 51 Alpha Park, Highland Heights, OH 44143, USA

Abbreviations: EDAB, ecosystem disruptive algal blomodifying, or modification; HAB, harmful algal bloom;NUE, nutrient use efficiency; RES, reduced ecosystem spressure.☆ The scientific results and conclusions, as well as anherein, are those of the author(s) and do not necessarilthe Department of Commerce. Approved for public reLaboratory; LA-UR-12-23750.⁎ Corresponding author. Tel.: +1 405 744 5956.

E-mail address: bill.henley@okstate.edu (W.J. Henley

2211-9264/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.algal.2012.11.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 August 2012Received in revised form 29 October 2012Accepted 3 November 2012Available online 26 November 2012

Keywords:AlgaeBiofuelsEcological riskGenetic modificationHorizontal gene transfer

Genetic modification (GM) of microalgae to improve commercial production of biofuels is underway. Inevitablegovernmental regulationswill likely address environmental, economic and human health impacts. Proactive ad-dressing of such regulatory protection goals should begin now, during early development of this new, potentiallylarge and transformative industry. We present strategies for ecological risk assessment of GM algae for commer-cialmass cultivation assuming that escape of GMalgae into the environment is unavoidable.We consider the po-tential ecological, economic and health impacts of GM algae that persist in and alter natural ecosystems.Horizontal gene transfer with native organisms is of particular concern for certain traits, especially when culti-vating GM cyanobacteria. In general, we predict thatmost target GM algal traits are unlikely to confer a selectiveadvantage in nature, and thus would rapidly diminish, resulting in low but nonzero ecological risk. Genetic andmechanical containment, plus conditional matching of GM algal traits to unnatural cultivation conditions, wouldfurther reduce risk. These hypothetical predictions must be verified through rigorous ongoing monitoring andmesocosm experiments to minimize risk and foster public and regulatory acceptance.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction: inevitability of GM algae, regulations and the needfor proactive research

It is nowwell established thatmicroalgae (hereafter algae for brevity)have among the highest biomass productivities of any autotrophicorganisms. Their high rates of biomass accumulation are attributedto several factors including: (1) all cells are autotrophic unlike ter-restrial plants which have a substantial amount of heterotrophicstructural tissues (roots and stems); (2) algae often have active inor-ganic carbon concentrating systems that reduce photorespirationand enhance photosynthetic efficiency; and (3) their doubling ratesare often measured in hours rather than days or weeks. Algae are

om; GM, genetically modified,HGT, horizontal gene transfer;ervices; RGP, reduced grazing

y views or opinions expressedy reflect the views of NOAA orlease by Los Alamos National

).

rights reserved.

particularly attractive as biomass feedstocks since many species consti-tutively or facultatively accumulate neutral lipids or triacylglycerol(TAG) as high energy density storage products that are drop-in compat-ible with new and existing liquid fuel conversion, transport and com-bustion infrastructure. With proper scale-up, biofuels derived fromalgae are arguably nearing the break-even point with respect to net en-ergy yield and economics, and eventuallymay become cost-competitivewith petroleum-based fuels [1–5].

To ensure economically viable production of algal biomass forbiofuels and other commodities, there is great, but not universal [6], in-terest in genetically modifying (GM) algae to improve, relative towild-type strains, production of feedstock compounds in high-densitycultures [2,7–12]. There are many non-GM genetic strategies to en-hance algal phenotypes, some of which could have ecological risks,but most countries only regulate transgenic/recombinant organisms,thus here we adopt that narrow definition of GM [13]. Requests forlarge-scale testing of GM strains have begun, and at least one approvalhas been granted.

Algenol apparently has received approval from the State of Florida toscale up cultivation of GM cyanobacteria in outdoor enclosed bioreac-tors to produce ethanol [14], after current USDA-APHIS and EPA regula-tions were deemed to exempt their process [15]. Based on the latter

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document, it seems that current federal regulations may exempt mostGM algae grown in an “enclosed” system.

To our knowledge, however, there are no approved releases of GMalgae in open-pond production systems, and little information on po-tential impacts [16]. These releases will fall under regulations relatingto GM plants under the U. S. Department of Agriculture [17] and regula-tions concerning intergeneric microorganisms by the U. S. Environmen-tal Protection Agency [18]. To our knowledge, no other countriescurrently have specifically addressed regulation of GM algae. Thus,this is an opportune time to incorporate ecological principles fromthe start, so as to minimize environmental risk and public concerns[16,19–21].

One important ecological and public health concern is how escap-ing mass-cultured GM algal strains may affect natural communities.Algae form the basis of aquatic food chains, which support crucialecosystem services such as commercial and recreational fisheries.The potential for accidently released GM algae to cause ecologicalharm cannot be ruled out, even with preventive safeguards [13].Given these concerns and the lack of data to directly assess the natureand magnitude of actual risk of GM algae, it is inevitable that govern-mental agencies will begin to issue regulations governing at leastsome aspects of producing GM algae, as they have for GM crops[22]. To inform regulations and balance the need for increased renew-able energy, greenhouse gas mitigation, and nutrient recycling versusthe statutory requirements to protect human and ecosystem health, itis critical to begin proactively evaluating the potential ecological andpublic health implications of industrial cultivation of GM algae.

2. Scope and problem formulation

We will focus on the potential ecological impacts specific to GMalgae, based on the types of traits and genes selected, both for lipidproduction and for biomass yield. Some general concerns of algalmass culture probably would exhibit no GM-specific impacts, andare thus beyond the scope of this analysis. For example, the likelihoodof escape from cultivation, and competition with agriculture for landarea, water and nutrients are not specific to GM algae. However, GMtraits resulting in higher yields per area or volume potentially mayhave the beneficial effect of reduced land and water needs. Moreover,algae engineered to grow rapidly in monoculture production systemsmay be at a disadvantage in the wild in terms of competitive fitnesswith wild algae in mixed populations (see Section 5). It is also difficultto envision any difference between the impacts of GM algae versuswild-type harmful algal blooms (HAB), which produce toxins thatcontaminate food supplies, disrupt food chains, and cause massivefish kills and the death of marine birds and mammals. Additionally,these blooms can result in large regions of hypoxia. Presumably, com-mercial producers of biomass and biofuel will choose to avoid engi-neering or cultivating known noxious or weedy algal species.

Other ecological impacts of GM algae could conceivably differqualitatively and quantitatively from those of wild-type algae, andthese must be evaluated as part of GM-specific environmental riskassessments. GM-specific (differential) ecological impacts may in-clude: (1) more successful competition with native phytoplanktonand formation of GM-HAB under certain environmental conditions;(2) altered phytoplankton species composition, dominance and di-versity, which in turn alters food web dynamics and biogeochemicalcycles; and (3) GM gene exchange with wild organisms via horizon-tal gene transfer (HGT) as a mechanism by which impacts mightoccur. Given that microalgae are the primary basis for most aquaticecosystems, the type of algae present dramatically influences ecosys-tem services such as the odor- and clarity-dependent recreationalvalue of lake and coastal waters, the safety of drinking water sup-plies, and the quantity and type of recreational and commercial ani-mal species available for harvest. Any GM alga considered forcommercial cultivation must be evaluated in the context of all of

those concerns, which represent potential bases for regulation,while ecological changes per se do not (at least in the United States).At this nascent stage of genetic engineering of algae, data are lackingon these aspects, and should be a top priority for near-term research.

To aid policy development and enforcement, researchers mustclearly define the potential harm, relevant to regulatory protectiongoals, associated with each of the three categories of impacts. Thisinitial stage in risk assessment protocols is generally referred to asthe problem formulation stage [23] and focuses on establishing riskhypotheses and assessment endpoints [24]. Fig. 1 shows a conceptu-al flow chart for environmental risk problem formulation for escapingGM algae, emphasizing the importance of regulatory protection goals.It also indicates our provisionally predicted level of risk for various sce-narios that we discuss below. Each arrow implies one or more hypoth-eses testable by rigorous monitoring protocols and/or experimentalmesocosms. We present here some examples of problem formulationin two steps: (1) identify the potential harm and (2) develop a scenariofor how that harm would come about (with implicit testable hypothe-ses). We leave to the scientific community the subsequent necessarysteps: (3) design experiments and monitoring protocols to test explicithypotheses and (4) develop biocontrol or mitigation strategies builtinto GM algae to address these potential concerns.

The goal of algal mass culture is analogous to that of crop systems:to maintain stable high yields of selected components of biomass. Ittherefore follows that some of the same types of GM traits are likelyto be pursued in algae as in crop plants. However, algae and plankton-ic communities are different in many respects from terrestrial plantcommunities, notably with respect to the role of physical forcing byhorizontal and vertical water currents, the rate of change in commu-nity dynamics, the ease of dispersal of algae, andmicroscopic size thatmakes low level detection challenging. Concerns about the potentialrisks posed by GM crop plants [25,26] provide a conceptual frame-work as a starting point to evaluate GM algae. The large body ofknowledge concerning GM crop plants has fostered the developmentof well-established methods for efficiently assessing risks with regardto making regulatory decisions [24,27–30]. Our goal is to provide apreliminary risk assessment of GM algae based on established risksidentified in GM crop plants and how those risks might apply to GMalgae given current knowledge concerning sexual reproduction, phys-iology and ecology in these organisms.

Ecological impacts per se are seldom considered in formulating riskassessments, because legislatively mandated protection goals are pri-marily intended to prevent or minimize economic losses and adversehuman health and environmental impacts. To inform current regulatoryprotection goals, risk hypotheses should be formulated to address eco-nomic loss, harm to human health, and potential disruption of normalfood web dynamics leading to loss of ecosystem services, such as thedegradation of drinkingwater sources, recreational value, and fisheries.When considered alone, these protection goals for assessing GM algaeignore their potential for causing harm to those vital ecosystem servicesthat are typically considered externalities. We recommend that regula-tory agencies also involve ecologists to evaluate broader impacts of es-caping GM algae on ecosystem services [21]. To address both currentregulatory structure and broader concerns about ecosystem services,we focus on how possible impacts of GM algae on natural planktoncommunities can serve as a proxy for ecosystem services as well as di-rect economic or human health consequences, which are extremely dif-ficult to predict.

This approach to problem formulation is supported by a largebody of knowledge concerning the economic, human health and eco-system impacts of HAB or ecosystem disruptive algal blooms (EDAB;[31–38]). Conservative estimates of HAB-associated annual economicdamages and increased health costs in the United States range from~$50 million to over $2 billion [34,39,40]. The selective traits thatallow these organisms to dominate aquatic ecosystems and causedamage are well understood at the ecological level (see Section 4),

Fig. 1. Conceptual diagram of the problem formulation stage of risk assessment for escaping GM algae. The box highlights current regulatory protection goals in the U.S., with thegeneral exception of ecosystem services, which have tended to be considered externalities. Predicted levels of risk relate to these regulatory protection goals. “Low to high risk” inlower left depends on recipient organism and GM trait. GM, genetically modified; HAB, harmful algal bloom; EDAB, ecosystem disruptive algal bloom; HGT, horizontal gene transfer.

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even when the exact molecular mechanisms are not. The degree towhich the modifications introduced into GM algae mimic these selec-tively advantageous traits is one measure of how likely those modifi-cations are to cause harm. This comparative approach has greatpower, and will lead to hypotheses applicable to most, but not allspecies.

In formulating risk hypotheses for GM algae, it is important to rec-ognize that many of the species being considered for large-scale pro-duction come from phyla which are much more distantly related toone another than are plants and animals [41]. Algal species varygreatly in biochemistry and ecology. Consequently, the potential eco-logical risks posed by GM algae will vary qualitatively and quantita-tively among species and perhaps among genotypes within aspecies. Another challenge to formulating risk hypotheses ispredicting how a released organism might affect the phytoplanktoncommunity. In addition to vast genetic diversity of algae, aquaticcommunity biomass and species composition responds dramaticallyon short time scales (days) to dynamic environmental conditions(nutrients, light, temperature, water currents, competitors, patho-gens and grazers). Given highly variable receiving communities, itis difficult to universally predict the impacts of escaped GM ornon-GM algae (Fig. 2). The known mechanisms by which harmfulalgal species come to dominate ecosystems can inform risk hypothe-ses concerning the extent and mechanisms of escaped GM algal per-sistence and harm to the environment.

The diversity of genetic modifications being undertaken or consid-ered also complicates formulating initial risk hypotheses. Many genesand associated traits are potential targets for modification, and therisks will vary qualitatively and quantitatively depending on whichgene/trait is altered. It is impractical to address here more than afew target traits, especially considering that much of the algal geneticengineering research is proprietary [42]. Despite this limitation, fourmajor classes of GM traits encompass most risks likely to be associat-ed with GM algae for biofuels. Three classes of potential target GMtraits deal with physiological enhancements that might affect the fit-ness of escaped GM algae: enhanced biofuel feedstock (e.g., lipid)

production, photosynthesis, and nutrient uptake and utilization. Thefourth class of possible target GM traits involves modifications to en-sure that GM algae will thrive in pond monoculture despite inevitablecontamination with unwanted organisms: reduced competition fromother algae and losses due to grazers and pathogens.

These four classes are discussed in Sections 5.1–5.4. Note thatthese trait classes are not fully independent due to reciprocal influ-ences. The specific risk hypotheses that should be applied to all fourtrait classes are: (1) released GM algae will have at the time of re-lease, or will evolve over time to have [43], a growth rate or other ad-vantage sufficient to alter normal healthy ecosystem function, causingeconomic losses or adverse public health effects [9,10,42,44–46];(2) released GM algae produce toxins or other compounds thathave direct or indirect adverse effects on human health [34,38,40,47]; and (3) a modified or selected trait in released GM algaewill be transferred by HGT to another organism, causing either(1) or (2) to occur via the recipient organism.

Inevitable escape of GM algae from any type of mass productionfacility is central to each hypothesis. It is unclear at present whetherthe economics of the massive scale required for commodity cultiva-tion of algae will favor open ponds or raceways versus expensiveenclosed bioreactors, or hybrid systems [3,4,7]. However, currenttrends in the industry favor open pond production systems [1,2]. Ul-timately, the economic implications of environmental impact andmonitoring must be considered in the life cycle analysis of algalbiofuels. Open systems in particular will foster routine release of cul-tivated algae to surrounding ecosystems (as well as routine contami-nation of the culture system with microbes, algae and grazers). Bothrisks are presumably much less, but nonzero, for enclosed bioreactorsor encapsulated algae.

Escape may occur through aerosol formation related to turbulenceand aeration necessary for cultivation and harvesting by dissolved airflotation; birds and other vectors; leakage, rupture, or overflow of con-tainment structures; maintenance draining of cultivation structures;and incomplete harvesting of biomass that allows some algae to leavewith effluentwater. Regardless of pathway, potential ecological impacts

Fig. 2. Conceptual diagram of the direct ecological consequences of escaping GMalgae. The upper panels contrast the natural phytoplankton dynamics of a potential receiving water bodywith the GM algal mass culture. The lower panels show two possible extreme outcomes for the natural receiving water body following escape of GM algae. This diagram ignores riskassociated with HGT of GM traits to other organisms.

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of escaping GM or wild-type algae will depend on air currents, algal vi-ability, and proximity to surface waters—lakes, streams or estuaries.Other than desert cultivation supported by groundwater, mass culturesof algaewill likely be near a source of surfacewater and potential recip-ient aquatic ecosystems.

Again, we stress that release of cultivated GM algae into surroundingaquatic ecosystems is unavoidable. The quantity of escaped biomass,whether it causes meaningful ecological impacts, and the nature thereof,cannot be predicted with any confidence. Ultimately this must be deter-mined empirically, initially by use ofmesocosms, but rigorousmonitoringwill be required after the onset of commercial mass culture. The questionis, howmuch does escaping GM algae matter: is the risk greater, equal toor less than the potential benefit of GM algae to the environment (e.g., byoffsetting CO2 emissions from displaced fossil fuel use) and human socie-ties? The risks should also be evaluated in the context of the already largeanthropogenic alterations in aquatic ecosystems through eutrophication,pollutants, overfishing, and climate change.

3. Risks to aquatic communities

For any escaped alga, GM or not, to have a substantial ecological im-pact inmost caseswould require that it is a successful invader, i.e., it ex-ploits an open niche to attain a persistent, as yet undefined, absolutebiomass or “significant fraction” of community biomass (Fig. 2) [32].We suggest addressing this issue as a near-term priority researchfocus, recognizing that the relative abundance of the biomass of the in-vading strain necessary to cause harmmay depend on the specific algaltraits and receiving community, and potential environmental perturba-tions such as anthropogenic nutrient loading. Invasiveness depends onpopulation stability characteristics, relative growth rates, and modes ofcompetition of resident and invading species [48].

Successful invasion does not necessarily require rapid growth rate ofthe invader. It is noteworthy thatfluctuating resident populations, char-acteristic of coastal phytoplankton communities, may predispose themto successful invasion [48]. The following discussion of potential ecolog-ical impacts assumes that critical relative biomass is persistentlyexceeded (Fig. 2), which is distinct from the probability that a givenGM alga will successfully become established in situ. Except in thecase of a massive catastrophic “leak” of cultured biomass, attainment

of a critical in situ relative biomass of a GM alga would require multiplegeneration times to develop. Thus, any cellular properties of GM algaewhose expression depends on unusual environmental conditions char-acteristic of mass culture systems (e.g., shallow, well-mixed, with highnutrient and CO2 loading) would likely diminish quickly in natural wa-ters. Dependence of GM trait expression on exceptional (unnatural) cul-tivation conditions could be a general requirement for all GM algaedesigned for mass culture. However, natural selection imposed by theculture system on spontaneous mutants may lead to potentially unde-sirable changes in algal traits [43].

Fig. 2 conceptualizes two extreme outcomes in the continuum ofecological responses to escaping GM algae. As will become evidentin Sections 4 and 5, we believe that the benign impact is most likely,in which the escaped GM alga may survive in situ, but at low level andwith negligible impact on native species, and consequently on highertrophic levels of interest to regulators. In the undesirable alternativeextreme outcome, the escaped GM alga dominates in situ biomass,completely displacing some native species and reducing the meanbiomass of others. This outcome alone is not a protection goal of con-cern for current regulations. Thus, this situation would need to be ofsufficient magnitude and duration to impact regulated aspects ofwater quality, economics or human health (Fig. 1).

GMorganisms that survive in natural ecosystems are potentially un-limited in time or space. Algal populations can grow explosively and ep-isodically through asexual, and in many cases sexual, reproduction.Indeed, rapid growth is one of the primary advantages of algae overplants for biomass production, but it alsomay represent a larger ecolog-ical risk. Knowledge of in situ algal sexual reproduction and populationgenetics is rapidly developing, but remains preliminary and limited torelatively few species [49–53], including nominally asexual species[54]. The emerging consensus from these studies is that sexual repro-duction and gene exchange are common among eukaryotic algae.Given the international interest in year-round commodity-scale pro-duction of algal biofuels, the potential for escape from cultivation ofa variety of GM algae is continuous and global. Moreover, algae arereadily aerosolized and dispersed, and many can remain dormantfor extended periods under unfavorable conditions [55,56], therebypersisting more or less indefinitely. These characteristics are commonto wild type as well as conspecific GM algae. The most general question

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of problem formulation follows: In comparison to wild types, to whatextent are GM algae more or less of a risk in these respects, and whatqualitatively different risks exist specifically associated with GM algae?

Another assumption underlying the risk hypotheses is that horizon-tal gene transfer (HGT) will occur either through uptake of DNA re-leased from the cells, or via viral mediation, or through hybridizationevents involving sexual exchange of genetic material among related or-ganisms. The mechanistic and genomic evidence for extensive HGT incyanobacteria [57–62] calls for extra caution with GM cyanobacteriafor mass culture. Based on this concern as well as metabolic advantagesof eukaryotic algae for biofuels production, our tentative recommenda-tion is that GM formass culture focus on eukaryotic algae, since they areless capable of integrating and transmitting foreign DNA present in theenvironment than are cyanobacteria. Furthermore, as discussed inSection 6, it is recommended that genetic traits that facilitate sexualtransmission of genetic material be inactivated in transgenic algae.This should greatly reduce, albeit not eliminate, the risk of HGT betweenthe mass cultured GM alga and environmental organisms. Eukaryoticalgae do show evidence of HGT, likely involving viruses and symbioses[63–65], and even transfer of DNA from cyanobacteria to eukaryoticalgae [66].

Given the immense population sizes of microbes, even statisticallyrare HGT events could occur regularly. For example, a population densi-ty of 107 algal cells ml−1 translates into 1018 cells in a 10-cm deep,100-ha facility. Dense microbial communities are most likely to resultin HGT if contaminating or recipient organisms are present [25]. Com-modity scale cultures would require multiple orders of magnitudescale-up from this, making the total cell numbers almost inconceivablylarge, and the chance of “rare” events nontrivial.We therefore urge cau-tion when considering deployment of engineered selection strategies.

For example, engineered resistance to the herbicide atrazine iswidely employed in terrestrial agriculture to prevent invasion of un-desirable species. Runoff from fields introduces low levels of atrazineto surface waters, where its impacts on phytoplankton have yet to befully established [67–70]. It is possible that environmental levels ofatrazine might represent an anthropogenic open niche that wouldconfer a selective growth advantage to escaped GM algae modifiedfor atrazine resistance. That being said, many of these potential selec-tion genes are unlikely to offer a selective advantage and in fact maybe disadvantageous to any recipient host, particularly if the selectiveagents are used sparingly and/or intermittently, or if we can developnew, short-lived herbicides matched to resistance genes, which are le-gally restricted to use in algal mass culture systems. Furthermore,many potentially desirable GM traits are likely to reduce fitness in thewild and thus are likely to be lost due to lack of selective advantage.The primary concernwould be indirect, via the transfer of, e.g., antibioticresistance genes to heterotrophic bacteria, further spreading resis-tance through the global bacterial community [13]. The extent towhich HGT occurs in eukaryotic (non-bacterial) species is not known.However, sexual reproduction is common throughout all algal groupsand the exceedingly large population sizes, rapid division cycle, andviral pathogens [64,71] of these microorganisms greatly increase theprobability of HGT occurring. Significantly, there are no known exam-ples of HGT from GM terrestrial crops to microorganisms under fieldconditions.

4. Harmful algal blooms (HAB) as an analog of mass cultivation

Globally, the number of documented HABs is increasing in marineand freshwater environments [32,34,39,40]. They vary greatly in spa-tial and temporal scales, but each is unique with respect to economicand human health impacts. For example, a 2005 bloom of the toxic di-noflagellate Alexandrium fundyense resulted in closure of most shell-fish beds from Massachusetts to the Canadian border, with anestimated US$2.4 million in lost revenue [72]. HABs are largely drivenby the interaction between physical processes (both natural and

anthropogenically altered) and increased anthropogenic nutrientloading, which elevates algal biomass and subsequent decomposition[73]. The recent boom in corn grain-based bioethanol may actuallyhave worsened this trend [74]. Use of nutrient-rich anthropogenicwastewater (rather than commercial fertilizer) in algal mass culturemay have the secondary benefit of reducing net nutrient loading tosurface waters [75–77]. The fact that algae efficiently sequester nutri-ents when available will allow commercial algal production facilitiesto take advantage of highly variable nutrient inputs from anthropo-genic sources as well as from natural sources.

Great advances have been made in understanding the causes andconsequences of wild-type HAB, including the ecologically and eco-nomically important subset of HAB known as EDABs [31,33,36,37].By definition, an EDAB dramatically and persistently reduces phyto-plankton diversity, often to near monoculture, and consequently im-pacts the food web and biogeochemistry [34,35,37,38]. Significantly,physical factors including temperature, light availability, currents,settling rates, and nutrient loading as well as biological factors suchas community species composition and herbivory all factor intoestablishing an EDAB. Dominance by EDAB species requires at leasttwo of the following traits: superior nutrient uptake ability at lowambient concentration, production of toxic compounds which inhibitgrowth of competitors and/or poison grazers, or a cell size or compo-sition which is poor food quality for grazers. As the biomass of anEDAB increases, these traits become even more effective, assuringthe competitive advantage of the EDAB. Furthermore, as EDAB algaldensities increase, they effectively shade out competitors. This posi-tive feedback mechanism of increasing competitive ability with in-creasing density allows EDABs to dominate. However, dominancerequires that environmental conditions remain reasonably stableand favorable to the growth of that EDAB species. Changing condi-tions result in the collapse of the bloom. This susceptibility of evenEDABs to changing environmental conditions is central to predictionsconcerning ecological harm from escaping GM or wild type algae.

Algal toxins [34,35,38] are best known from dinoflagellates [78]and cyanobacteria [79], and a few diatoms [80], haptophytes [37]and euglenoids [81]. Dinoflagellates, haptophytes and euglenoids donot appear to be priority subjects for biofuels, and earlier we urgedcaution in using GM cyanobacteria because of the high risk of HGT.However, it is conceivable that some algal biomass producers mayconsider designing GM algae to possess toxins to maximize competi-tive advantage and grazer-resistance in culture. Such an approachwould require rigorous testing and monitoring for potential humanhealth and environmental impacts.

The biomass dominance desirable in mass cultured algae (GM orwild-type) for commodity production will in many respects need tomimic the characteristics of EDABs. The aim is to enable dominanceunder cultivation while precluding development of GM-EDAB beyondthe confines of cultivation. We can address this concern through re-ciprocal optimization of culture conditions and GM algal traits. Likelymass culture conditions include high light, nutrient and CO2 loadingin shallow, well-mixed vessels, a combination of conditions thatprobably would occur only rarely in nature. EDAB species, oftensmall and slow growing, dominate by tying up nutrients in biomassdue to minimal grazing, and shading other phytoplankton due todense biomass [37,82]. Small size and low maximum growth rateare not characteristics typically sought in algae for mass culture,where rapid continuous biomass production and easy harvesting areprimary goals.

It is likely that the nutrient loading regimes, e.g., intermittent nu-trient pulses [83], in mass cultures would be optimized for the nutri-ent uptake characteristics and requirements of the cultivated alga soas to reduce competition from contaminating algae. Furthermore, in-ducing acute nutrient deprivation stress to induce facultative lipid ac-cumulation prior to harvest could result in reduced biologicalcontamination. None of these peculiar conditions are likely to persist

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in natural receiving waters, thus there is a relatively small probabilityof a GM alga attaining exceptionally high and/or persistent biomassunder in situ conditions, i.e., becoming an EDAB. Although potentialdevelopment of an ecosystem-level GM-EDAB would be difficult totest at a small experimental scale, because it would lack the full spec-trum of physical forcing, a demonstrated loss of GM expressed traitsin field mesocosms would indicate minimal risk.

5. Risk assessment of specific classes of GM traits

The potential adverse environmental consequences of GM algaewill be intrinsically linked to how the organism has been modifiedto simultaneously outcompete other algae, prevent grazing losses, re-sist disease and produce useful chemical feedstocks. This type of engi-neering sounds challenging, but in reality is currently feasible. Forexample, modern GM cotton and maize can be herbicide resistant,allowing for control of plant competitors, and can also contain anengineered toxin that kills the primary limiting pest. The potentialrisk of GM biofuel algae will depend on the degree to which releasedGM algae will affect ecosystem services or public health when re-leased, or what may happen if the genes they carry are transferredto other organisms.

Multiple genetic engineering approaches are being pursued to en-hance production of eukaryotic algal biofuels [10]. Targets include genesrelated to lipid metabolism; direct biosynthesis of biofuels (e.g., fattyacid ethyl esters, alkanes, alcohols, and isoprenoid derivatives); secretionof biofuel feedstocks; carbohydrate metabolism; enhanced nutrient up-take; hydrogen production; enhanced photosynthetic efficiency; stresstolerance; bioflocculation; and enhanced cell disruption for oil harvesting.Mechanistic approaches may involve (1) altering an existing functionalgene, (2) altering an existing regulatory sequence, or (3) inserting agene or regulatory sequence from a different organism that is new tothe recipient alga. Only approach (3) is generally considered GM in thenarrow sense of being transgenic, and is currently regulated by the U. S.Department of Agriculture and theU. S. Environmental Protection Agencyin accordance with the established Coordinated Framework for Regula-tion of Biotechnology [84]. USDA and USEPA are currently consideringwhether approaches (1) and (2) will be regulated, and under whatconditions.

Major physiological changes induced by any of these approachesare likely to alter the rate of growth/productivity and biochemicalcomposition of GM algae, thereby potentially affecting their compet-itive fitness under natural conditions and possibly their role in thefood web, when inevitable escape occurs. Each specific GM traitmust be independently evaluated for potential ecological impacts, aswell as its enhanced value for feedstock production, carbon mitiga-tion, and nutrient recycling. It is also important to consider potentialecological impacts of selection marker genes that have been used inGM algae to date, although they may not affect metabolism of GMalgae. These typically include antibiotic resistance or fluorescent orbiochemical markers [10]. Importantly, none of these areas of activeresearch address the dynamic, inherently unstable nature of plank-tonic communities. The most productive GM alga will be useless ifcontaminating competitors, grazers and pathogens preclude itssustained dominance in mass culture [76]. The four classes of GMtraits mentioned in Section 2 are presented here as exemplars ofthe problem formulation stage of risk assessment for GM algae.Table 1 summarizes the specific concerns and predicted risk levelsfor these as well as selected non-GM approaches to improving algalmass culture productivity and stability.

5.1. Enhanced lipid production

Nonpolar storage lipids, specifically triacylglycerides (TAG), are a pri-mary desired algal feedstock for the production of biofuels. Thus, strainselection, culture protocols and genetic engineering have focused on

altering carbon metabolism to favor accumulation of TAG [10]. HighTAG content, at least at the time of harvest, is desirable regardless ofwhether GMorwild type algae are used inmass culture. To date, accumu-lation of high cellular TAG content has required cessation of growth (celldivision) by stressing cultures, usually bywithholding a key nutrient suchas nitrogen [85], or silica for diatoms [86], or by providing a source of re-duced carbon to support mixotrophy [87–89]. At the time of harvest, GMstrains presumably would have higher biomass and/or TAG content. Fur-thermore, more reduced and shorter chain fatty acids (C10–C12 FA) aremore desirable feedstocks for fuel production [90]. This raises anothergeneral problem formulation question that could be refined into testablehypotheses concerning an alleged harm. Could TAG/FA content have anyGM-specific implications for ecological impactswhenGMalgae enter nat-ural surfacewaters? The answer(s) are inherentlymultidimensional, con-ditional and not obvious, but are experimentally testable. Intuitively, thewell-documented wide range of TAG/FA content in wild type algae, itsvariation with growth conditions, and the apparent lack of evidence forhigh-TAG strains forming EDABs implies that natural plankton communi-ties have successfully “managed” this variability.

The batch culture inductive oil accumulation approach features initialnutrient replete growth conditions to allow rapid biomass production,followed by nutrient starvation or heterotrophic growth (“boosting”)shortly before harvest to trigger TAG accumulation [87–89,91]. GM cellsthat escape prior to this trigger point would not contain “abnormal”TAG at the time of entering a natural ecosystem, thus to grazers thecells would not be nutritionally different than wild type. In contrast,cells escaping just before or during harvest would have unusual TAG/FAcontent, as well as elevated C:N and C:P ratios. Such cells may besuboptimal food sources to sustain consumers, a trait common in EDABspecies, which could alter predation pressure and consumer physiology.The samewould be true forGMalgae engineered to constitutively containaltered TAG/FA content in continuous growth mode.

Grazers may select prey based on “taste” as well as size [92]. Evenabsent selective grazing, algal nutritional quality differentially affectsreproduction and population growth of various grazer species, with po-tential community consequences for phytoplankton and higher trophiclevels. Algal biochemical composition is a major concern in aquaculture[93–95], and also is important towild grazers and filter feeders [96–98].Producer nutritional quality impacts trophic structure, although lessstrongly in aquatic than terrestrial ecosystems [99]. However, this con-cernmust be balanced by the general inverse relationship between bio-mass accumulation rate and lipid accumulation [6,86,100,101]. Thepotential ecological effectswould be in the realmof community dynam-ics, and thus require long-term tracking.

This raises another question that may serve as a testable risk hy-pothesis. To what extent is the population biology and species com-position of local consumers altered by palatability, nutritional valueand relative abundance of the GM algae, compared to non-GMalgae? The alleged harm of relevance to governmental regulation is:do any effects on grazers adversely impact higher trophic levels in-cluding shell- or finfish?

In addition to their potential effects on grazers and indirectly onpred-ators, would GMalgaewith altered TAG/FA content likely have any com-petitive advantage in situ? If anything, diverting a large fraction ofmetabolic energy to a storage compound such as TAG (or ethanol inthe Algenol system) intuitively would be disadvantageous, in that it cor-relates with reduced cell division rate. Even if the TAG content detersgrazers, the GM alga could persist and dominate native phytoplanktononly if it were also a superior light and nutrient competitor under insitu conditions, which as previously noted are unlikely to resemblemass culture conditions. Experimental mesocosms can be used to testwhether biochemical differences between GM and wild type algae per-sist once the GM cells experience prolonged suboptimal natural condi-tions. It seems unlikely, thus we predict that the probability is very lowthat GM algae modified in TAG/FA content would form an EDAB, oreven persist at a high level.

Table 1Summary of predicted ecological risks of selected major approaches to improving success of mass algal culture for biofuels. Both GM and non-GM approaches are included for comparison and thoroughness. Target traits discussed in the textare indicated with the corresponding section number. NA, not applicable. RES, reduced ecosystem services. RGP, reduced grazing pressure.

Potential approach Target GM trait Pros Practical consequencesof the approach

Selective advantage for GMalgal dominance in situ

Potential harm if releasedGM alga thrives in nature

Predicted risk — should be testedexperimentally in mesocosms, andtied to regulatory protection goals

1. Physiological enhancementsSelect for or GM-based highTAG content (Section 5.1)

High TAGaccumulation

Higher yields of biodiesel None None — high TAG contentwould slow growth in situ

None unlessconstitutive high TAGsdeter grazing

Very low — biotic, chemical and physical factorswould likely control GM alga in situ.

GM enhanced photosynthesis(Section 5.2)

Smaller lightharvesting antennasize

Higher cell density, Cuptake in optically densecultures

None None None Very low — unlikely to be competitive under insitu light conditions.

Select for or GM in optimalnutrient efficiency (Section5.3)

Nutrient useefficiency (NUE)

None Low probability of success None — natural algalassemblages already havediverse NUE strategies

None Very low

2. Counteract contaminating algaeGM in herbicide resistanceand apply herbicide tocultures (Section 5.4)

Reduced competition Reduced broad spectrumcompetitors

Small metabolic cost todetoxify herbicide; mustcontain herbicide

Would be favored byambient residual herbicidesin surface waters

Reduced ecosystemservices (RES) if ambientherbicides favor GM alga.Higher risk if HGT to EDABalgal species

Low if little herbicide release to surface watersand avoid environmentally common herbicidessuch as atrazine. Low probability of HGTto EDAB algal species or algae toxic to humans.

Select for natural or GMin allelopathy

Allelopathy Reduced broad spectrumcompetitors

Metabolic cost reducesbiomass production

Reduced competition RES Low if an existing algal allelopathiccompound is used. Moderate if a novelallelopathic compound is introduced orupon HGT to EDAB algal species.

3. Reduce grazingSelect for preexisting toxinproduction (Section 4)

NA Reduced macro- andmicrozoo-plankton grazers

Metabolic cost reducesbiomass production

RGP RES Low — some native algae are toxic andit is unlikely that GM alga morecompetitive than native toxic algae.Health risk to workers?

GM in a novel toxin Reduced grazing Reduced macro- andmicrozoo-plankton grazers

Metabolic cost reducesbiomass production

RGP RES Moderate — novel toxin(s) more likelyto provide a competitive advantage forreleased GM algae. Possible HGT tonative EDAB algae or other organisms. Healthrisk to workers?

Select for or GM in poorfood quality

Reduced grazing Reduced macro- andmicrozoo-plankton grazers

None if no effect onbiofuel production

RGP RES Low — escaped algae GM for biofuel productionare unlikely to have other competitive advantagesin situ.

Apply pesticides to masscultures

NA Reduce macro- but notmicro-grazers (physiologysimilar to algae)

Toxic waste issue wheneffluent released

Selective advantage only ifpesticide pollution persists

None unless effluent createslocal environments favorablefor the GM algae

Low if the waste effluent is treatedproperly.

Biological control:zooplanktivorous fishtrophic cascade(Section 5.4)

None Reduce macro-, but notmicrograzers

Partial control of grazing.Attract birds as vectorsfor microbes

None None unless an invasivespecies is used as the controlagent and it escapes

Very low

Physical separation(size-cutoff filtration)

None Exclude macrograzerswithout pesticides

Economically andenergetically impractical?

NA NA NA

4. Enhance pathogen resistanceSelect for natural pathogenresistance (Section 5.4)

None Help reduce losses due topathogens

Must rotate or add newgenotypes of GM algae toresist new pathogens

NA NA None — resistance is selected from natural variationalready present in wild type algae.

72W.J.H

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5.2. Enhanced photosynthesis

Increasing the efficiency of photosynthetic light utilization or bio-mass productivity is one trait likely to be targeted for improvement innext-generation GM algae. Here, we discuss the biological rationalefor whether GM algae with altered light harvesting systems are likelyto dominate in nature, based on preliminary information. We focus onchlorophyte algae as an example, noting that all classes of algae andcyanobacteria have some sort of light harvesting pigment proteincomplexes that could be manipulated genetically. Thus, the principleswe present should apply generally.

Enhancing the efficiency of carbon fixation per unit chlorophyllcould provide a selective advantage to those algal strains due to fastergrowth rates. Over 50% of the energy losses associated with photo-synthesis are attributed to inefficiencies in light capture by the lightharvesting chlorophyll (LHC) antenna pigment protein complexes.This is due to the fact that at 25% of full sunlight intensity the migra-tion of excited states from the antennae chlorophylls to the reactioncenters exceeds the rate at which charge separation can be supportedby the photosynthetic electron transfer chain [102]. The excess ener-gy captured by the antennae chlorophylls is then dissipated as heat orfluorescence and does not do useful work. In chlorophytes and plants,most of the chlorophyll (all of the chlorophyll b) is involved in lightcapture by the LHC. Blocking chlorophyll b synthesis in green algaeprevents assembly of the LHC complex, reducing the optical cross sec-tion of the antennae and the efficiency of light capture [44,103,104].The reduction in antenna size better couples the kinetics of light cap-ture to the downstream slow photochemical processes, resulting in1.5–2× increased efficiency of CO2 fixation and growth per unit chlo-rophyll [44,105]. Algae lacking LHC complexes, however, cannotcarry out state transitions to optimize energy distribution betweenthe two photosystems and they have a reduced capacity to preventphotoinhibition due to an impaired xanthophyll cycle [103].

Inmanagedmonocultures, using algal strains engineered to have re-duced antennae size results in increased biomass productivity [103]. Tocapture all of the available solar energy, however, algae with reducedLHC optical cross sections must be grown in deeper ponds to interceptmore light. Algae with reduced antennae sizes (cbs3 mutants) do notoutcompete wild-type algae when grown in shallow (8 cm) cultures(Brad Postier, Phycal, Inc., personal communication; see description ofmutants in [103]). In mixed cultures, algae with small antennae haveno competitive advantage, because wild type algae having large ineffi-cient antennae effectively shade competing algae [102,106]. In addition,wild type algae are able to carry out photosynthesis at greater depthsin the water column, providing a greater probability of surviving inlow light environments. Thus, GM algae with reduced antennae sizedesigned to enhance growth and productivity in intensively managedmonocultures are expected to have reduced fitness in comparisonto their non-GM counterparts in complex algal communities andunmanaged environments due to reduced ability to compete for light.This again illustrates the need to co-engineer algal genetics and cultiva-tion systems, such that competitive advantages in production systemsdo not transfer to natural ecosystems. All of this should be verifiedin mesocosm experiments.

5.3. Enhanced nutrient uptake kinetics

The three most critical and costly nutrient inputs required for max-imal productivity in autotrophic algal culture systems are CO2, nitrogen(N), and phosphorus (P). Among the environmental and economic ad-vantages of algal cultivation systems are their abilities to capture CO2

from point sources, and to recycle N and P from nutrient rich, lowercost municipal sewage wastewater and feedlot runoff [75,107]. Algalmass culture should reduce CO2 emissions and wastewater N and P ef-fluent, and thereby indirectly reduce the likelihood of HABs or EDABs

associated with anthropogenic release of these nutrients into theenvironment.

GM-enhanced nutrient uptake efficiency and sequestration could,hypothetically, provide increased fitness and growth potential to GMalgae in the wild where these nutrients are often limiting. Significant-ly, enhanced nutrient uptake traits are typically not single gene traitsand if randomly distributed (unlinked) in the genome would reducethe likelihood of successful HGT of the engineered complex of traits.However, this complexity also complicates development of such aGM alga and its physiological response. N and P maximum uptakerate (Vmax) and slope of the saturation curve (Vmax/Km) allometricallyscale with cell/organism size, i.e. SA:V ratio and concomitant bound-ary layer thickness [108,109]. Swimming can produce sufficientshear to partially reduce this boundary layer [110]. Evolutionarily,algal cells have reached an optimized balance between maximalgrowth rate and the ability to obtain nutrients, which is determinedby external factors including nutrient concentration, temporal avail-ability [83], and turbulence, as well as biotic factors such as cell sizeand shape, motility, and the density of cell surface nutrient trans-porters and their specific reaction rates [110–112]. Moreover, thesefactors have tradeoffs with grazing susceptibility [113]. Without al-tering the size or mobility of the GM alga, it is difficult to envisionany genetic modification that would significantly improve nutrientuptake kinetics of the cell to confer a growth advantage in cultivationand simultaneously restrict its ability to thrive upon escape in thewild. A more effective strategy would be to optimize nutrient supplyregime in the cultivation system to favor the cultivated (GM) alga, ineffect making it an EDAB analog under the cultivation conditions only,recognizing that nutrient uptake traits alone are unlikely to ensure amonoculture long-term (see Section 4). Given these constraints, itfollows that nutrient uptake characteristics of mass cultured GM orwild type algae, accidentally released to natural surface waters, areunlikely to confer a competitive advantage over native algae with di-verse nutrient uptake characteristics. The larger challenge probablywill be to maintain nutrient competitiveness of the GM alga in culturewith contaminating algae.

Mesocosm experiments in a contained environment are essential toassess the effects of increased nutrient uptake and sequestration poten-tial on competitive fitness and growth rates, and thereby potential eco-system level impacts. As discussed in Section 6, the feasibility andefficacy of physically linking enhanced fitness and growth traits withbiocontainment traits will need to be assessed as a potential mitigationstrategy.

5.4. Achieving commercial scale production: risks associated with theneed to address losses due to herbivory, competitors and pathogens

Both open and closed mass cultivation systems will prove chal-lenging in that they will require some method of preventing thealga of interest from being outcompeted by other organisms as theyare inevitably introduced. This is particularly true of open pondsystems, which receive a continual input of competing organismsthrough vectors such as wind, rain, birds and insects. Competitorsare minimized in terrestrial agriculture by preparing fields to initiallyeliminate competitors, followed by application of herbicides/pesticides/fungicides to resistant crops, or the use of organic methods to minimizelosses toweeds, insects and various pathogens. The use of prepared fieldsalso makes it possible to control the application of nutrients, herbicide,pesticides and water to favor the growth of the desired crop plant overcompetitors. Given that algae are subject to analogous losses due tocompetitors, grazers and pathogens as terrestrial plants, similar manage-ment strategies must be applied if commercial scale production of algalbiofuels is to become a reality.

Terrestrial agricultural researchers have several hundred years ofexperience to draw upon when engineering large-scale productionoperations. In contrast, we have virtually no experience with

74 W.J. Henley et al. / Algal Research 2 (2013) 66–77

managing and stabilizing mass cultures of algae. For example, dis-ease resistance is one aspect of modern terrestrial agriculture thatmust be considered for both pond and closed algal biofuel systems.Plant breeders are often only one or two steps ahead of pathogens indeveloping sufficient disease resistance to prevent major crop losses[114]. Sometimes this effort fails and a breakthrough pathogencauses large-scale crop loss. Algae suffer from analogous viral[115,116] and bacterial [117] infections, thus selecting or engineeringfor disease resistance in mass cultured algae will be crucial for the suc-cess of the industry.

Based on ecological studies, we predict that stable production ofmicroalgae in ponds can fail unless the production system is engineerednot only to maximize biofuel feedstock production, but also tooutcompete invading “weedy” algae, to minimize selection against de-sirable traits [43], and to minimize losses to grazers and pathogens[118,119]. Though introduction of competing organisms can be bettercontrolled in closed systems, it will be virtually impossible to perma-nently eliminate all competitors, grazers and pathogens, so thesesystems are subject to the same types of potential loss factors. Fortu-nately, both biological and mechanical engineering strategies can beused to control potentially harmful biocontaminants. For example,the use of zooplanktivorous fish in trophic cascades [76], circulatingpumps that shear rotifers, nutrient pulsing technologies to controlweedy algae [83], and the application of other transiently applied bio-control agents (herbicides) can be used to reduce biocontaminant loads.

To date, little effort has focused on ecosystem-based or GM technol-ogies to control biocontaminants, herbivores or pathogens. Significantly,technologies that are dependent on the addition of a xenobiotic tocontrol weedy algae (e.g., herbicide), pathogens (e.g., anti-microbialpeptides), or herbivores (e.g., pesticide) in the pond are potentiallyattractive because resistance mechanisms to these xenobiotics are un-likely to enhance fitness or growth in the wild [120], although this pos-sibility cannot be ruled out. Ultimately this hypothesis must be testedempirically, initially by the use of mesocosms in contained environ-ments. In addition, the degree to which the feedstock compounds pro-duced by the GM algae could serve the dual function of inhibitingcompetitors or grazers in mass culture and/or in natural ecosystemshas yet to be determined.

6. Recommendations on containment options

Given the high likelihood of escape of GM algae, it is recommendedthat biocontainment strategies be engineered into production systemsthat both reduce thefitness of GMalgae and the likelihoodofHGT. Ideally,multiple containment strategies should be stacked in GM algae to reduceor prevent their potential for weedy growth in the wild. Potential GMbiocontrol strategies include introduction of traits or mutations thatconfer: (1) reduced growth fitness in the wild, (2) conditional lethality,and (3) impaired ability to transfer genes sexually or asexually. Each ofthese reduces the risk that escaping GM algae could occupy any emptyniche that may exist in situ. One potential strategy to reduce fitness inthe wild is reduction of the photosynthetic light harvesting antennaesize to reduce the ability to compete for light with wild algae(Section 5.2). Another strategy is elimination of inorganic bicarbonatepumps to reduce photosynthetic CO2-fixation efficiency at atmosphericCO2 concentrations, but allow normal growth in CO2-enriched ponds. Itmay be feasible to inactivate nitrate reductionpathways to induce depen-dence on reduced nitrogen sources for growth.We believe, however, thatthis would not be simultaneously effective tomaintain GM algal compet-itiveness in cultivation andpreclude survival in nature, for the reasonswediscuss in Section 5.3. Moreover, at least one EDAB species, Aureoumbralagunensis, dominates despite being incapable of utilizing nitrate [121].

Ideally, gene knockouts or gene deletion technologies should beused tominimize potential genetic complementation and provide effec-tive reduced fitness traits. Additional GM biocontainment strategies in-clude conditionally expressed lethality traits that would disrupt central

metabolism if expressed. Lethality traits could be expressed under thecontrol of gene switches that are repressed by xenobiotics not foundin nature, but which would be applied to ponds. Examples include con-ditional repression of engineered algae co-expressing barnase, whichdegrades cellular RNA, and barstar, which inhibits barnase, expressedunder control of a gene switch [122]. Significantly, barstar/barnasewould be unlikely to confer a selective advantage to HGT recipient or-ganisms. To reduce further the possibility of HGT, multiple containmentstrategies should be stacked. Knocking out essential genes for geneticrecombination, mating, or meiosis in sexual algae would reduce thelikelihood ofHGT towild relatives. Using split gene or split protein tech-nologies for the expression of transgenes would also reduce the likeli-hood of HGT of an intact functional gene [123]. The incorporation ofselenocysteine encoding amber codons into essential genes of algaethat can make selenoproteins (e.g., Chlamydomonas) could preventtransgene expression in unrelated organisms [124].

Finally, building physical barriers or berms around cultivationareas could minimize the risk of catastrophic release of large volumesof algae into the environment. These spill containment strategiescould be complemented by the addition of low toxicity biocidesshould a spill occur, to reduce the potential impact of an uncontrolledrelease of GM algae into nearby waterways.

7. Conclusions and recommendations

We have presented examples of some likely GM algal traits formass cultivation, conducted “thought experiments” to discern theprobable ecological risks, and suggested problem formulation ques-tions to guide development of rigorous risk assessment protocolsfor GM algae that are relevant to regulatory compliance. Prior toimplementing mass culture of GM algae, the scientific community,in consultation with regulatory entities, must (1) develop criteriafor realistic open mesocosm experimental testing of hypothesesconcerning potential ecological impacts of GM algae [21], to testour general prediction of low but nonzero ecological risks that pre-sage potential economic or human health impacts; and (2) adaptthese general testing protocols to specific GM algal traits and ecolog-ical contexts relevant to the sites of mass cultivation. As with GMcrops, however, monitoring and adaptive management may be re-quired during commercial scale-up of GM algal cultivation [25]. GMalgae may be detectable in situ by unique intrinsic DNA sequencesand/or inserted genetic markers [16]. Other traits might enable effi-cient automated detection, for example unique fluorescent tags de-tectable by flow cytometry in flow-through or discrete samples ofnatural surface waters adjacent to cultivation facilities. This wouldallow tracking by flow cytometry of significant quantities of escapingGM algae and potentially the HGT-dependent appearance of GM traitsin wild algae of receiving waters. Monitoring is essential to distinguishcommunity/ecosystem impacts of GM algae from effects of other HAB,climate change [125,126], eutrophication, and other anthropogenicstress. Ultimately, the question will come down to a risk–benefit analy-sis. The potential benefits for carbon capture and nutrient recycling, aswell as sustainable fuel production, need to be weighed against the po-tential risks. Ecologically-minded GM design is also essential, as advo-cated by the Ecological Society of America: “sterility, reduced fitness,inducible rather than constitutive gene expression, and the absence ofundesirable selectable markers” [25]. The scientific community shouldinsist on ecological monitoring and research concerning mass culturedGM algae [19,21], even in the absence of relevant regulations.

Acknowledgments

WJH and LN received support from the Oklahoma Center for theAdvancement of Science and Technology, grant PSB11-013. RWL re-ceived support from NOAA program funds. RTS received supportfrom the U.S. Department of Energy, National Alliance for Advanced

75W.J. Henley et al. / Algal Research 2 (2013) 66–77

Biofuels and Bioproducts Consortium, and from the Los Alamos Na-tional Laboratory, Laboratory Directed Research Program. The manu-script was greatly improved by the constructive criticisms of the threeanonymous reviewers.

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