The responsible production of genetically modified (GM) microalgae and its appropriate regulation in many ways parallels the previous emergence of GM crops utilised in terrestrial crop-based systems. GM crops have been in field testing for approximately three decades now and with their global scale now approaching almost 200 million hectares, their benefits have been demonstrated, although they have been beset by much controversy, and there are also some cautionary lessons learned. There are some important distinctions between the two forms of production (i. e. aquatic versus terrestrial), and microalgae systems are generally capable of much greater containment than conventional cropping systems. In order to preface this discussion, it is important to first examine the current issues with wild (non — GM) algae, both in the environment and in commercial production systems, and the current state of regulatory oversight.

Wild Algae in Aquatic Ecosystems ‘Toxic algae blooms’ are a regular headline in the mainstream media resulting in a public perception that algae are a menace. In water treatment industries, this fear of algal toxins is also relatively well established. In reality, the number of algae that produce any toxins is a tiny fraction of the existing biodiversity. Almost all of the known toxins attributed to algae are actually found in certain types of cyanobacteria and dinoflagellates, with a much smaller representation from some bacillariophytes (diatoms), haptophytes, pelagophytes and euglenoids. In some cases, there are groups who are cultivating specific species, e. g. dinoflagellates, to utilise toxic compounds for applications such as biomedical cytotoxins (generally under laboratory conditions), but this is the exception rather than the rule, and the overwhelming majority of the industry is focused upon avoiding toxic species. For example, the cultivation of the cyanobacteria Arthro — spira (Spirulina) for human food consumption must be free from the cyanobacteria Microcystis.

Environmental algal blooms, while an ongoing concern, are usually the result of anthropogenic nutrient outflows or natural processes of nutrient cycling. They are not generally the result of well-managed microalgae farming practices. Such blooms can occur during periods of elevated nutrient levels due to either natural processes (e. g. weather effected nutrient run-off from land or oceanic currents and upwellings) or from anthropogenic nutrients (e. g. municipal, agricultural or industrial waste waters), with the latter being more closely correlated with the increase in the frequency and the intensity of environmental algal blooms.

Algal blooms can be broadly divided into classes as (1) blooms that are transient and innocuous (2) both transient and persistent blooms that are generally considered to be harmful, and (3) blooms that are clearly detrimental and disruptive to eco­systems. As the algae themselves are by and large ubiquitously present, the primary underlying issue is the management of nutrients and eutrophication processes. While innocuous algae blooms are generally rapidly consumed by organisms higher up the food web (e. g. plankton and filter feeders) and are therefore transient, harmful algal blooms (HABs) (Anderson 2009; Anderson et al. 2002; Van Dolah et al. 2001) and ecosystem disruptive algal blooms (EDABs) (Sunda et al. 2006) can be comprised of algal species that are generally unpalatable to aquatic herbi­vores or that contain toxins. This is important because it disrupts the food web and the concordant transition of nutrition and chemical energy to higher trophic levels which can result in a loss of ecosystem biodiversity (with ecosystem biodiversity being closely correlated with ecosystem resilience). Historically, these problems are largely caused by agricultural nutrient outflows, and there has been significant analysis of how outflows of nutrients and chemicals from agricultural production can vary greatly in their ‘pollution footprint’, e. g. (Hill et al. 2006). The potential for reducing the pollution footprint is one of the strong benefits of microalgal production systems (Smith et al. 2010) in that they generally have no chemical outflows, and due to greater containment relative to fields of crops in soil, they can have much lower nutrient outflow, and in some cases a negative footprint where they utilise anthropogenic nutrients from other systems, e. g. wastewater integration and bioremediation systems. Nevertheless, forward thinking risk management strategies are needed to ensure that microalgae production systems at very large scale do not induce similar concerns to those experienced in traditional agriculture.

Proper Management of Microalgal Production Systems Proper management of microalgal systems is an important aspect of any commercial operation. This will be increasingly important as systems are scaled for large-scale production and the varieties of engineered strains used increase. The establishment of production models aiming to exploit the benefits of GM microalgae contributes additional complexity to prudent regulatory frameworks. There is a duality to the responsible management of GM microalgae production systems in that (1) from a product perspective, farmed microalgae cultivations must be maintained at adequate purity and free from contaminants that can compromise product quality (e. g. in the Ar — throspira example given above), and (2) from an environmental perspective, the release of nutrients or microalgal biomass must be properly managed in order to mitigate any risk to local ecosystems. Given that for the production of biocrude, biomass will be subjected to thermochemical processing, it is the latter point which is central to this discussion.

Both the type of release (nutrients or biomass) and the scale of release are important parameters in a proper risk assessment. Gressel et al. (2013) have added to the discussion on mitigating spills and propose that spills from large-scale cul­tivations will be inevitable—however, there is an important consideration here regarding the terminology moving forward in this discussion. We expect that implementation of proper standards in prudent farm management should be able to mitigate the chances of large-scale spills into the environment; however, it is widely agreed that microalgae have a relatively high capacity for dispersion (e. g. micro­scopic size, and potential to form aerosols). Thus, if some aerosolised cells escape to the environment, it is certainly a release, but is this considered a spill? In terms of nutrients, the scale and/or persistence of release is generally the most important variable in terms of subsequent eutrophication potential and the corresponding risk assessment, but in terms of biomass, a single cell escaping as an aerosol particle has the potential to establish itself outside of the farm boundary even if there is no ‘spill’. Thus, in this respect, species release is indeed inevitable, and it is in this context that any discussion of GM strains must be conducted. Hence, if small-scale release cannot ultimately be avoided, then the discussion is inexorably dependent upon the biological character of what is released.

GM Microalgae and Their Regulation Considering the inevitability of release, risk assessments of GM microalgae must be conducted on a case-by-case basis, with specific attention to the nature of the modification and whether it actually conveys a competitive advantage of some kind to the strain in question when it is relocated within a natural ecosystem or whether the modification can result in disruption to ecosystems in some other way. Henley et al. (2013) have recently published an excellent examination of GM algae risk assessment which should serve well as a foundation study for this evolving discussion. They rightly stipulate that for a GM-specific environmental risk assessment, primary considerations of potential ecological impact include the following:

1. The potential of GM microalgae to be more highly competitive in natural ecosystems.

2. The potential of GM microalgae to result in altered communities of aquatic herbivores in terms of composition, dominance or biodiversity.

3. The potential of GM microalgae to be involved in horizontal gene transfer (HGT) to other micro-organisms.

Given that it is anticipated that, for the most part, new algae producers will not be cultivating microalgal species that are invasive or toxic—the primary consid­erations will be the genetic modifications themselves rather than the host strains (indeed popular host strains such as C. reinhardtii are quite easily outcompeted by many wild-type species); however, it has previously been seen that some potential production candidates have already been involved in bloom events that have resulted in their classification as EDABs (Sunda et al. 2006). Thus, we encourage a careful and iterative investigation of all aspects of microalgae production, but emphasise that in this discussion it is the specific genetic modifications relevant to high-density microalgae cultivations that is in need of far greater discussion in the literature. Consequently, we discuss here the implications of the engineering applications highlighted in section two, with respect to associated risk of species establishment, dominance and ecosystem disruption. Much can be gleaned from the parallels with GM crop species, especially pollen dispersal; however, there are distinct differences between terrestrial crops and communities of aquatic micro­organisms.

For microalgal strains engineered to have varied light-harvesting and photo­synthetic efficiency, the general desire is to increase net biomass productivity. As discussed above, this can be achieved through different methods. The down-regu­lation of LHC proteins or pigmentation can provide an overall net benefit to high- density cultures in high-light conditions (i. e. the artificial farming environment), e. g. (Oey et al. 2013); however, this generally makes individual cells less com­petitive in natural ecosystems where competing wild-type cells retain the capacity to modulate their antennae size and pigmentation levels. In theory though, genetic modifications that unilaterally increase total productivity (e. g. a higher efficiency rubisco enzyme or strains that can utilise a wider range of the spectrum) could potentially convey a competitive advantage irrespective of the growth environment.

Where strains are photosynthetically superior irrespective of environment, they could potentially affect ecosystem dominance and diversity, and while microalgae composition might not be significantly changed, the increased availability of these microalgae could result in additional effects like changes in plankton composition. In contrast, LHC/pigment-reduced cells could lead to some immediate composi­tional changes when consumed, but this would be insubstantial at the community level, and as these strains are outcompeted by wild-type organisms, there would be no net change to dominance or biodiversity. Again, the real concern for HGT would be that microalgae with superior generic photosynthetic efficiency would be capable of transferring this trait to other phototrophic organisms enabling them to also have greater competitiveness in the natural ecosystem. The transfer of disabled antennae/ pigment modulation would not convey an advantage to other species.

GM strains that have a greater capacity for nutrient scavenging may have an increased competitiveness if released, but there is already a diverse range of strategies for nutrient uptake and usage among naturally occurring algae (Henley et al. 2013). Thus, while the predicted risk for these modifications is considered to be low, there has not yet been sufficient data from field trials to properly draw a conclusion.

Metabolic engineering is intended to alter the composition of microalgal bio­mass. While for biocrude producing systems, this will ideally result in strains that have higher overall carbon content, and it is not producing strains outside of the range of what occurs in nature. Nevertheless, if the available proportion of the population containing high carbon (i. e. abundance of GM microalgae relative to wild-type microalgae) is shifted, there is potential for an effect even if the conse­quences are low. If the nutritional value of the microalgae is altered, then this could also lead to changes in the nutritional value of plankton and filter feeders and subsequently lead up the food web to higher trophic levels. By the ecological risk assessment proposed by Henley et al. (2013), this risk is considered to be very low; however, this should be monitored in the longer term to obtain confirmation. In general, the accumulation of energy storage products in the form of reduced carbon molecules does not convey a competitive advantage to GM microalgae and it is likely that they will also be outcompeted by wild types within natural ecosystems.

GM traits that enhance the capacity of a microalga to remain dominant in the presence of predators, pathogens and competitors are varied in their approach and range from resistance to chemicals (e. g. herbicides and pesticides) to the use of allelopathy and toxins to maintain dominance. The use of chemicals is unlikely to become widespread for low-value commodity products such as biocrude due to the economic pressure it places upon business models; however, the engineering of endogenous chemicals into GM microalgae that prevent contamination is a potential risk that must be properly examined. Henley et al. (2013) propose that the risk of this approach is low to moderate depending upon whether the allelopathic chemical is naturally occurring or novel; however, we suggest that the range of potential risk assessment outcomes can be as variable as the potential allelopathic chemicals that can be engineered and that even for relatively low-level allelopathy, at the very large scales of production proposed for addressing fuel demand, even mild allelopathy could have ecosystem disruptive effects. Thus, we advise a strict examination of these strategies; though to the best of our knowledge, these strat­egies have not yet been employed. We do agree though that where traits are selected for from large populations and then elucidated and reproduced through engineering (rather than engineering of novel chemicals), the risk will be attenuated.

Other GM strategies to increase the harvestability and processability of micro­algae are unlikely to affect their dominance in natural systems, and the risk for these traits is considered very low. Similarly, where protein expression is used to create a primary revenue stream from a high-value product before HTL of residual biomass, these strains are unlikely to compete in natural systems due to diversion of much of their energy flow towards a product that is not useful to the microalga.

The theoretical risk assessment discussed here and that presented by Henley et al. (2013) can be quite informative, relying on an analysis of whether similar traits are already part of the ecosystem. However, a physical risk assessment strategy will be more convincing where laboratory-scale simulated ecosystems are developed from natural water bodies and the long-term survivability of GMO algae in mixed culture can be evaluated, e. g. by PCR.

11.4 Conclusion

The commercially profitable production of algal biocrude, at scale, will represent the culmination of a long and parallel development of algal agronomy, biology, GM, bioreactor engineering, harvesting and chemical conversion processes and the development of suitable sensors and control systems, along with their associated modelling and control software. No one innovation will suffice to overcome the formidable challenges faced by this nascent industry, and no actor will have ownership of all the important intellectual property. Since the most significant competitive challenges are between algal technologies and other fuel systems and secondary markets, the field of algal biotechnology stands to benefit greatly from relative openness of sharing data, technology and experience. This suggests that the modified algal strains used for biocrude production in the future will be heavily modified fuel factories equipped with streamlined metabolism, externally control­lable cellular programs, and both sensors and reporting systems for monitoring the state of the system. Biocrude production appears, at this stage, to offer one of the most promising production pathways for algal biofuel production, and genetic manipulation offers a powerful tool for fine-tuning microalgal biofuel production all the way along the development pathway.

Acknowledgments The authors would like to acknowledge support from Australian federal research grants: NHMRC Project Grant APP1074296, ARC Project Grant DP130100346 and the Queensland State Government NIRAP Grant High Efficiency Microalgal Biofuel Systems.