Fourth generation biofuel: A review on risks and mitigation strategies

Renewable and Sustainable Energy Reviews 107 (2019) 37–50

Contents lists available at ScienceDirect

Renewable and Sustainable Energy Reviews journal homepage: www.elsevier.com/locate/rser

Fourth generation biofuel: A review on risks and mitigation strategies a

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d

Bawadi Abdullah , Syed Anuar Faua’ad Syed Muhammad , Zahra Shokravi , Shahrul Ismail , ⁎ Khairul Anuar Kassime, Azmi Nik Mahmoodb, Md Maniruzzaman A. Azize,

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Chemical Engineering Department, Universiti Teknologi PETRONAS, Bandar Seri Iskandar, 32610 Tronoh, Perak, Malaysia School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, UTM Skudai, 81310 Skudai, Johor, Malaysia Department of Microbiology, Faculty of Basic Science, Islamic Azad University, Science and Research Branch of Tehran, Arak, Iran d School of Ocean Engineering, Universiti Malaysia Terengganu, 21030 Terengganu, Malaysia e School of Civil Engineering, Universiti Teknologi Malaysia, UTM Skudai, 81310 Skudai, Johor, Malaysia b c

ARTICLE INFO

ABSTRACT

Keywords: Fourth generation biofuel Microalgae Genetic Legislation Water footprint Disposal

Fourth generation biofuel (FGB) uses genetically modified (GM) algae to enhance biofuel production. Although GM algae biofuel is a well-known alternative to fossil fuels, the potential environmental and health-related risks are still of great concern. An evaluation of these concerns and accordingly devising appropriate mitigation strategies to deal with them are important to a successful commercialized production of FGB. While extensive research has been carried out on genetic modification and other technologies that aim to increase the productivity of algae strains, only a handful of them deal with the legislative limitations imposed on exploiting and processing GM algae. This paper examines this legislation and the mitigation strategies to meet potential risks associated with the exploitation and processing of FGB. Open-pond system is an economic solution for largescale cultivation of microalgae; however, the concern regarding the health and environmental risk of cultivating GM algae and the associated stringent regulations is considered as the main barrier of FGB production. Disposal of the residue is another important issue that should be considered in FGB production. The byproducts obtained from energy extraction step and residual water from the harvesting process may contain plasmid or chromosomal DNA that may cause the risk of lateral gene transfer. Hence an appropriate mitigation practices should be used for replacement of the hazardous water residue and by-products with more environmentally friendly alternatives. The results obtained from several field testing projects for open-environment exploitation of GM algae show that under the various conditions used, there was no apparent proof to support possible horizontal gene transfer in release of GM algae.

1. Introduction Consumption of fossil fuels is a major source of greenhouse gas (GHG) emissions. Biofuel is a promising alternative to meet the needs for a clean energy source in the transportation, power-generation and heating sectors. Biofuel can be used as a substitute for fossil fuels or can be blended with fossil fuels [1]. The GHG emissions resulting from biofuel combustion are much lower than those produced by conventional fuels. Hence, biofuel can be used to promote climate-change mitigation [2,3]. Based on the feedstock, biofuels fall into four groups: first, second, third, and fourth generation biofuels (FGBs) [4]. Of these, first and second generation biofuels are the only options which are commercially produced (see Fig. 1). Large-scale commercialized production of third generation biofuel and FGB is not carried out yet due to the insufficient

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biomass production, high production cost as well as environmental and health concerns [5]. First generation biofuels are made from oil-based plants as well as sugar and starch crops. Production of genetically modified (GM) crops has been continuously increasing from the time it was started in 1996. Genetically modified soybeans, maize and rapeseeds biomass occupies 73·3 × 1013, 46·8 × 1013 and 7 × 1013 m2 of the global area [6]. The usefulness of first generation biofuels has been increasingly questioned due to concerns about competition for arable lands and raw materials [7,8]. To address these concerns, second generation biofuels have been introduced. These biofuels are derived from non-food crops. Second generation biofuel is made mostly from agriculture and forestry residues. However, the production of second generation biofuel could become unsustainable if competition for available land were to arise [9,10]. Third generation biofuels, which are derived from algae, have

Corresponding author. E-mail address: [email protected] (M.M.A. Aziz).

https://doi.org/10.1016/j.rser.2019.02.018 Received 22 November 2018; Received in revised form 1 February 2019; Accepted 18 February 2019 1364-0321/ © 2019 Elsevier Ltd. All rights reserved.

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B. Abdullah, et al.

Nomenclature

FGB GFP GHG GM GMO pDNA TAGs TALEN TSCA USDA UV WF ZFN

AD Anaerobic digestion CRISPR/Cas9 Clustered regularly interspaced palindromic sequences DGAT Diacylglycerol acyltransferse DHA Docosahexaenoic acid DIR Dealings involving release DNIR Dealings not involving release DPA Docosapentaenoic acid EPA Environmental Protection Agency EU European Union FAO Food and Agriculture Organization

Fourth generation biofuel Fluorescence protein Greenhouse gases Genetically modifies Genetically modifies organism Plasmid DNA Triglycerides Transcription activator-like effector nucleases Toxic Substances Control Act US Department of Agriculture Ultraviolet Water footprint Zinc-finger nuclease

Fig. 1. Commercial production of biofuel based on generation [11].

attracted enormous attention due to their high yield, assimilation of carbon dioxide (CO2), and relatively simple processing. Algae can be cultivated in wastewater and seawater as well as in unproductive drylands and marginal farmlands. Thus, they do not compete with food crops on arable land or in freshwater environments [12,13]. FGB is sourced from GM algae biomasses to achieve enhanced biofuel production. Improving photosynthetic efficiency, increasing light penetration, and reducing photoinhibition are common strategies used in the genetic modification of microalgae [14]. One of the solutions for enhancing light penetration into dense microalgae cultures is to use the truncation chlorophyll antenna of chloroplast [15]. The photosynthesis efficiency of GM microalgae can be improved by the expansion of the absorbing spectrum range of microalgae in photosynthesis [16]. Enhancing the penetration of light into microalgae culture by reducing the size of the chlorophyll antenna [17,18] as well as minimizing the light absorption and manipulation of the pigments [19,20] are also of the main strategies used for genetically modification of microalgae. Additionally, the metabolic engineering of microalgae can lead to significant increases in lipid or carbohydrate content [21]. Lipid and carbohydrate maximization are among the most attractive factors that can enhance the efficiency of the yield of a microalgae biomass [22]. The advantages and disadvantages of different biofuel generations as well as the comparison among them are presented in Table 1. Microalgae are a large group of eukaryotes and cyanobacteria with a high adaptability to extreme environmental conditions, including salinity, drought, photo-oxidation, osmotic pressure, temperature anaerobiosis, and ultraviolet (UV) radiation [36]. Nitrogen and phosphorus are the main nutrients in microalgae. accounting for 10–20% of its biomass. Algae possess a wide range of strategies for composition, food reservation, photosynthetic pigments, cell wall chemistry, and reproduction [37]. The available microalgae are not equally important in

terms of their potential use in biofuel production. Bacillariophyceae (diatoms), Eustigmatophyte, Chlorophyceae and, Chrysophyceae microalgae classes have been identified as the most appropriate for biofuel production. As such, most of the research in this field has been devoted to microalgae of these classes [38,39]. Production of biofuel from genetic engineering of algae is discussed under the fourth generation biofuel term. The biomass supply in fourth generation biofuel comes from microalgae, macroalgae and cyanobacteria. Microalgae and macroalgae are eukaryotes – i.e. containing a nucleus surrounded by a membrane – belonging to kingdom Protista. Cyanobacteria are prokaryotes – i.e. lacking membrane-bound organelles – belonging to the kingdom bacteria that is referred to as algae in this study [40]. Table 2 lists the most researched microalgae strains in the field of biofuel production based on their lipid, protein, and carbohydrate contents, as well as their attributed classifications. Bacillariophyceae, also known as diatoms, are mostly unicellular with silicate cell walls which have carbohydrates and triglycerides (TAGs) storage compounds. Chrysophycea are golden-brown unicellular algae that inhabit both freshwater and marine environments. Similarly to diatoms, Chrysophyceae’s main storage compounds are oils and carbohydrates. Eustigmatophytes are yellow-green unicellular eukaryotic algae that have promising potential to be used in biofuel production. Eustigmatophytes have mostly small cell sizes and high amounts of essential fatty acids. Chlorophyceae is a major group of green algae with rigid cell walls. Most of the Chlorophyceae algae contain proteins and starch as storage compounds [38]. The cultivation of GM microalgae can be carried out in contained and uncontained systems. However, the challenges created by each of these methods are significantly different from each other. The contained cultivation system has a more tightly controlled condition, while contamination and environmental exposure are minimized. Despite offering better protection, the capital expense of the contained cultivation system is high. 38

Potable water is required for cultivation Using pesticides and fertilizers are of the main concerns

Water footprint

39

The capital cost is fairly low Parameters such as temperature and humidity must be within a suitable range

Financial input Environmental condition

Regulation

Harvesting is done by hand or machine picking The regulations are fairly clear

Harvesting

Nutrient requirements

Commercialization Sustainability

Commercially produced Is not conservative in use of natural resources such as water and land Using pesticides and fertilizers are of the main concerns

Made from edible oil and starch feedstock Requires arable land Easy conversion

Competition with food crops Land footprint Conversion to biofuels

Environment-friendliness

First generation biofuel

Topic

The capital cost is fairly low Parameters such as temperature and humidity must be within a suitable range

Harvesting is done by hand or machine picking The regulations are fairly clear

Commercially produced Do not preserve ecology due to deforestation concerns No need for any fertilizer treatment

No expenditure on fertilizer, or pesticides; however deforestation is a concern

Non-arable land can be used for cultivation Easy conversion due to increased hydrolysis and/or fermentation efficiency

Require arable land or forests Need sophisticated downstream processing technologies due to high contents of hemicelluloses and lignin Potable water is required for cultivation

The initial cost for large scale cultivation is too high Can be cultivated in harsh environmental condition such high pH, salinity high light intensities

Large carbon and nitrogen sources are required. Solar energy is only available at day time. Nutrients can be recycled in the process Harvesting of microalgae is expensive, and complicated No regulation is available for marine cultivation

CO2 fixation, waste water treatment, no expenditure on fertilizer are pros and ecological concerns such as marine eutrophication cons Insufficient biomass production for commercialization Do not have favorable economics

Waste, saline and non-potable water also can be used

No food-energy conflict

Third generation biofuel

No food-energy conflict

Second generation biofuel

Table 1 Advantage and disadvantage of each biofuel generation with comparison.

Medium (CO2 fixation, waste water treatment, are of the pros but release of GM organisms, is the main concern Insufficient biomass production for commercialization There are concern about leaking of GMO to environment and ecological risks Large carbon and nitrogen sources are required. Solar energy is only available at day time. Nutrients can be recycled in the process Harvesting of microalgae is expensive, and complicated No regulation is available for marine cultivation furthermore strict regulation are for the intended release of GM algae The initial cost for large scale cultivation is too high Can be cultivated in harsh environmental condition such high pH, salinity high light intensities

Waste, saline and non-potable water also can be used

Non-arable land can be used for cultivation Easy conversion due to increased hydrolysis and/or fermentation efficiency

No food-energy conflict

Fourth generation biofuel

[5] [34,35]

[5,33]

[32]

[31]

[5] [28–30]

[27]

[25,26]

[23] [24]

[5]

Reference

B. Abdullah, et al.

Renewable and Sustainable Energy Reviews 107 (2019) 37–50

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B. Abdullah, et al.

Table 2 Properties of the most widely used microalgae in algal biofuel production [38,39]. Class Eustigmatophyceae

Chlorophyceae

Bacillariophyceae Cyanophyceae

Microalgae strain

Lipids (%)

Proteins (%)

Carbohydrates (%)

Chlorella Chlorella Chlorella Chlorella Chlorella

41–58 22–24 2 40–60 14–57

51–58 40.5 57 10–28 47.89

12–17 26.8 26 11–15 8.06

Botryococcus braunii Scenedesmus obliquus Dunaliella tertiolecta Dunaliella salina Scenedesmus dimorphus Tetraselmis suecica Haematococcus pluvialis Scenedesmus quadricauda

25 30–50 11–16 6–25 16–40 15–23 25 1.9

– 10–45 20–29 57 8–18 – – 40–47

– 20–40 12.2–14 32 21–52 – – 12

Phaeodactylum tricornutum Thalassiosira pseudonana

18–57 20

30 –

8.4 –

Spirulina platensis

4–9

46–63

8–14

vulgaris sorokiniana pyrenoidosa protothecoides minutissima

Fig. 2. Schematic of the FGB production and the related mitigation strategies for disposal of the GM microorganisms.

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Uncontained systems are generally raceway ponds. Uncontained cultivation systems have lower operating costs than contained systems, but the potential for the GM algae to be released from these systems is much higher, as uncontained systems are prone to leakage, animal interference, and aerosol dispersal [41]. Other concerns, such as diffusion into the environment and user safety, have caused much of the research to have remained within research laboratories. Hence, the exploitation of GM microalgae requires a stringent assessment of the associated risks and the proper management of environmental impacts [42]. Much research has considered the environmental advantages of using GM algae – such as CO2 sequestration and assimilation [43,44], wastewater treatment by heavy metal bioremediation [13,45], and GHG emission reduction [46]. Other articles have considered the environmental and health risks associated with GM algal biofuel. Mazard et al. [47] reviewed the cyanobacteria’s metabolism and discussed the environmental impact of cyanobacteria in the formation of coastal blooms as well as toxin production. The concerns about the potential environmental harm caused by genetically engineered algae and cyanobacteria, along with the potential horizontal gene transfer between unrelated microbes, have been addressed by Snow and Smith [48]. Savakis et al. [49] introduced a method for direct biofuel production from cyanobacteria. Fig. 2 shows a Schematic of the FGB production and the related mitigation strategies for disposal of the GM microorganisms. Hewett et al. [50] analyzed the human health and environmental risks associated with synthetic biology in energy applications. This review was based on risk-related literature and interviews with the biosafety professionals. Szyjka et al. [51] evaluated the ecological risks related to the open-pond cultivation of genetically engineered algae, stating that the uncontained cultivation of GM microalgae does not cause adverse effects on the environment or the surrounding native algae population in the experimental period. Henley et al. [52] conducted a risk analysis of GM microalgae in the cultivation stage for large-scale biofuel production. Table 3 presents a summary of the literature on the environmental and health risks associated with FGB. Hence, a complete study of the production-related aspects that contribute to the growth, water management, and disposal of FGB is needed. As such, the authors have set the following objectives:

secretion, lipid and carbohydrate metabolism, improved nutrient use efficiency, hydrogen production, improved photosynthesis efficiency, higher stress tolerance, enhanced cell disintegration, and bioflocculation [63–65]. These mechanisms can significantly improve the production of algal biofuels. However, genetic modification cannot be applied to all algae species mostly due to a lack of available genomic data, the complexity of transgenesis, and difficulties establishing a competence equilibrium between metabolic and energy storage pathways. Enhancement of productivity and lipid accumulation is the easiest way to reduce the cost, nutrient consumption and water footprint. Genome editing methods are widely used for increase the productivity and lipid content in microalgae. Presently, three types of genome editing tools: zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced palindromic sequences (CRISPR/Cas9) [66]. The first genome editing experiment in microalgae was reported on Chlamydomonas reinhardtii using ZFN [67]. Genome editing of Phaeodactylum tricornutum (with TALEN [68] and with CRISPR/Cas9 [69]), Nannochloropsis oceanica (with CRISPR/Cas9 [70]), Chlamydomonas reinhardtii (with TALEN [71] and CRISPR/Cas9 [72]) has been successfully demonstrated. The mechanisms of genome editing have been reviewed elsewhere [61,73]. Table 4 shows the most common genetic modification methods applied for enhancement of algal biomass production in FGB. The applied genetic engineering methods for biofuel enhancement may include (1) modifying an the sequence of an existing functional gene, (2) modifying an available regulatory sequence, or (3) replacing a regulatory sequence or gene with one from another strains or organism [52]. The microalgae resulting from approaches (1) and (2) are free from any foreign DNA and could be exempted from the legislation of GMO products [82,83]. The details for the genetic modification of algae strains are beyond the scope of this paper and can be found in [84–86]. 2.1. Health and environmental concerns High-throughput genetic engineering techniques are becoming increasingly affordable and efficient, and many microalgae strains have been studied for their potential use in biofuel production. However, GM microalgae can easily invade ecosystems due to their small size, rapid growth, and enormous number. The main environmental concerns regarding the uncontained exploitation of GM algae relate to competition between the introduced microalgae and native species, changes in natural habitats, horizontal gene transfer, and toxicity [50]. Nonnative species have more potential to invade and spread in native communities. Therefore, the risk posed by GM microalgae mainly depends on their persistence against biotic and abiotic stresses in the presence of native species [51]. Thousands of algae strains have been engineered to

i. Discover the major environmental and health risks of GM algae and the related legislation ii. Examine the production-related risks that contribute to the growth, water management, and disposal of FGB iii. Suggest ways in which FGB production can meet the practical aspects of strain growth and by-product disposal The literature in the field of FGB focuses mainly on the genetic modification and strain enhancement of microalgae. Few researchers have covered the practical aspects of meeting environmental and health concerns. No paper has investigated in detail the potential risks of using GM biomass in the cultivation, harvesting, and processing steps of FGB. A few reviews have discussed the risk of genetically modified organisms (GMOs) but have failed to provide a solution to the enforced regulatory framework on the exploitation and processing of GM strains. In this study, using open-pond system as an economic solution for large-scale cultivation of GM microalgae as well as the associated risks and regulations is discussed. In addition, potential risk attributed to disposal of the byproducts obtained from energy extraction step and residual water from the harvesting process is evaluated.

Table 3 Summary of the literature on the environmental and health risks of FGB.

2. Health and environmental risk of FGB and the contributed regulations Several genetic engineering techniques have been introduced to enhance algal biomass. These enhancement strategies are mainly based on the target genes for the direct biosynthesis of biofuels, feedstock 41

Year

References

Cultivation

Harvesting

Processing

2011 2011 2012 2012 2013 2013 2014 2015 2016 2016 2016 2016 2016 2017 2017 2017 2018

Sharma et al. [29] Van Asselt et al. [53] Snow et al. [48] Menetrez [54] Wijffels et al. [55] Henley et al. [52] Usher et al. [56] Hegde et al. [57] Misra et al. [58] De Farias Silva et al. [59] Zeraatkar et al. [45] Mazard et al. [47] Ghosh et al. [60] Beacham et al. [42] Chu [61] Szyjka et al. [51] Hess et al.[62]

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✓ ✗ ✗ ✗

✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✗ ✓ ✗ ✗ ✗

Renewable and Sustainable Energy Reviews 107 (2019) 37–50

grow quickly and survive in highly unlikely environments; all risks should be addressed before these microalgae are dispersed from their open or contained cultures [12]. The risk associated with GM algae is presented in Table 5. Releasing toxic algae strains into the environment can pose severe risks to humans’ health [97]. Concerns about environmental and health risks related to GM algae strains have been raised based on examples of invasive aquatic species, such as the spread of the toxic algae Alexandrium minutum in algae blooms along the French coast, which began in 1985 [98]. Dinoflagellates are natural producers of biotoxins and can synthesize toxic compounds. The synthesis of toxic compounds is the major cause of algal blooms and the discolouration on sea surfaces. The scientific consensus is that harmful algal blooms events have been increasing globally over the last 40 years [42]. Horizontal or lateral gene transfer is defined as a mechanism by which the genetic material of one organism is transferred to another in a non-genealogical manner [99]. Cyanobacteria are important organisms for industrial applications due to their fast cell growth, high potential for genetic modification, and simple nutrient requirements. However, the growth-monitoring of cyanobacteria is a challenging task due to the potential horizontal gene transfer between different cyanobacterial taxa and between cyanobacteria and eukaryotic algae. Furthermore, some cyanobacteria can take up DNA through viral vectors [48]. It is noteworthy that the term ‘genetic modification’ is used to address genetic engineering methods that are applied to add, remove, or modify specific parts of an organism’s genome. Hence, natural replication methods, such as random mutagenesis, could be exempted from GM regulations. The outdoor cultivation of GM microalgae is crucial to the sustainability and commercialization of FGB production. However, before introducing the GM algae into the environment, risk assessments should be performed to minimize the potential environmental and safety concerns associated with the escape of GM traits from cultivation, harvesting, and processing facilities. Accordingly, regulatory frameworks have been developed in several countries to ensure that the exploitation and processing of GM algae biomass do not cause irreversible damage to the environment through competition with native species, changes in natural habitats, horizontal gene transfer, or toxicity. In the next section, some important regulations and legislation that have been developed for FGB production are presented.

[75] [76] [77] [68] [78] [79] [80] [80] [81] Targeted gene knockout and knock‑in via NHEJ in Chlamydomonas RE assay, Sequence Analysis were the detection methods P. tricornutum FcpB & FcpF promoter was used. PCR and southern blot, western blot was used for detection. PCR, Southern blotting, Western blotting, Sequence analysis were used for detection PCR around nuclease target site and sequencing is used as detection technique. RE assay, PCR, sequence analysis for mutant detection Genome editing using CRISPR/Cpf1‑based nucleases Genome editing using CRISPR/Cpf1‑based nucleases Detection was withRE assay, RT-PCR, sequence analysis, deep sequence analysis MAA7, CpSRP43, and chlM FKB12, Ku70, ALS, ARG Urease PtAureo1a UDP-glucose pyrophosphorylase Urease psbA1, nblA nblA nifH Nitrate reductase Electroporation CRISPR/Cas9 Electroporation CRISPR/Cas9 Particle bombardment TALEN Particle bombardment TALEN meganuclease Biolistic Particle bombardment CRISPR/Cas9 Conjugation CRISPR/Cpf1 Conjugation CRISPR/Cpf1 Electroporation CRISPR/Cas9 Chlamydomonas reinhardtii Chlamydomonas reinhardtii Phaeodactylum tricornutum Phaeodactylum tricornutum Phaeodactylum tricornutum T. pseudonana Synechococcus UTEX 2973 Synechocystis 6803 Anabaena 7120 Nannochloropsis oceanica IMET1

Comment Gene/marker Delivery/ Nuclease Species

Table 4 The most common genetic modification methods applied enhancement of algal biomass production in FGB [66,73,74].

Reference

B. Abdullah, et al.

2.2. Regulations on cultivation and processing of the GM algae The release of GM algae into the environment is regulated because of the possible damage it could cause to land water, protected species, and natural habitats [100]. While the intentional release of GM algae into the environment is a necessary step for introducing new products, any release of GM algae must be approved by the authorities prior to the commencement of the production of GM algae or their derivatives [101]. In most countries, clear regulations are in place for practices involving modified microorganisms that are applicable to algae without any ambiguity. Based on a ruling of the Environmental Protection Agency (EPA) of the US, any modified algal biomass is subject to the regulations under the Toxic Substances Control Act (TSCA). The authorities must be notified in advance of any commercial, importation, or outdoor research and development (R&D) of the modified microorganism. Any non-commercial R&D activities conducted under suitably contained conditions are not juried by the TSCA. The potential impact of the large-scale uncontained cultivation of modified algae on agriculture or the environment is covered by “7 CFR Part 340” of the regulations outlined by the US Department of Agriculture (USDA) [102]. The use of the modified algal organisms in a contained bioreactor is not within the scope of USDA’s regulations due to the low possibility that such practices will create any environmental hazards [52]. The regulations on aquaculture, which are governed by 42

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Table 5 The health- and environment-related risk of GM algae [50,54]. Topic

Risk contribution

References

Effect

Allergies Antibiotic resistance Carcinogens Pathogenicity or toxicity Change or depletion of the environment Competition with native species Horizontal gene transfer Pathogenicity or toxicity

Human health Human health Human health Human health Environment Environment Environment Environment

[54,87,88] [88,89] [54] [48,52,54] [90,91] [92,93] [94,95] [96]

Dermal, ingestive, respiratory exposure Reducing the effectiveness of medical treatments Carcinogenic residues Pathogenicity of some strain to human; toxic blooms; chemical transfer; toxic residues Removal of nutrients from ecosystem; reducing biodiversity of the flora and fauna Outcompete native organisms; changing aquatic ecosystems Transfer of genetic organisms Pathogenicity of some strain to human; algal blooms; generating genetic–related toxins

the Food and Agriculture Organization (FAO), are applied in many countries. Where applicable, these regulations may lead to additional requirements in the way of permits or further environmental assessments [103]. The sanction on genetic modification of the European Union (EU) mainly relies on two directives (i.e., 2009/41/EC and 2001/18/EC) that pertain to the contained utilization of GM microorganisms and the intended release of GMO into the environment, respectively [104]. Directive 2009/41/EC aims to conserve the environment and protect human health through regulations on the contained utilization of genetically manipulated algae strains. Its primary focuses are to assess and classify the risks associated with genetic engineering and to provide containment [105,106]. In contrast, the regulation for the intended release of GMOs into the environment is stipulated in Directive 2001/18/EC and applies a stepby-step risk assessment approach [107]. The biotechnology safety directive regulated by the European Union (EU) is stringent on the production, labelling, importing, and authorization of GM products. The shipment of ingredients made from GM algae or their by-products from the US to Europe is currently not allowed. Hence, these regulations have virtually eliminated the EU as a GMO market. Table 6 shows the regulatory sanctions for production of FGB. The contained and uncontained use of GM algae is subject to notifying and, in some cases, obtaining approval from a country’s federal government. Under Japan’s biotechnology regulatory regime, two types of uses for modified organisms (“Type I” and “Type II”) are introduced. The “Type I” use is applicable to the deliberate release of modified organisms, which is different from the contained “Type II” use [109]. The Australian regulation for the contained and uncontained use of modified microorganisms was implemented in 2011 under the name “Gene Technology Regulation” [111]. A distinction is made between contained and uncontained uses of microorganisms, referred to as ”dealings not involving release (DNIR)” and “dealings involving release (DIR),” respectively. Based on the Canadian Environmental Protection Act, the use of contained and uncontained modified microorganisms needs approval from the federal government [110,117]. In response to the concerns raised by the potential environmental

and health impacts of releasing modified biomass, government regulatory requirements are defined for contained and uncontained uses. The sanctions on the production and processing of these organisms are particularly stringent for both deliberate and accidental releases. The cultivation of the microalgae in open ponds has been widely practised worldwide [118,119]. However, using open-pond reactors for growing GM algae would pose additional risks in comparison with unmodified algae species. The field testing of these organisms has been proposed as a way to assess the potential risk of the growth and processing of the GM microalgae on health and the environment. Within the coordinated framework of the EPA and USDA, the implementation of field tests and commercial uncontained production’s environmental effects have been proposed [33]. In addition, the EPA has sponsored projects for the open-environment exploitation of algal biomass to assess the attributed risks [51]. Environmental and health risks as well as the public acceptance and attitude toward genetically modified products are of the main driving forces for policy makers to set national target and benchmarks of future development. Generally, the regulations on discharge of GM algae are divided into two main classes of deliberate or unintended release. Industrial use of GM algal biomass for FGB production need clear regulations without any ambiguity to deal with deliberate or unintended release. The regulation on release of GM algae for several countries is presented and their specification is discussed. The sanctions on genetic modification in some countries such as the EU are stringent and import of the GM algae or their by-products is not allowed. Some countries such as the US have less stringent criteria to accept or reject a plan of production. In the following sections, the risks associated with biomass exploitation and the processing of GM microalgal biofuel are further discussed. 3. Mitigation strategies of the FGB Because of growing objections due to the drastic previous experiences of invasive species on native biodiversity and allergic reactions to transgenic foods, a science-based and transparent assessment of GM algae is required to support or refute its potential future outdoor

Table 6 Regulatory sanctioned for production of FGB. Reference

Legislation

Topic

Glass [33] and Bergeson et al. [83] Trentacoste et al. [102], and Henley et al.[52] Glass [33] Food and Agriculture Organization [103] European Union [105,108], Yamanouchi [109], Darch et al. [110], and Tribe [111] Glass [33] USDA [112,113] Glass [33] USEPA [114] Strauss et al. [115] FDA [116] Glass [71]

EPA regulations under TSCA USDA biotechnology regulations Food and Drug Administration (FDA) regulations Aquaculture regulations International biotechnology regulation EPA regulation USDA regulations International regulation EPA regulation USDA regulation FDA regulation International regulation

Exploitation Exploitation and applicability Applicability Cultivation – Research uses Research uses Research uses Commercial uses Commercial uses Animal feed uses Commercial uses

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cultivation. There are limited studies and assessments on the large-scale outdoor cultivation of GM microalgae and its impact on the environment. An important limitation preventing the implementation of largescale experiments is the stringent laws and regulations imposed by authorities [120]. Kenny et al. [121] modelled the growth mechanism of microalgae strains in accumulation, structuring, and changes in cellular content under nutrients and light availability. Real-life datasets from a laboratory-scale bioreactor were used for validating the presented model. Using the proposed validated model, the author tested a wide range of scenarios to identify the optimal solution for industrial production. Based on the economic models, life cycle analyses and practical considerations ca. 6000 L ha−1 year−1 were advised for industrial-scale operations. The study shows that open-pond cultivation is the only choice for such massive production. According to the life cycle analyses, limitations in microalgal physiology (e.g., CO2 fixation and photosynthesis), genetic or allied biological modifications are necessary solutions for making algal biofuels economically viable. The majority of the commercially produced microalgae are cultivated in outdoor open-pond systems. Uncontained cultivation had the advantages of low operating costs and low energy requirements. Except for a few companies, such as TerraVia Holdings, Inc. or Algatechnologies Ltd., that are in contained bioreactors the rest use open-pond raceway systems [42]. The enhancement of lipid accumulation in GM algae by providing the appropriate growth condition could significantly affect the viability of biofuel production. Hamilton et al. [120] explored the effects of the culture condition on transgenic strains of Phaeodactylum tricornutum. The P. tricornutum is modified to poses higher amounts of docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). Different cultivation conditions were investigated, including the laboratory-scale growth of cultures in a flask, a bubble column bioreactor, a contained photobioreactor, and a raceway pond using artificial illumination. The transgenic strain grown in the open-pond system yielded the highest partitioning of EPA. However, the highest level of DHA was produced in the photobioreactor. Chen et al. [122] evaluated the performance of genetically engineered Scenedesmus obliquus in open-pond and photobioreactor systems. Enhanced results were achieved by using GM strains; however, a better yield was achieved by using a closed system than by using an open-pond system. Although using an outdoor pond is the best choice for algae exploitation, there is much uncertainty and risk surrounding the cultivation of GM algae in uncontained outdoor ponds due to a lack of knowledge about the potential process performance. To better understand the performance of GM algae in outdoor cultivation, the EPA conducted an experiment in which genetically engineered Acutodesmus dimorphus was grown within an open-pond system over a period of fifty days [51]. In this experiment, dispersal traps were arranged within a defined distance from the cultivation ponds to prevent the GM algae from migrating into nearby natural water bodies. The dispersal traps were filled with freshwater containing algae growth media as a supplementary source. The samples taken from five local lakes showed that the effect of the GM algae on the biodiversity and species composition of the pond was negligible; the introduced microalgae were not able to outcompete native strains. Mutant escape can pose a serious threat to a local ecosystem’s biodiversity, potentially increasing the risk of the creation of an algal bloom. The invasion of a wild type of microalgae into a cultivation pond can impair its productivity [123]. De Mooij et al. [124] simulated an outdoor mass culture of modified Chlorella sorokiniana algae to investigate the competition between the wild and mutant types. The literature-derived simulation model of the wild type and modified Chlorella sorokiniana mutants in open-pond culture demonstrated a good productivity potential of the mutant type. However, in a competitive environment contaminated with the wild type, the mutants were rapidly overgrown, resulting in a loss in

productivity. Murphy et al. [125] reported a numerical study to predict the photosynthetic rate of an open-pond photobioreactor in the cultivation of wild and GM Chlamydomonas reinhardtii (CC125) strains. Russo et al. [123] carried out a competitive growth experiment to study the risks of the open-pond cultivation of mutant strains. Chlamydomonas reinhardtii strains (CC-124) and mutant cells with high lipid yields (CC4333) were used in this experiment. The results show that the growth rate of the mutant was twice the exponential growth rate of (CC-124) in the monoculture. The lipid content of mutant strain contained 200% more TAGs per cell than the wild type. However, due to the slow transition phase, the mutant cells outcompeted the wild strains in coculture and posed an insignificant risk to the environment in an escape case. Matsuwaki et al. [126] investigated the spread of genes from an uncontained cultivation system into the surrounding environment using a Pseudochoricystis ellipsoidea MBIC 11,204 strain. Several water samples were taken from eight locations around the cultivation pond to evaluate the spread risk of the P. ellipsoidea algae. Identical psbA sequences of P. ellipsoidea were detected even in vessels that were 150 m from the open pond. Based on the findings, wind flow was deemed an important factor in the spread of algae strains from the outdoor culture. In the next steps of the experiment, the survival of the P. ellipsoidea in the water samples was studied. The growth was not observed in waters with low nitrate concentrations. In an attempt to reduce the containment measure of GM algae cultivation, Kasai et al. [127] developed a self-cloning-based positive selection process for the genetic transformation of Pseudochoricystis ellipsoidea algae strains. The self-cloning-based positive selection system is an attractive option for the large-scale cultivation of microalgae. This method does not involve the stringent regulation of GM algae biomass and can be cultivated at a large scale in outdoor open ponds. Table 7 shows a number of studies in which an uncontained cultivation system was used for the exploitation of GM microalgae biomass. Aravanis et al. [92,128] introduced an end-to-end algal biofuel production process using a genetically engineered Chlamydomonas reinhardtii strain. In the production phase of the proposed biofuel known as “Green Crude,” the C. reinhardtii strain was cultivated in a raceway pond with an area of around 400.000 m2. The harvesting and dewatering processes were done by membrane and disc stack centrifuges, respectively. Szyjka et al. [51] evaluated the ecological risk of the openpond cultivation of genetically engineered algae. The finding shows that outdoor cultivation of GM microalgae did not create any adverse effects on the environment or the surrounding native algae population. The use of the genetic or allied biological modification of algae is widely proposed for the sustainability of FGB production. However, the literature suggests that the open-pond cultivation of the GM algae is the necessary choice for the large-scale cultivation of GM algae to be economically viable. The number of the precedents and the amount of research on the commercial cultivation of GM microalgae are relatively small due to the stringent legislation and rigorous review process imposed by the authorities. However, recent research has shown promising results, so it is hoped that exploitation of GM strains will soon gain the support of the public and of policymakers. Another challenge in producing FGB pertains to the disposal of residue that results from the energy-extraction process. The next section of this paper discusses potential disposal techniques. 3.1. Residue from biofuel extraction The residue of algal biomass after extraction process is valuable due to their rich nutritional components. The residues that result from biofuel production can be used for animal consumption in various industries (e.g., fish aquaculture, cattle and poultry industries). Algal coproducts are not only a source of animal feed, but they also promote eco-friendly technology [53]. Even though the algae are a relatively new and promising product in 44

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the market, the possible hazards of their consumption are largely unknown. This scenario might be due to the large variety of microalgae species and their different characteristics [129]. In order to better use of the remains of the biofuel production process as feed, the origin and nature of the microalgae must be specified. Concerns about feed safety risks are raised when there is not enough transparency regarding the origins of microalgae [130]. Genetic engineering aims to enhance the production of algal biofuel. However, one of the major concerns of using GMOs is their disposal. The applied disposal methods should destruct the microorganism and the genetic element to minimize the risk of lateral gene transfer [131,132]. DNA release in microorganisms generally occurs via cell lysis. The mechanisms of microorganisms remain active even after their death. This phenomenon may cause a release of plasmid or chromosomal DNA. The deliberate or accidental release of chromosomal or plasmid DNA at certain concentrations could result in horizontal gene transfer by transformation; hence, there are strict regulations for the disposal of these products [133]. Waste reduction practices are aimed at minimizing the environmental effects associated with GM residue and at the replacement of hazardous by-products with more environmentally friendly alternatives. Several strategies could be applied to reduce waste (e.g., waste separation and concentration, waste reduction, energy/material recovery, waste exchange, incineration/treatment, and secure land disposal) [134]. Composting is one of the most reliable options for the safe disposal of GM organisms [135]. The by-products of genetically engineered biomass must be decontaminated to prevent cross-pollination with wild species in surrounding ecosystems [136]. Both the organism and the genetic material must be deconstructed simultaneously to decontaminate GM algae. Temperature and pH are two important factors in DNA degradation and in reducing the rate of horizontal transfer in genetically engineered species. Due to high concentrations of microbes in active compose and subsequent increases in nucleases concentration, the DNA degradation is higher in compost than in soil [137]. Two classes of composting are generally used for the degradation of GMO strains: open turned windrow systems and enclosed bioreactor systems. Using an enclosed bioreactor is recommended because it has a significantly faster composting time while ensuring that the materials are composted in an optimal condition [138]. Reusing the residues of algal biomass after processing its lipid, carbohydrate, and protein contents is a well-known solution for eliminating concerns regarding the horizontal gene transfer [139]. Ueda et al. [140] proposed burning dried residue from the biofuel extraction process to recover energy and CO2. The CO2 generated from the burning process was added to the culture medium by a gas diffuser. The energy obtained by the combustion of the dried residue can cover the operating energy of the process. Hence, the proposed method has three advantages: the recovery of energy, the recovery and reuse of CO2, and the reduction of waste. Huo et al. [141] proposed an approach to extract biofuel from the residue that is produced from the fermentation of algae. GM algae is one of the most favorable sources for the presented method because it allows for the reuse of waste materials, it involves the eco-friendly disposal of environmentally detrimental remains, and it achieves a maximized biofuel yield. Reusing the algae residue in cultivation media is a solution that makes biofuel production sustainable by minimizing the input of commercial fertilizers [142]. While much research has documented the optimization of biomass exploitation by improving processing conditions and media composition, little research has investigated the possibility of recycling cultivation medium after energy extraction [143]. The main constituents of algal biomass are lipids, carbohydrates, proteins, inorganic compounds, and nucleic acids. The solid material extracted from recycling culture media after the processing stage can be used as a secondary source of

Photoautotrophic Photoautotrophic

Pseudochoricystis ellipsoidea Chlorella sorokiniana Scenedesmus obliquus

Pseudochoricystis ellipsoidea Chlamydomonas reinhardtii

Matsuwaki et al.[126] De Mooij et al.[124] Chen et al. [122]

Kasai et al. [127] Murphy et al. [125]

Self-clonning of P. ellipsoidea strains Chlammydomonas reinhardtii (CC125) and the truncated chlorophyll transformant tla1

Chlamydomonas reinhardtii Chlamydomonas reinhardtii Acutodesmus dimorphus Russo et al. [123] Aravanis et al. [128] Szyjka et al. [51]

High-lipid accumulating mutant (CC−4333) Optimizing thioesterase gene and integrating into the chloroplast genome Adding enhanced fatty acid biosynthesis, and recombinant green fluorescence protein (GFP) expression New genus, Pseudochoricystis ellipsoidea (MBIC11204) Antenna size mutation The overexpression plasmid

Mesotrophic Photoautotrophic Autotrophic

Several closed photobioreactors and a raceway pond Centrifuge tubes culture Open raceway ponds carboys, hanging polybags and outdoor air-lifted pond An outdoor raceway pond A simulated outdoor microalgal raceway pond Tubular photobioreactor and open pond outdoor system Outdoor open ponds Numerical model of an open pond photobioreactor Mixotrophic, heterotrophic and photoautotrophic Mixotrophic Photoautotrophic Photoautotrophic Overexpressing heterologous genes Phaeodactylum tricornutum Hamilton et al. [120]

Genetic modification Microalgae strain Reference

Table 7 Experiments that considered the uncontained cultivation of GM microalgae biomass.

Culture condition

Cultivation system

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defined as the freshwater required to dilute polluted water to reach the standard quality levels of freshwater [153]. Zhang et al. [154] compared total direct WFs for three microalgae-containing terrestrial plants (sweet sorghum, cassava, and Jatropha curcas). It was stipulated that the direct green WF of the microalgae was almost a quarter of the average direct green WF of the three terrestrial plants. Freshwater, seawater, and wastewater can be used as sources for algae production [26]. The use of undiluted wastewater is a promising substrate for microalgae cultivation due to its high level of nutrients and because it saves freshwater resources. Hence, the use of wastewater for the cultivation of algae can potentially make biofuel production economically attractive and environmentally sustainable [155]. Open-pond cultivation has a higher footprint than contained systems. It is estimated that the WF for microalgae production in an open pond can reach 5.5 ha-ft/acre/year [156]. Yang et al. [157] conducted a case study to quantify the WF of the production of biodiesel from microalgae. It was found that around 3700 kg water, 0.7 kg phosphate, and 0.3 kg nitrogen are required to produce 1 kg of microalgal biodiesel. Fig. 3 shows the schematic WF and water demand associated with the production of algal biofuel. Harvesting is the most important phase in the biofuel production process. There are two main potential choices regarding the WF of algal biofuel: (1) culture with recycling and (2) culture with the disposal of the harvested water. Recycling the discharged water from the harvesting phase can reduce the amount of water consumed during biofuel production by up to 90.2% [158]. The WF of biofuels from microalgae biomass has two components of grey and blue waters. When all the water discharged from the algae culture medium is recycled using the best practices possible, it is assumed that there would be no more water pollution, and the amount of grey WF would be zero. Recycling wastewater from the harvesting stage of GM algae decreases the need for fresh media [159]. Adopting a nutrient-recycling system in large-scale microalgae production could improve the efficiency of algal biofuel production in terms of conserving blue water, enhancing economic opportunities, and minimizing material input [143]. AD has been the most widely studied technology for nutrient recycling in algae exploitation. This method can be applied to the largescale recycling of a variety of types of organic waste [160]. Crofcheck et al. [142] studied the use of the recycled biomass residue of anaerobic decomposition to enrich the cultivation medium. Algal digestate in AD is an available source of nitrogen and phosphorus that has the potential to replenish nutrients. It has been stated that the media used to exploit biomass can be recycled back into the process and reused up to four times. Barbera et al. [161] assessed the feasibility of using the aqueous phase obtained from flash hydrolysis for nutrient recycling purposes. The growth performances of the flash hydrolysis media were compared to those of a standard substrate; the flash hydrolysis was able to recycle nutrients. Zhang et al. [162] conducted a comparative study of on-site nutrient recycling technologies used in algal biofuel production. Two

nutrients in cultivation systems [144]. Particularly, using the recycled biomass residue of anaerobic decomposition in biogas production is well studied. Digestate in anaerobic digestion (AD) is rich in mineralized nutrients such as nitrogen and phosphorus [145]. Thermal treatment is a widely used method for the degradation of DNA in GMOs. Hrnčírová et al. [146] studied the effects of thermal degradation on the quantity of the extracted DNA. The results show that increasing the temperature significantly reduced the DNA content in GMOs in a time-dependent manner. The level of DNA degradation at a temperature of 200 °C within a period of 240 min was significantly increased. Bergerová et al. [147] studied the effects of thermal processing on transgenic and non-transgenic DNA. The size of the matrices of the extracted DNA was found to be an important parameter in degradation. For example, DNA in fine matrices degraded faster. Thermal treatment could be an effective solution for DNA degradation; however, the temperature and exposure time must be evaluated for each individual GM algae strain [42,148]. Table 8 presents some strategies used for waste management in the disposal of FGB production residues. Residue of the GM algae biomass obtained from energy extraction process may contain plasmid or chromosomal DNA that can be harmful to health and ecosystem. Hence, the treatment and handling of disposals must meet the requirements of the GM algae releasing regulations. Disposal methods that destruct the microorganism and the genetic elements are the most effective choices to minimize the risk of lateral gene transfer. The applied disposal method may have direct impact on economic viability and sustainability performance of the FGB. Saving in energy can be achieved through burning the residue of the biofuel production process to cover the operating energy of the process. Based on the economic viability of FGB, the appropriate wastereduction method could be adapted. Water consumption during the production of FGB is another challenge that needs to be addressed to ensure the sustainable use of resources and biodiversity. To this end, the next section deals with WFs and the associated environmental impacts in the production of FGB. 3.2. Water footprint Ensuring safe and clean water is an overriding global concern due to water pollution and water scarcity. The concept of WF was first introduced by Hoekstra [150], and this concept is useful in addressing these concerns and relating the production of terrestrial and aquaculture crops to water pollution and scarcity. The concept of WF led to the idea of sustainable water consumption over the full supply chain [31]. WF is a multidimensional indicator that assesses the water consumed by its source, by the amount of polluted water, and by the type the pollution [151]. Three independent components of WFs (green, blue and grey) determine the water consumption involved in algae cultivation [152]. The consumed volumes of rainwater and groundwater in the production process are referred to as green and blue WFs, respectively. Grey WF is

Table 8 Strategies and applied methods for waste management in the disposal of residues of FGB production. References

Method

Applied strategy

Description

Singh et al. [135] Epstein [138]

Composting

Secure land disposal

Hrnčírová et al. [146] Bergerová et al. [147] Ueda et al. [140]

Thermal treatment

Treatment

Degradation of DNA by applying temperature and pH stresses for minimizing or eliminating horizontal transfer Reduction of the DNA content in GMO through thermal degradation

Ethanol production cycle

Energy recovery

Huo et al. [141] Möller and Müller [144] Lowrey et al. [143] Lowder and Herbert [149]

Protein extraction Nutrient recycling

Material recovery Material recovery

Autoflocculation

Waste separation and concentration

46

Burning dried residue of the biofuel extraction process to recover energy and carbon dioxide Extraction of biofuel from the residue of fermentation process Recycling of the culture medium after processing stage to be used as the secondary source of nutrient in a cultivation system Spontaneous flocculation of microalgae from the culture medium

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Fig. 3. Schematic representation of the WF and water demand in GM algae biomass exploitation.

AD and hydrothermal liquefaction (HTL) methods were shortlisted after a thorough review of available methods. The results for a Scenedesmus dimorphus microalgae strain showed that AD performs better than HTL in term of nutrients and energy recycling as well as technology maturity. Talbot [163] compared nutrient recovery via rapid and conventional HTL in microalgae cultivation. Nutrient recovery performed better in rapid HTL; nearly 50% of recycled phosphorus and nitrogen were replaced. The disposal of the GM algal feedstock is a concern in the production of FGB. The residual water from cultivation cannot be disposed of into environment without additional treatment. Wastewater discharged from the harvesting and dewatering steps is channelled through a drainage system into the treatment sump which is disposed of following treatment. Different strategies are applied to eliminate the inhibitory and remediation of aquaculture in GM microalgal growth. These strategies include UV treatment, pasteurization, dilution, filtration, chemical deactivation, and heat deactivation [31,42]. Patzelt et al. [31] applied several treatments (UV, carbon filtration, and dilution) to remediate the residue medium of algae growth. The load of harmful substances in an aqueous phase was minimized by using photodegradation of UV radiation [164]. Of 28 potential toxic compounds, 23 were eliminated via the irradiation of UV light. Additionally, four new compounds were generated during the treatment process. Twenty-six out of 28 toxic substances were eliminated during carbon filtration [165]. The exploitation of algal biofuel has a lower WF when compared with other biofuel feedstock. The reusability of the discharged water from the harvesting process offers an additional benefit in terms of WF. However, the culture medium can be recycled a limited number of times and should be disposed of after this limit has been reached. Discharging the culture medium from the GM algae cultivation could pose environmental and health-related risks. Specific water treatment processes must be devised before wastewater can be discharged safely. The risk of releasing wastewater into the environment – and the associated treatment option – directly affects the economic viability of the production phase. In the next section, the economics of FGB production are further discussed.

The enhancement of algal strains through genetic modification and the metabolic engineering of algal strains has been widely studied, and many studies on the topic are ongoing. the aspects concerning legislation issues and environmental risks related to the production of GM algal biomass are two important topics that need further attention. Major concerns in this field include the risk of the introgression of GMO strains into the natural environment, the large capital investments required, and the high operational costs of operating photobioreactors. Such drawbacks limit the commercial exploitation of FGB. Therefore, conducting study to overcome these drawbacks is an important step toward the sustainable and commercialized production of FGB. Higher productivity could be achieved by improving reactor designs, using high-throughput genetic modification strategies, and effective waste management. Based on the current legislation, additional costs are incurred to the biofuel production process dealt with the waste management facilities and treatment of the disposals which was covered in this study. The number of studies that deal with the economic aspects of using GM in biofuel production and applying the appropriate economic solutions to meet legislation-based sanctions is very limited. This is mainly due to the stringent regulations imposed on the exploitation of GM algal feedstock and lack of case studies on the large-scale cultivation of GM algal crops in aquatic cultures. The future is promising for FGB, and genetic modification is a necessary element for an effective transition from fossil fuels to biofuel as our main energy source in the 21st century and beyond. This potential adds to the already great importance of considering the sustainability implications of conserving the environment and health safety. This can be achieved through thoughtful and comprehensive assessments of the risks associated with the production and utilization of FGB. The first step toward this is identifying the right questions to ask. 5. Conclusion Algal biofuel is a promising alternative for fossil fuel owing to its advantages of high energy content, low emission, and non-polluting nature. However production of the algal biofuel is not economically viable due to the low yield and high production costs. Hence, in recent years, many studies are conducted on FGB. The available literature in the field of FGB is mainly concerned with finding strategies to produce GM biomass from various algae species and the studies on how the environmental and health risks in production process can be mitigated, is rather few and scattered. To the best of author’s knowledge, no study has been undertaken to review and investigate the risks of using GM biomass in production stage (i.e. the cultivation, harvesting, and processing steps of FGB) and to discuss the associated mitigation strategies. Using open-pond cultivation is the necessary step for success in industrial-scale production of FGB. However, the concerns regarding the health and environmental risk exert huge pressure on governments

4. Recommendations and future works The future of GM algae biofuel depends on improving the efficiency of its cultivation, enhancing the algae strains used, and promoting the commercialization of biomass production. These advancements can be achieved by lowering the cost and increasing the output of cultivation systems as well as by introducing more efficient genetic modification or allied methods to increase the yield and quality of the products. However, the commercial cultivation of GM algae is currently hampered by the risks involved in the deliberate and unintended release of the modified strains into the environment. 47

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to implement stringent regulations on both deliberate and accidental releases of GMOs. Several field testing project for open-environment exploitation of GM algae were conducted to assess the attributed risks on health and the environment. The result shows that under the various conditions used, there was no apparent proof to support possible horizontal gene transfer in release of GM algae. Disposal of the residue is another important issue that should be considered in FGB production process and appropriate mitigation practices should be used to reduce the risk of environmental and health harms. Several waste reduction strategies are aimed at minimizing the environmental effects associated with GM residue and at the replacement of hazardous by-products with more environmentally friendly alternatives. Disposal methods that destruct the microorganism and the genetic element are the most effective choices to minimize the risk of lateral gene transfer. It has been stated that the media used to exploit biomass can be recycled back into the process and reused up to four times. The residual water from the harvesting process of the FGB production contains genetic elements and need to be treated carefully. The treatment process should be conducted in parallel with the nutrient recycling to ensure that resources are used sustainably. The treatment of the byproducts and discharged water from the system incur additional cost into production process that must be accounted for.

[17]

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Acknowledgements

[27]

The authors would like to thank the Ministry of Higher Education of Malaysia for their financial support through the University research grants, GUP (15H32 and 19H98), without which this study would not have been possible.

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