Creative solutions

Current industrial sized cultivation systems are designed for high value products rather than cheap bulk biomass. Future bioreactors will demand innovative solutions for efficiency in energy and light use, gas transfer and dewatering (Morweiser et al., 2010) (Table 1.8).

Research suggests focusing development activity towards technologies which improve algal yield without a cost increase, yet there is great promise in reducing capital costs though novel culturing and harvest technologies (Brown, 2009).

Table 1.8: Summary of proposed solutions to issues surrounding microalgal biodiesel production.

Adapted from van Beilen, (2010).

Issue Potential solutions

Light Pulsing LEDs, microlensed PBRs

Temperature IR glass/plastic, use extremophillic species, warmed using cooling water Nutrient Wastewater, utilise flue

Dewatering Filamentous species, self-flocculating/settling species Oil extraction GM of excretory pathways

Costs Co-production of a valuable product, novel PBR designs

Rapid light-dark cycling (>1Hz) can improve photosynthetic efficiency and increase algal productivity (>5-fold compared to continuous illumination) (Love, 2011) by reducing photoinhibition and ‘wasted light energy’. This can be achieved with pulsing LEDs which can be further optimized (using light intensity and wavelength) (Gordon and Polle, 2007) or

‘microlensing’ of the surface of a closed PBR, converting cheap ‘continuous’ sunlight into a

‘stroboscope’ (Love, 2011). Both methods have been shown to achieve much higher biomass densities when compared to a normal PBR.

Culture systems that utilise natural sunlight are prone to temporal and spatial patterns of growth due to light or temperature fluctuations (Mutanda et al., 2011). In regions where sunlight is most abundant, increases in temperature can slow productivity (Morweiser et al., 2010). One way to reduce detrimental heating is to block infra-red wavelengths (which constitute 40% of solar radiation), using infra-red reflecting glass and plastic, yet this would

37 add to capital costs (Stanghellini et al., 2011). In Europe low temperatures provide poor reaction kinetics and slow growth in outdoor culture systems, making them viable for production only between June-October (Sandefur et al., 2011). Warmed cooling water from powerplants could be used to keep cultures warm (Morweiser et al., 2010). Extremophillic algae may be beneficial for industrial applications even if their growth rates are slowed, as contamination risks are lowered if open ponds run hotter (or saltier or at an extreme pH) (Pulz and Gross, 2004). In addition, extremophiles often have unusual metabolites or enzymes which may be of biotechnological interest (Pulz and Gross, 2004). Ecological studies on algal communities may help achieve better yields, as not only are mixed communities more productive (in terms of biomass) but they are also less prone to invasion by airborne microorganisms compared to a monoculture (Smith et al., 2010). Co-culturing may also reduce the nutrient requirements for microalgae. For example, nitrogen biofertilisation using a strain of hyper-ammonium-excreting bacteria (Azotobacter vinelandii) has been shown to support the growth of an oil rich green microalga without the need to add nitrates (Ortiz-Marquez et al., 2012).

Savings can be made by coupling algal culturing to other processes. It takes 3t CO₂ to produce 1t of algal biomass (Parker, 2012). Flue gas usually contains CO₂ at 3-15%, with combined cycle gas turbine (CCGT) and coal fired power plants emitting 370t GWh^-1 and 912-1280t GWh^-1 of CO2 respectively (Packer, 2009). The supplementation of CO2 in microalgal culture increases the rate of growth and biomass accumulation (Packer, 2009, He et al., 2012 and González-López et al., 2012). Warm flue gas may also help raise the temperature of outdoor culture systems. Flue gas however also contains nitroxides, sulphoxides (which can affect pH) and may require filtering prior to use (Packer, 2009). Nutrient removal by algae could be a less expensive and ecologically sound way of scrubbing wastewater (containing nitrates and phosphates), recovering resources, offsetting the cost of fertilizers (Pulz et al., 2009).

Phosphorus is vital to any biological growth (synthesis of DNA and ATP) (Wang et al., 2008), yet bioavailable reserves are also being depleted and it is often overlooked as a dwindling resource (Sivakumar et al., 2012). Wastewater treatment is a promising platform for the commercialization of algal growth for biofuels, as domestic wastewater contains nutrients beneficial to microalgal growth (Chistenson and Sims, 2011). Bioremediation of both waste water and flue has yielded some encouraging results. For example, algae cultured in wastewater have shown marked improvements in lipid accumulation when supplemented with CO₂ from flue gas (Devi and Mohan, 2012). However, trace metals can accumulate and

38 become toxic to algae cultivated using wastewater or flue gas. Yet some studies have shown certain species to have scope for bioremediation of these metals (Bozarth et al., 2009).

Dewatering of dilute microalgal cultures is energy and cost intensive with many established commercial methods being unsuitable for a large facility dealing with enormous effluents (van Beilen, 2010). Filamentous, self-flocculating or settling algal species would be beneficial in greatly reducing costs (van Beilen, 2010). Immobilization techniques already used commercially can often be applied to algae with few modifications. Alginate beads, carrageenan, chitosan and polymers such as polyvinyl foam and polyurethane are suitable materials (de-Bashan and Bashan, 2009). Scenedesmus dimorphus has successfully become immobilised onto stainless steel textured metal sheets are textured specifically to allow algal attachment. Sheets are subsequently driven though a conveyor belt to ‘mow’ the algal lawn, with residual algae serving as an inoculant for subculture (Cao, 2009). Similar promising results have been found using polystyrene as a supporting material (Johnson and Wen, 2010).

Novel, low cost PBR designs can have a large effect on the overall energy demand of an algal culturing system. Many unusual designs are already being used on small commercial scales.

For example, the V-shaped bag reactor (Novagreen), flat panel airlift reactor (Subitech Germany) and various designs utilising permeable membranes (Morweiser et al., 2010), (Kumar et al., 2010). One notable design is the Offshore Membrane Enclosure for Growing Algae (OMEGA), which eliminates the use of freshwater and fertilizers by delivering wastewater and flue CO2 via a permeable membrane system. Additionally the OMEGA system utilises sunlight and is floated offshore, the sea not only controlling the temperature but also mixing the cultures by wave energy (Figure 1.10). Membrane systems do require measures to control the level of fouling and will require replacing periodically (Pulz et al., 2009).

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Figure 1.10: Schematic of the Offshore Membrane Enclosure for Growing Algae (OMEGA).

This system is an excellent example of creative low energy solutions to a variety of problems associated with microalgal culturing. Adapted from Pulz et al. (2009).

Based on current technology alone, it is not feasible to grow microalgae solely for biodiesel production. It needs to be more economically viable with a positive energy return (Pittman et al., 2011). At present there are many small start-up companies, from which some larger companies will potentially near commercialisation. This could make market entry more difficult for smaller local businesses (Parker 2012). Research seems to suggest that integration of small local plants will be vital to the success of algal biodiesel, in which case it will become important for them to be supported (Wagner, 2007), depending on availability and suitability of land (Parmar et al., 2011).

1.3.3 Legislation

Several external factors such as oil prices, stricter GHG emission targets and carbon trading schemes (currently ~$200t CO2) will also affect the future viability of algal biodiesel (Stephens et al., 2010). Devices such as tax credits or exemptions could incentivize and greatly influence the development of particular technologies (Carriquiry et al., 2011). Carbon tax (an economic device for incentivising the reduction of carbon dioxide emissions from fossil fuel sources) can promote the use of biofuels. If carbon tax on fossil fuels as used to subsidy biofuel costs it could significantly aid the penetration of biofuels into the energy sector (Timilsina et al., 2011).

Governments could also help with underfunding from the private sector in order to deploy certain technologies. Investments part financed by the US Department of Energy have been very effective at reducing the cost of enzymatic digestion of cellulosic ethanol biofuels.

40 Biodiesel still more expensive than regular diesel and therefore will need to remain subsidized (Razon and Tan, 2011).

Policies regarding the way in which biofuels are produced, consumed and traded are fast changing (Carriquiry et al., 2011). Previous legislation is often modified and now many countries have policies to incentivize supply and use of biofuels regardless of the environmental costs of production or relative benefits they provide (i.e. net energy ratio) (Carriquiry et al., 2011). There are some exceptions notably in the US and EU. For example tax credits are preferentially given to second generation biofuels than first generation (Carriquiry et al., 2011). It is important to consider trends in regional and international development to exploit synergies and maximize benefits (e.g. biofuels that do not match international standards would not progress beyond a domestic market) (Carriquiry et al., 2011).

An important question to ask when considering investment into microalgal biofuels is; what is the priority for cost reduction? For cellulosic ethanol the major cost is conversion, yet for microalgae capital cost of plant development is often highest (Leh and Posten, 2009). Given current research status and uncertainties of any major developments what would help make algal biofuels cost-viable, would be to give different support depending on nature of the production process (Parker 2012). In particular an integrated approach combining rural development, climate change, and energy provision could prove very valuable when considering biodiesel production from microalgae (Carriquiry et al., 2011). There are likely to be method preferences for different locations (certain growth methods will vary in different geographical regions) and local legislation should reflect this (Shirvani et al., 2011).

41 1.4 Aims and objectives

The project began with the purpose of improving algal strains for the chemical conversion of CO₂, with the intention of initiating and developing algal research at the University of Bath.

Broad aims included; investigating methods to lower the energetic cost of microalgal product extraction and the isolation of novel strains from environmental samples. Objectives remained flexible, due to the infancy of algal research at Bath.

Objectives included:

 Method development of basic laboratory techniques for maintaining and culturing microalgal stocks, isolation of algae from environmental samples, mutagenesis, DNA extraction and molecular identification of microalgae.

 The size and strength of microalgal cells, renders many lipid extraction methods inefficient, energy intensive and expensive. Characterisation of the cell wall with the intention of simplifying or de-energising product extraction was investigated using enzymatic digestion, staining, sonication and electron microscopy.

 The genetic modification of microalgae is believed to have the largest impact in their development as a commercial organism. Mutagenesis and screening for the generation and isolation of novel strains underwent development using the oleaginaceous model alga C. emersonii.

 Microalgae are incredibly diverse group of organisms. However only a select few species are studied and used commercially. Bioprospecting is believed to become an invaluable tool for the development of algae-based bioresources, in particular species isolated from extreme environments. The city of Bath is home to one of the only ‘hot’

springs in the UK, the Roman Baths. This unique natural habitat was the focus for isolation and identification of novel thermo-tolerant microalgal strains.

 Interesting or unusual microalgal species may behave entirely differently when under certain culture conditions. The temperature tolerance, lipid quantity and profile of microalgal isolates from the Roman Baths were investigated.

 Solvent extraction is the standard method for lipid extraction from microalgal samples, however it is a time consuming process delaying data analysis. Various laboratory lipid extraction methods were also investigated in an attempt to accelerate experimental analysis.

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2. MATERIALS AND METHODS