INTRODUCTION

1.0 In recent years bioremediation has been set to replace the tertiary stage of wastewater treatment as a more sustainable and environmental friendly process. Currently the tertiary stage utilises various chemicals and machinery which can be considered harmful to the environment (Gray, 2010; Pentecost, 1984; Schmidt, 2010). This avenue of research has been heavily invested in and extensively studied in Australia and the United States One major difficulty facing this new avenue of research is the freeze tolerance of the algae used within bioremediation. The most popular species to use within this new process is Chlorella vulgaris (Hegger, 2010; Lighton, 2012). C. vulgaris along with other green algae species is widely available in many freshwater sources, produces high amounts of 0₂ while reducing C0₂ and has rapid growth.

1.1 With these benefits phycologists have stipulated that green microalgae could be used for wastewater whilst being harvested for biofuels. Potentially this could mean that algae used in bioremediation could reclaim land which has been used for ‘energy crops’ (Brown, 2009; Daigger and Hanrikson and Edwards, 2012). These crops have been controversial as they take up much needed land which could be used for food. Additionally it has been reported that the lipid or oil content of these land crops such as Brassica napus (Rapeseed) is 40% lower than it is in most species of microalgae. As a result the EU has placed a cap on a member state’s output of energy crops to 5% of a country’s total crop turnover (Deutsch, 2008; Lawlor, 2000). Microalgae have also been shown to be a viable source of organic fertiliser for land based industries. Together the above factors demonstrate the economic ability of microalgae as a source of bioremediation, fertiliser and fuel. With this in mind, why has bioremediation not been implemented throughout the UK? One significant challenge facing the commercialisation of algae is the low freeze tolerance of the strains used as stated by 4^th World Algae Europe Conference (2012).

1.2 A UK parliamentary report on Science and Technology (2011) stated that the UK’s interest in developing bioremediation to enhance the country’s ‘green’ technologies charter and other related targets tied in with environmental policies. Since the UK annually has over 60 days of sub-zero temperatures as indicated by the Met Office (2012) this makes microalgae an unreliable method of treatment. The optimum temperature required for many green microalgae is ~30˚C. Much of the research compiled thus far has been limited as it occurred in tropical regions or within laboratory conditions (Etnier and Guterstam, 1996). This shows that more research is required to develop the freeze tolerance of many algal strains. According to Friday et al. (2012) many freeze tolerant organisms such as Chlamydomonas nivalis (Ice algae) exhibit high lipid levels to manipulate the permeability of their cell membranes. This is achieved by increasing the internal osmotic pressure to protect against external pressures developing microcrystals. By adding glycerol to a sample of algae, the cells plasmodesmatas will absorb the fats due to the polarised nature of the membranes and fat monocles (Gacheva and Polarski, 2008; Graham et al., 2008). This will result in higher internal osmotic pressure, thus increasing freeze tolerance.

HYPOTHESES

1.3 The aim of this study is to increase the freeze tolerance of 3 commonly used microalgae for bioremediation. If proven to be effective these microalgae could then be used within the UK horticultural industry as a natural and sustainable source of wastewater treatment. The following are to be investigated throughout this study:

Alternative Hypothesis

When supplemented the glycerol will alter the concentrations of chlorophyll a and phaeophytin a, cell counts and biovolumes of the green microalgae strains.

Null Hypothesis

When supplemented the glycerol will not alter the concentrations of chlorophyll a and phaeophytin a, cell counts and biovolumes of the green microalgae strains.

LITERATURE REVIEW

Membrane Structure

2.0 It is known that all algae species have a membrane structure dividing the cytoplasm complex from the cell wall. The principal function of this membrane is to protect the integrity of the interior compartment within the cell. This structure allows certain substances to enter whilst inhibiting others. This also serves to reinforce the cytoskeleton and cell wall (Hoek et al., 1996; Ke, 2010).

2.1 The membranes are comprised of a double layer of fluid lipids in which proteins float laterally forming a changing mosaic pattern as demonstrated by the Fluid-Mosaic Model (Kim, 2011; Wiessner et al., 2012). This bilayer lipid layer is primarily formed from positively charged phospholipids and a small number of stigmasterols. They orient with their hydrophilic heads toward the outside and their hydrophobic tails towards the inside, thus increasing the strength of the membranes (Luz et al., 2000; Watanbe et al., 2011). Embedded in this layer are proteins that function in 4 cellular activities: transportation, reception, communication and protection.

2.2 Transport proteins regulate the transferal of molecules across the membranes through facilitated ‘diffusion’. Essential monocles manipulated for energy generation such as Na^- and CI^- are unable to pass through the membrane without assistance (Leegood and Lea, 1998; Barbar and Anderson, 2004). Within the membranes are ion channels which are specific to a certain ion. These ion channels are regulated by the nucleus and either open or closed to control the passage of these substances. Carrier proteins bind to the specific ions, change shape and then deposit the monocles across the membrane (Seckbach and Gordon, 2012; Bajguz, 2012). Once the transaction has concluded the proteins return to their original shape to continue this process. However glycoproteins which are short chains of carbohydrates control and monitor cell communication (Sanghera et al., 2011; Sharma, 2009). This is conducted through the chemical messager channels and molecule transport chains (Tiquia-Arashiro, 2012).

Mechanisms of Osmosis

2.3 One process which cannot be controlled by the membrane is osmosis. Osmosis is the movement of water molecules from a higher concentration to lower concentration through a semipermeable membrane (Allaby, 2008; Blankenship, 2001). The purpose of osmosis is to reach a state of isotonic displacement as stimulated by the second law of thermodynamics. When a membrane has a volume of pure water on both sides, water molecules pass in and out in each direction at the same rate. There is little net flow of water through the membrane (Uttley et al., 2010; Wang and Lan, 2011). However when concentrations of ions are higher outside the cell the water monocles are drawn out of the cell thus the cell dehydrates. The same process occurs essentially when water freezes (Brown, 2012. Pers. Comm. Prof Murray Brown is the leading microalgae expert at Plymouth University).

2.4 A special adaptation of many psychrophilic organisms such as Chlamydomonas nivalis includes their ability to maintain fluidity of their membrane at low temperatures, even to -40˚C (Meimers et al., 2009). This mechanism is caused by a high portion of unsaturated phospholipids which are double bonded hydrocarbons attached to the outer layers. Another effective mechanism is the excretion of antifreeze exopolymer proteins which are produced within the interior of the membrane and then transported to the outer regions of the cell wall (Bayer-Giraldi, 2010). This mucus disrupts the osmotic pressure which surrounds the cell and enables water monocles to remain within the cytoplasm (Melmikov, 1999; Russel, 2000). An alternative method of this is to disrupt the water molecules themselves without changing the osmotic pressure; this pressure utilises more energy. Some psychrophilic bacteria exhibit water replacing proprieties. A trehalose sugar replaces the internal water and thus preventing vital organelles, enzymes and proteins from denaturing (Obata and Taguchi, 2009; Robinson et al., 1997; Cooper, 2009).

2.5 Most membranes are positively charged due to the bilayer lipid layer which needs to attract ions. This interaction between lipids and ions suggests that increasing the lipid and fatty acid content of the external layer of the membrane may increase the freeze tolerance as lipids have a lower freezing point, up to -10˚C (Borowitzka and Moheimani, 2012; Hall and Rao, 1999). This goal could be achieved by using glycerol (C₃H₈O₃) which is a natural source of fatty acid. However in the long term by using fast evolving techniques green algae could increase lipids through biofuel research programs as stipulated by a parliamentary report on Science and Technology (2011).

Review

2.6 Lim et al. (2010) conducted a study into the use of Chlorella vulgaris and 9 other microalgae species for bioremediation of textile wastewater. This study used 5 varying dilutions of wastewater ranging from 20% to 100% to examine the effectiveness of the green algae species. The wastewater was collected from local garment factories and was tested before and after the trials. 10ml of C. vulgaris were inoculated in 90ml of textile wastewater in 250ml flasks in triplicate. The cultures were grown for 10 days in an incubator shaker at 150rpm set at 25˚C, with an irradiance of 40-60µmol m^-2s^-1 on a 12:12 hour light-dark cycle. Initial pH of the wastewater was adjusted to 7.0 prior to inoculation Lim et al. (2010). Chlorophyll a determination, cell biomass, NH₄, PO₄ and N and initial colour were tested before and after the trials. A cell count was measured every 2 days to determine the growth rate. This was repeated a further 3 times.

2.61 After laboratory testing the investigation moved outside by using 5 high rate algae pools set to 15rpm using 5 variants of dilution as noted above. 10% of the total wastewater made up the algae concentration. The outdoor experiment reflected what had occurred within the laboratory. Nutrients which had derived from Bold Basel’s Medium were supplemented within the pools. Chlorophyll a determinations and cell counts were carried out every day. The pools were aerated by using paddles. This was repeated with 3 trials. After each trial dry weight and NH₄, PO₄ and N determinations were carried out.

2.62 This research revealed that the use of C. vulgaris could remove 85% of contaminates from the wastewater, NH₄, PO₄ and N by between 78 and 93% after 10 days. The high C:N:P ratio of 8470:45:1 was an indication that the wastewater was high in carbon but low in nitrogen. This was much higher than the recommended ratio of 56:9:1 for algal development (Liu and Liptak, 1999; Lobban, 1996; Whitton and Brook, 2011). Although this demonstrates high proliferation of C. vulgaris it also uncovered that C. vulgaris had a limitation of growth in low temperatures. When the temperature reached 5˚C the efficiency of C. vulgaris decreased by 50.3% and a further 4.2% for every 2˚C decrease in temperature. The study concluded that Chlorella vulgaris had grown 80% more when in a 100% dilution of wastewater. Up to 40% of algal biomass was found to be made up of lipids.

2.71 Gronlund (2002a) led a study into microalgae’s ability to proliferate in the cold climates of Sweden. When subject to depths between 30 and 50cm microalgae could develop and remove 78-97% of inorganic N and P from the wastewater. Gronlund’s (2002a) team also discovered that the level of nutrients was a major factor in conjunction with freeze tolerance. The higher the nutrient levels the higher amount of growth. This would inevitably create a biofilm which can increase cold by 37.5% due to insulation properties. Over the pools there were pulsating cycles of light and dark. Over a 6.5 month period: light, temperature, C0₂ and N:P ratio was monitored daily and a biomass weekly. Oxygenation was encouraged by utilising paddle wheels (Hannan et al., 2003; Carrington, 2010). This was found to encourage a group of nitrogen-fixing bacteria (Phyllobacterium myrsinacearum) to work in symbiosis with the microalgae which further improved the filtration rates.

2.72 Although this study cultivated microalgae in cold climates it was only tested between 5 and 10˚C. Additionally the research tested microalgae’s ability to filter heavy metals. Only 2 microalgae were found to be effective against them: Chlorella vulgaris and Phormidium murrayi. The study finally concluded that P. murrayi had the more enhanced filtering capabilities under low temperatures. It was established that P. murrayi could also remove 82-94.5% of heavy metals, 93.6% of inorganic N and P, and was discovered to have doubled its growth every 2.3 days. C. vulgaris was proven to have a strong capability to filter pollutants; however from the biomass readings the efficiency of the strain decreased by 56% when the temperature reached 5˚C.

2.73 This research by Gronlund (2002a) establishes the limitations of Chlorella vulgaris as a source of bioremediation. However it was uncovered that nutrient levels within the pool influenced the levels of biomass. As algal biomass increased this formed a biofilm across the surface of the wastewater which then insulated the lower layers of microalgae. To build upon the previous work of Lim et al. (2010) this research found that microalgae, especially C. vulgaris could grow (18g m² day) to depths between 30 and 50cm where light levels can be as low as 65%. This phenomenon was also supported by Furuya (2003) and Allen (2012) who both stipulated that many microalgae species could tolerate between 75 and 82% of terrestrial light levels. This illustrates microalgae’s ability to tolerate extreme light conditions, although freeze tolerance still requires more research.

2.81 One investigation carried out by Bhatnagar et al. (2010) focused its attention to Chlorella minutissima as a source of biofuel from wastewater bioremediation. The aim of this research was to see if biofuel costs could be lowered by using an unconventional microalgae strain. The experiment took a year to perform and within that time inorganic N, P levels and biomass were measured weekly. To monitor the environment daily temperature, light and C0₂ readings were collected. 3 large ponds were filled with 80% wastewater from the local water treatment plant with 3 samples of C. minutissima.

2.82 Over the year patterns emerged from the data with algal growth through the seasons. Bhatnagar et al. (2010) also installed an oxygenation pump for each pond. The algae samples were also placed within an anaerobic chamber with 5% NaCI and under ^-10 bars of osmotic pressure. As a note this pressure is similar to icy waters during freezing (7.3 bars) (Gray et al., 1997; Kanno, 2005; Kirk, 2004). The samples were found to have survived from utilising NH₄ and N from the waste heterotrophically. This illustrates the ability of the alga to reside within a light-absent environment. Moreover it was found to have performed more efficiently when compared with traditional methods of anaerobic conversion of lipids. C. minutissima could tolerate acidic conditions of 4.0. As well as being heterotrophic in the dark it had similarities of a mixotroph in the presence of light. By raising the pH from 4.0 the researchers discovered that it resulted in long term gigantism within the cells which improved filtering efficiency (Bhatnagar et al., 2009).

2.83 From the aeration, several nitrogen-fixing bacteria including Phyllobacterium rubiacearum and P. myrsinacearum had developed a symbiosis with the C. minutissima, increasing production. However to the researchers’ surprise C. minutissima had only removed 45-63% of inorganic N and P. Conversely lipid levels were 27% greater in comparison to other commonly used species, for instance Scenedesmus subspicatus and Nannochloropsis oculata (BBC, 2011; Kay, 2010; Gronlund, 2002b). According to the temperature readings there was a correlation between increased temperatures and growth increases. One possible confounding variable could have been the high temperatures as the experiment occurred in Southern Greece. The research concluded with the recommendation of testing C. minutissima under cooler climates to examine the effects that it would have on filtration, nutrient intake, lipid production and sourcing of nitrogen-fixing bacteria.

2.91 Daligault et al. (2003) inoculated 6 250ml flasks of Chlorella vulgaris within an incubator with weekly temperature decreases of 2˚C from 25˚C to -3˚C. Samples were taken from each flask for enzyme analysis. The analysis uncovered that when the

temperature reached the freezing point of water, microcrystals split the hydrogen atoms from the carbon atoms within the C-H bonding which forms the membrane’s structure. Furthermore the external osmotic pressure resulted in increasing the rate of breakdown of the C-H bonds. The experiment also exposed Arabidopsis thaliana to the same conditions to provide a terrestrial comparison of freeze tolerances. The study found that the microcrystals had the same effects on the membrane structure of A. thaliana. This was an indication that algae and plants share similar freeze tolerance strategies and systems.

2.92 After data collection, deuterium was added to the C. vulgaris samples at a rate of 50ppm and the experiment was repeated. It was witnessed that the heavy hydrogen bonds from the isotope were attracted to the C-H bonding within the membranes. This attraction strengthened the bonds, thus increasing freeze tolerance. Conversely when the deuterium sample was added to the A. thaliana this resulted in a lower freeze tolerance. A further trial continued with C. vulgaris. The temperatures were again reduced by 2˚C a week from -3˚C to –11˚C. This time the C. vulgaris tolerated temperatures of -5˚C and still grew. Nevertheless efficiency had reduced by 54.8%. Although deuterium increased freeze tolerance by an extra 10˚C the cost involved would make using deuterium on an industrial scale economically unviable. On the other hand this research revealed that the C-H bonding within the membranes greatly impacted upon freeze tolerance. By increasing the amount of C and H this would significantly increase freeze tolerance.

2.93 According to Kirke (2006) glycerol contains a long chain of covalently bonded carbon atoms with nonpolar bonds to hydrogen atoms along a carbon chain, which could increase the strength of the C-H bonding. Daligault et al. (2003) concluded with the recommendation to investigate the effects of natural sources of C and H such as refined fats.

2.10 Makareviciene et al. (2011) examined the growth of Chlorella sp. and Scenedesmus sp. in several Lithuanian lakes to obtain optimum conditions for future biomass cultivation. Weekly samples were taken from each of the lakes over a year. The samples were analysed to determine the biomass content, N and NaCI levels, C0₂ concentrations and the presence of other algae. The above lakes were previously tested to determine the concentrations of the required microalgae. The lakes were spread across a variety of conditions and more importantly temperatures. Lake Vistytis, Sartai, Plateliai and Rekyva were the main sources of sampling and where average temperatures can fluctuate from 7.2˚C to -2.4˚C. The aim of the study was to increase the production of biofuels without using arable land.

2.101 The testing revealed that microalgae would not be a viable source of biofuels as for every 1.5˚C decrease in temperature, lipid conversion decreased by 3.6%. The researchers after testing set up an experiment to find a simple and cheap method to increase freeze tolerance. Makareviciene et al. (2011) stated that there was a correlation between light levels and freeze tolerance. When light levels increased it stimulated the production of mucus, which increased the strength of the membranes. After a 6 month period of culturing Chlorella sp. and Scenedesmus sp. each with 10 250ml flasks within a Percival growth chamber, their theory was unverified. However when they reviewed the conditions of the smaller lakes they realised that the salt levels varied. They found that salt concentrations within the lakes were at: 6.2%, 12.7%, 18.6% and 36.9%. As the salt concentrations increased so did the freeze tolerance.

2.102 With regard to Makareviciene et al. (2011), one limitation of their findings is that most lakes are derived from freshwater. To introduction salt concentrations into a lake for the production of biofuel could negatively impact upon local ecologies. If their salt treatments were to be implemented it would have to be by artificial means, resulting in higher costs. Within their discussion the researchers reviewed the monocular structures and similarities of the salt deposits and the algae membranes. They argued that the monocular bonds within the salts could improve the strength of the C-H bonding within the algae membranes. The study recommended that a further study was necessary to find a source of higher concentrations of C and H atoms.

METHODOLOGY

3.0 Many strains of microalgae can be used within horticultural wastewater bioremediation. However without constant tropical conditions these microalgae fail to be effective in the UK By increasing the freeze tolerance, these microalgae could become a low cost and environmentally friendly method of water treatment.

Samples

3.1 Chlorella vulgaris, Scenedesmus subspicatus and Nannochloropsis oculata were chosen from previous studies which had been proven effective at bioremediation (UK N.C.C., 2001; Lee and Lee, 2001). The species were obtained from the Marine Science Laboratory at Newcastle University at 100ml each. This experiment occurred within the Microbiology Laboratory of Newcastle University due to the availability of resources required. Chlamydomonas nivalis was used as a comparison to provide an insight into the interactions of a membrane which is adapted to control osmosis in sub-freezing temperatures. The microalgae were cultured within a photobioreactor for 2 weeks prior to this study at 30˚C. To gain viable samples for analysis, a Fisher loop was used to scrape a small group of algae cells from the plates. Subsequently the cells were resuspended in a solution and stored until needed.

Procedure

3.2 During the experiment species were transferred to a Sanyo bacterial incubator. The incubator was left to run for 2 weeks prior to this investigation to examine the environmental variances. As a note the microbiology laboratory has a backup power source in case power cuts arise. There was a 12:12 day and night cycle programmed. The control comprised of 5 weeks with weekly temperature reductions of 5˚C from 10˚C to -10˚C. This was to test the algae prior to adding the 20% glycerol stock solution and to act as a comparison to the trials. During the trials weekly temperature reductions of 5˚C from 5˚C to -10˚C occurred. This was to reflect the temperature fluctuations of UK winters. Once the samples reached -5˚C a 50mm section of frozen algae was cut off for testing (Hack, 2012. Pers. Comm. Dr. Ethan Hack is a marine doctor at The School of Biology of Newcastle University). The glycerol was derived from a mixture of 20ml of 100% glycerol and 80ml of dH₂0. This mixture was then autoclaved for 30 minutes. During the trials 500µl of glycerol was pipetted into a 10ml concentrate sample of each species within a petri-dish. This was repeated a further 17 times to complete the test groups within each trial. This procedure was repeatedly carried out under an Esco hood to prevent contamination. To further reduce changes of contamination 70% ethanol solution was used to sterilise work surfaces, materials and equipment. See Appendix 1 for trial design.

3.3 90mm x 15mm petri-dishes were filled with 30ml of Modified Chu-10 Medium with an added metal solution: H₃BO₃, MnCI₂, ZnCI₂, CoCI₂ and CuCI₂ which was brought to pH 7.0 as recommended by Acreman (2010) and Sharma et al. (2011). A Gorman and Levine Modified media brought to pH 7.0 was required to culture the C. nivalis (Gorman and Levine, 2005; Oilgae, 2010). To remove bubbles which had developed during media pouring under an Esco Laminar Airflow cabinet, a Wall Lenk handheld blow torch was used. To prevent partial loss of media integrity, the plate lids were closed to reduce chances of drying out and rupturing.

Apparatus

3.4 The plates were inoculated within an Esco hood to prevent contamination. For each group 6 petri-dishes were required. A total of 18 plates was utilised for each trial and 24 plates for the control, totalling 78. Each trial was cultured for 4 weeks and the control an additional 5 weeks, totalling 17 weeks.

3.5 9 250ml conical flasks were half filled with a Richmond (2003) general algae broth with algae sample. This was required for a continuous supply of 10ml algae concentrate for the trials and the control (Gotingen, 2008a and Gotingen, 2008b). The broths were placed within another Sanyo incubator at 20˚C. When transferring the algae from the broth to the media some may be lost by using the traditional method of loops. A P1000 Fisher pipette was used to transfer samples to safeguard against wastage. The ice algae were directly cultured within their medium and did not require a broth as they were not needed for the trials.

3.6 During the testing period the temperatures were recorded by using a Lascar 4 USB data logger. The average light intensity levels were measured at the outer wall of the culture plates using a Sekonic L-358 Luxmeter. A GX-Optical compound microscope was used to photograph the cells by up to x100. Although this is not enough to take detailed photographs of individual cells, it can show conglomerations of algae. See Appendix 2 for environmental specifications. As a summary there were 3 trials each consisting of 3 different species of green microalgae to test the modifications made to the permeability of osmosis. The control tested the cold tolerance prior to these alterations as shown in Table 1 below.

3.7 To determine the effects of glycerol, a cell count and biovolume were used as a measure of growth. Chlorophyll a and phaeophytin a determinations were used as an indication of health (Humana, 2004; Papageorgiou and Govindjee, 2009). All 4 measurements were recorded weekly alongside observations by using a Nikon x2000 compound microscope. A hemocytometer was used to calculate the number of cells per plate. The experimenter was the only one who carried out each of the measurements. This was to reduce chances of human error. The total number of cells was determined using the following calculations:

4,000 x average count = Total cells per ml

3.8 A photospectrometer was used for the chlorophyll a and phaeophytin a determinations. Chlorophyll a concentration was an indication of algal biomass.

The phaeophytin a was used as an indication of the degradation of chlorophyll a. The following equations were used to calculate the concentrations:

3.9 The biovolume was also used as an indication of the algal biomass and cell density (Carpentier, 2004; Saha, 2007). The following calculation was used to determine the biovolume where Sb equals the total volume per unit of water, C equals the number of organisms per ml and B¹ the mean biovolume per cell:

Sb = CB¹

Statistical Analysis

3.10 The data from the flask culture studies at week 1 and 4 were analysed by using an Anderson-Darling test which examined the normal distribution within the data. Subsequently a Levene’s test was carried out to identify any significant variances in the data. A One-way Independent ANOVA test occurred to identify the P-value of the trials when compared with the control. Finally a Tukey Post-hoc test was required to identify significant differences between the groups (Clewer and Scarisbrick, 2001; Adams, 2003). The statistical program Minitab 16 was used to execute these tests. When examining differences between each trial and control, the trial data was averaged at week 4 so that an equal number of data was compared.

RESULTS

Cell Count

4.0 From the descriptive statistics it was found that when glycerol was supplemented to Chlorella vulgaris, the cell count increased by 65.69% when compared with its control. When Scenedesmus subspicatus was compared with its control it had increased by 97.03%. Furthermore Nannochloropsis oculata cell count had incraede by 75.16% when subject to glycerol, as demonstrated in Figure 1.

4.1 Two inferential analyses occurred to determine normality and equal variance. The Anderson-Darling tests show that there was normality within the trial data: C. vulgaris P=0.231, S. subspicatus P=0.412 and N. oculata P=0.231. Furthermore it revealed there was normality within the control as demonstrated by: C. vulgaris P=0.0349, S. subspicatus P=0.415 and N. oculata P=0.534. The Levene’s test returned a P-value of 0.418 which showed there was equal variance within the control and trial (P=0.108). A One-way Independent ANOVA test occurred to due to both data being parametric. This test was to determine if there was a significant difference between the control and trial:

One-way Independent ANOVA Test F= 5448.87, P>0.001, n6=6

4.2 This establishes a significant difference within the cell counts. To further investigate, a Tukey’s Post-Hoc test occurred to determine where the differences lay:

From this test there were significant differences between all the trial and control data, though there was no significant difference found between 2 control groups as demonstrated by their differing letters.

Biovolume

4.3 C. vulgaris when subject to glycerol increased in biovolume by 96.56%. Likewise when glycerol was added to S. subspicatus its biovolume increased by 95.28% and there was a 75.16% biovolume increase in N. oculata, see Table 2.

4.4 The Anderson-Darling tests verified that there was normality within the trial data: C. vulgaris =0.408, S. subspicatus P=0.246 and N. oculata P=0.190. Likewise it also revealed that there was normality within the control: C. vulgaris P=0.534, S. subspicatus P=0.415 and N. oculata P=0.534.The Levene’s test provided a P-value of 0.220 which demonstrates that there was equal variance within the control and trial data (P=0.483). A One-way Independent ANOVA test was undertaken to determine if there was a significant difference between the control and trial data:

One-way Independent ANOVA Test F= 10683.92, P>0.001, n6=6

Grouping Information Using Tukey Method

FACTOR N Mean Grouping

CHLOR TRIAL 6 92984326 A

NANN TRIAL 6 85605401 B

SCEN TRIAL 6 47394720 C

NANN 6 22136810 D

CHLOR V 6 3187022 E

SCEN 6 2357960 E

Means that do not share a letter are significantly different.

4.5 This demonstrates a significant difference within the biovolumes. To further investigate, a Tukey’s Post-Hoc Test occurred to determine where the differences were:

From these tests there were significant differences between all the trial and control data, though there was no significant difference found between 2 control groups.

Chlorophyll a determination

4.6 This study revealed that when glycerol was inserted, the chlorophyll a content of C. vulgaris grew by 74.58%. There was a 66.62% inflation of chlorophyll a in S. subspicatus. Furthermore N. oculata increased in chlorophyll a content by 66.62%. See below for further details.

4.7 The Anderson-Darling tests verified that there was normality within the trial: C. vulgaris P=0.389, S. subspicatus P=0.420 and N. oculata P=0.604. The control data was also found to have normal distribution: C. vulgaris P=0.078, S. subspicatus P=0.446 and N. oculata P=0.382. The Levene’s test provided a P-value of 0.932 which demonstrates that there was equal variance within the control. However there was no equal variance within the trial (P=0.007). In turn a Kruskal-Wallis test occurred to determine if there was a significant difference between the control and trial due to the data being nonparametric:

Kruskal-Wallace Test H= 31.44, P>0.001, n6=6

4.8 This demonstrates a significant difference within the chlorophyll a determinations. To further analyse, Mann-Whitney U tests were carried out to determine where the differences were:

Mann-Whitney U test: W=57.0, P=0.005 (N=6,6) when comparing C. vulgaris with its control.

Mann-Whitney U test: W=57.0, P=0.005 (N=6,6) when comparing C. vulgaris – N. oculata.

Mann-Whitney U test: W=57.0, P=0.005 (N=6,6) when comparing S. subspicatus – N. Oculata.

From these tests there were significant differences between all the trials and controls.

Phaeophytin A determination

4.9 C. vulgaris increased in phaeophytin a content by 49.39% when glycerol was added; phaeophytin a also increased by 45.44% in S. subspicatus. When N. oculata was subject to glycerol, phaeophytin a levels rose by 66.53%, see Table 3 for further details.

4.10 The Anderson-Darling tests verified that there was normality within the trial data: C. vulgaris P=0.390, S. subspicatus P=0.636 and N. oculata P=0.516. Moreover the control data was found to have normality: C. vulgaris P=0.114, S. subspicatus P=0.899 and N. oculata P=0.066. The Levene’s test provided a P-value of >0.001 which demonstrated that there was no equal variance within the control. However there was equal variance within the trial data (P=0.059). A Kruskal-Wallis test was undertaken to due to the control data being nonparametric. This data was to determine if there was a significant difference between the trial and control:

Kruskal-Wallace Test H= 31.09, P>0.001, n6=6

4.11 This demonstrates a significant difference within the phaeophytin a determinations. To further analyse, Mann-Whitney U tests were carried out to determine where the differences lay:

Mann-Whitney U test: W=21.0, P=0.005 (N=6,6) when comparing C. vulgaris with its control.

Mann-Whitney U test: W=21.0, P=0.005 (N=6,6) when comparing S. subspicatus with its control.

Mann-Whitney U test: W=57.0, P=0.005 (N=6,6) when comparing S. subspicatus – N. Oculata.

From these tests there were significant differences between all the trials and controls.

Refer to Appendix 3 for Raw Data.

Refer to Appendix 4 for Observations and Algae Slides.

4.12 Figure 3 compares cell counts and biovolume readings of the control and trials. As demonstrated, biovolume readings peaked at 95,913,967.67µm³ ml^-1 when C. vulgaris was subject to glycerol. Additionally the cell count peaked at 39,586,840 per ml when glycerol was supplemented to N. oculata. However cell count and biovolume were constantly at their lowest during the control of S. subspicatus averaging 226,300.03 and 2,357,960.31.

4.13 Figure 4 illustrates the fluctuations in chlorophyll a and phaeophytin a determinations of the trial and control. There appears to be more of a difference between chlorophyll a and phaeophytin a within the trial. Within the control this difference appears to be closer as in the growth of S. subspicatus and N. Oculata. Chlorophyll a was at its highest concentration when N. oculata was subject to glycerol at week 3.

4.14 Figures 5 to 6 represent the end result of the trial algae species being subject to -10ºC. Chlamydomonas nivalis was added as a comparison to the green microalgae species. As seen in Figure 5, C. vulgaris saw a 57.27% increase in phaeophytin a and 64.84% increase in chlorophyll a levels when compared with C. nivalis. In Figure 6, C. vulgaris had a 2.09% increase in biovolume and a 30.73% decrease in cell count when compared with C. nivalis when subject to -10ºC.

Figure 5 Chlorophyll a and phaeophytin a determinations, green algae species comparison with C. nivalis taken at -10ºC (mg m-3) (n6=6)

DISCUSSION

5.0 There was a significant difference found in the cell counts of all 3 green microalgae samples when subject to 20% glycerol. When further analysed, Scenedesmus subspicatus was found to have increased by 97.03%, which was the largest increase; refer to Chapter 4, page 4.1 for further information. From the P-value of >0.001 the null hypothesis can be rejected.

5.01 This is supported by the notion that glycerol which is a type of lipid, has a lower freezing point up to -15ºC. Furthermore glycerol emits an overall positive charge which is then attracted by the overall negative charge of the algae’s outer membranes (Li et al., 2009; Lavoie, 2005). From this, both glycerol and the outer membranes can be attracted and glycerol can remain on the surface of the membranes until it is absorbed. Absorption can occur due to glycerol having a smaller molecular structure when compared with an alga’s outer membrane (Laginestra and Van-oorschot, 2011). Most cells like algae require lipids to be able to create hydrophobic barriers that allow portioning of the aqueous contents of the cells and subcellular structures. In addition, lipids are required to able to access proteins for energy production and energy storage (Newcombe et al., 2010; Warr et al., 1985).

5.02 As supported by Gonzalez-Bashan et al. (2000) when a cell is in contact with nutrients it requires that the cell’s outer membrane will start to allow them through by means of facilitated diffusion or by carrier proteins. Using this theory, when algae cells are subject to high levels of lipids they will utilise them to be able to catalyse and storage more energy. Within this study there was a clumping progression which was observed; refer to Appendix 4 Observations, page 3.0-5.0. This may be explained by the surplus supply of glycerol left floating in the samples, sticking cells together. This clumping may also indicate why the algae treated with glycerol was so successful at tolerating subfreezing temperatures.

5.03 This notion was supported by Gronlund (2002a) who observed algae biofilms forming which acted a bit like a ‘blanket’ insulating the algae below. This was demonstrated to increase cold tolerance by 37.5% due to this ‘insulation’ effect. Although in this investigation algae did not create a biofilm, it did however create small clumps of algae. When algae were grown in the control this clumping did not occur, which could be an indication that glycerol is the main cause. An additional benefit to this clumping could be that the algae have higher amounts of lipids, which have been metabolised by the cells. Lim et al. (2010) realised that the application of C. vulgaris to wastewater treatment could remove 85% of contaminates from wastewater. After each trial an average ratio of 8470:45:1 (C:H:N) was present, demonstrating the numbers of C. vulgaris cells. This demonstrates the potential use of C. vulgaris in bioremediation without any modifications at an optimal temperature of 20ºC. When Lim et al. (2010) reduced the temperature to 5ºC the effectiveness of the algae reduced by 50.3%.

5.04 Although this investigation revealed significant improvements in freeze tolerance in cell counts of 3 major green algae species, it did however occur within a controlled environment. As such this may not precisely reflect the outside environment. In contrast Gronlund’s (2002a) and Lim et als’. (2010) studies both occurred in an outside pool which was exposed to external conditions. They both monitored algae development throughout the year to gain an acute outlook. Though the outcomes of these studies concluded that a controlled environment would have formed a more precise measure, they did reinforce the need for research to be undertaken under controlled conditions. In isolation this research did actually remain at the set temperatures within a 5% margin of error. Although this research was undertaken within laboratory conditions it does nevertheless support the findings of Gronlund’s (2002a) study. When green microalgae were subject to temperature reductions of 2ºC, algae cell count reduced by 4.2%.

5.1 This study found significant differences in cell biovolumes of all 3 microalgae samples when glycerol was supplemented. On further scrutiny, C. vulgaris was found to have increased in biovolume by 96.56% which was the largest alteration. From the P-value of >0.001 the null hypothesis can be rejected. As the amount of glycerol increased so did the volume of the algae cells; see Chapter 4, page 4.2 for further information. This may be an indication that the glycerol was absorbed by the cells at such a rate that the cells began to swell. As the cells utilise lipids for energy production and subcellular compartmentalisation this may have been an effect caused by glycerol (Stolz, 2010; Wiencke and Gruyter, 2010).

5.12 To function, cells must avoid excessive alterations of their volume which could endanger their structural integrity (Scarella et al., 2010). As a cell increases in volume its surface area increases but at a slower rate, resulting in a surface area which is stretched and unable to process and exchange ions efficiently. The increases in cellular volumes were confirmed by this study. Refer to Chapter 4, page 4.4. This may be an illustration of the rapid growth but at a cost of reducing the efficiency of processing ions. Glycerol by its very nature sticks to negatively charged membranes which could obstruct cell openings and thus reduce efficiency of ion exchange (Wiencke and Gruyter, 2010). If correct, cell count would inevitably reduce as cells could not process enough ions used for their growth. However this study demonstrated that cell count increased when compared with the control.

5.13 Bhatnagar et al. (2010) placed 3 species of green algae within an anaerobic environment which was put under ^-10 bars of pressure. They revealed that the biovolume increased by 76.3%. Comparable to this study, Bhatnagar et al. (2010) revealed that the cell count of the algae remained constant and in some cases had increased. On further analysis, it was found that algae cells could tolerate high levels of pressure within in their cytoplasm. This is similar to the case of Chlamydomonas nivalis where the species can survive under pressures of up to ^-7.3 bars, which is known to increase intracellular compression and external tension owed to forming ice crystals from the surrounding water. This species of ice algae which shares a common ancestor to C. vulgaris has specialised openings within the outer membranes which could protect against these pressures (Osterhout, 2005). This pressure on the cell can result in increasing or decreasing the volume of C. nivalis without reducing the cell count.

5.14 Daligault et als’. (2003) study added deuterium to a sample of C. vulgaris at a rate of 50ppm and it was found to have strengthened the C-H bonds within the outer membranes, thus increasing freeze resistance. Although this study increased the freeze tolerance of C. vulgaris by 57.6%, it found that their algae’s volume increased rapidly. Similarly to Bhatnagar et als’. (2010) study, the cell count of their algae was found to have increased and not decreased which further supports the concept that algae cells can naturally tolerate high levels of volume and surface tension.

5.15 One limitation to testing the biovolume in this study may have been due to its controlled environment. As previously discussed a controlled environment may not be a good representation of the external environment. Nonetheless, in this example biovolume would most likely be different when under control conditions. A cell’s volume is subject to more pressure when cultured outdoors; this is due to temperature variations as water could freeze and thaw in the same day (Li et al., 2009). In contrast, within an incubator temperature variations are low and water within samples does not generally freeze and thaw so fast, thus the outer membranes are subject to more pressure.

5.2 This research revealed significant differences in chlorophyll a determination of 3 microalgae species when glycerol was added. On further inspection, C. vulgaris was found to have increased in biovolume by 74.58% which was the largest alteration. From the P-value of >0.001 the null hypothesis can be rejected.

5.21 Within most algal studies, chlorophyll a content is used as a measure of health and the levels of algae present in any given sample. Chlorophyll a is the principal pigment which is used in the process of photosynthesis. Thus as the concentration of chlorophyll a increases, the absorption of light increases which generates more energy (Metcalf and Tchobanoglous, 2002). In this study, chlorophyll a content increased by 70% with the supplementing of glycerol. This increase supports the theory that algae have robust mechanisms to cope with pressures related to their outer membrane, including osmotic tension. When this data is combined with the biovolume it verifies that the algae’s outer membranes were able to cope with the pressure created by the glycerol, potentially blocking the cell’s pores (Kaiser et al., 2011). If the cell’s openings were unable to cope with this, then logically the cell count and chlorophyll a content would decrease.

5.22 One factor which may have reduced the likelihood of these pores getting congested could have been the concentration of glycerol utilised. Using 100% concentration would have most likely obstructed the pores. However 20% concentration was selected due to Daligault et als’. (2003) research. Their experiment recommended investigating the effects of using natural sources of carbon and hydrogen, such as refined lipids to increase membrane strength. This research went further, stating an optimal concentration which could be used.

5.23 Makareviciene et al. (2011) and Lim et al. (2010) carried out research to investigate microalgae’s effectiveness of water filtration. They both used pools filled with freshwater, however Makareviciene et al. (2011) used salt pools as well as freshwater pools. Makareviciene et al. (2011) over a period of a year uncovered that as the NaCI concentrations increased so did the freeze resistance. On further analysis, they realised that this was due to 2 factors. Firstly, saltwater tends to have a lower freezing point when compared with freshwater. Secondly, algae subject to saltwater encounters automatically high levels of osmotic stress (Kaiser et al., 2011). This stress causes a slight imbalance of water and salts within the cell causing chlorophyll a concentration to increase.

5.24 This process is similar to dehydration, however as supported by Wong et al. (1994) and Wegmann et al. (1980) does not adversely affect the cells like dehydration would. To most other cells this process would result in rupturing the outer membranes, because the cytoplasm present within the cells would contract so much as to pull the membranes apart (Dainty et al., 1986; Sugiyama et al., 1991). Algae cells present in saltwater undergo a process in which chlorophyll a begins to become more concentrated, to prevent the cells losing ions. As the concentration of chlorophyll a increases and the surrounding fluid does not, this creates a sink where salts or ions cannot be removed osmotically (Chitnis, 1996; Pyle et al., 2008). This process may explain why the concentration of chlorophyll a increased during this investigation.

5.3 This research uncovered significant differences in phaeophytin a levels within 3 species of green algae when subject to glycerol. When further investigated, N. oculata increased in phaeophytin a levels by 66.53% which was the largest increase. From the P-value of >0.001 the null hypothesis can be rejected. Although the levels of phaeophytin a increased within the test groups, they were matched by the increases of chlorophyll a. Phaeophytin a is the measure of chlorophyll a pigment which has been damaged or made dormant. On average phaeophytin a levels were 22.63mg m³ lower than chlorophyll a levels, see Chapter 4, page 4.5 for further information.

5.31 Lim et al. (2010) and Gronlund (2002a) both used chlorophyll b which is the subsidiary photosynthesising pigment used in green algae. Using this pigment as a comparison to chlorophyll a can indicate the levels of productivity of a target sample. Chlorophyll a is the pigment which harvests photons to spilt H₂0 and chlorophyll b is the pigment which assists in the P650 reaction centre in photosystem I. Both studies found that when the concentration of chlorophyll a increased, chlorophyll b either remained constant or decreased. This is quite similar to phaeophytin a; however as the concentrations of phaeophytin a decrease, the more chlorophyll a there is likely to be. As stipulated by Lim et al. (2010) with temperature decreases of 2ºC the levels of phaeophytin increased by 8.4%. This figure resembles the control of this experiment. When all 3 microalgae samples were subject to 5ºC decreases in temperature during the control, phaeophytin a levels increased by 17.8%. When Daligault et al. (2003) injected deuterium into their samples of algae they noted that phaeophytin a levels decreased at the same rate as samples which had not been subject to deuterium. Daligault et als’. (2003) findings reinforce the results of this investigation and even go further by developing a chlorophyll a: phaeophytin a ratio. This ratio was 10:6 when C. vulgaris was exposed to -5ºC before deuterium and 32:11 after deuterium. In this research it was found that when C. vulgaris was subject to -5ºC the ratio before glycerol was 10:7 and 36:11 after glycerol.

5.32 Bhatnagar et al. (2010) demonstrated microalgae’s ability to grow heterotrophically in the absence of light. Though this seems highly advantageous to the algae’s survival Bhatnagar et al. (2010) uncovered that the levels of phaeophytin a were elevated when compared with their control. Nonetheless when water begins to freeze light levels naturally decrease causing this effect. This was a confounding variable which affected this trial; however it could not have been prevented as freshwater freezes below 0ºC. Using milli-Q water which has a freezing point of -5ºC would have prevented algae growth as suggested by Wiencke and Gruyter (2010).

5.4 Within this study there were 3 varieties of green microalgae which were recommended for study by Reza (2005) and Hegger (2010). This investigation however could have focused more on one specific genus. Chlorella would have been the logical choice as they were popularly used in biofuels and many of the species within this genus have a high potential for use in bioremediation. This focus on one genus could have allowed more detailed study of the mechanisms involved, the filtration efficiency and particular adaptations of them. If this was to be carried out C. minutissima and C. rehinhardtii as well as C. vulgaris would be interesting specimens to examine due to the high lipid content, ability to process wastewater and rapid growth patterns. Both these species are able to convert into a heterotroph in the absence of light and have low cold tolerances which need to be enhanced for industrial application. The genus Chlorella has been noted as a curial species which could enhance the processes involve in bioremediation, biofuel production and use of as a natural photovoltaic generator.

5.5 Another improvement to this investigation could have been to extend the times of the trials, more specifically between the temperature reductions. Instead of allowing 2 weeks per 5ºC decline, a 4 week period between these declines could have been allocated. Additionally the temperature could have been reduced at a smaller rate for instance, instead of 5ºC declines, 2.5ºC declines may have been applied. This slower decline may have had less of a sudden impact upon the algae cells, thus reducing confounding variables. In addition increasing the amount of plates used within each test group may have increased the viability of this experiment. As with many investigations, increasing the amount of subjects would understandably increase the significance of the results. As an alternative to using 6 plates per test group, 12 could have been used. This would have resulted in over 36 plates per trial and 108 per experiment. With both improvements the experiment would occur over an extra 8 week period with an additional 50% more samples.

5.6 More research is required to determine what concentrations of glycerol could be used to sustain this new freeze resistance. 20% glycerol was utilised in this experiment but reducing the amount of lipids used, could decrease the industrial cost of application. Refer to Appendix 5 for industrial scaling. A possible minimal glycerol concentration could be between 5-8%. This would reduce the amount and thus the cost of glycerol required to sustain algae freeze tolerance. Another area for research is to uncover what other effective species could be used for bioremediation. Such species as Lemna major or ‘duckweed’ have a high potential for wastewater filtration but share the same issues in regards to freeze resistance to microalgae (Watanabe, 2001; Ben-Amotz and Avron, 1973). This simple 3 leafed aquatic plant has a higher rate of water filtration when compared with microalgae and research is being undertaken for its use in biofuels. A possible natural solution could be to enhance the thickness of the cuticle by means of genetic modification or by heightened selective breeding techniques.

5.7 In summary, the cell counts, biovolumes, chlorophyll a and phaeophytin a determinations all have demonstrated significant differences, verifying the alterative hypothesis and the theory that 20% glycerol improves the freeze tolerance of the microalgae. Increasing the amount of samples used over a longer period of time could have improved the reliability of this experiment. Focusing on one genus such as Chlorella may generate understanding of species which had not been studied thoroughly enough for industrial application. Avenues for future research could include determining what minimal concentration would be required to ensure that the algae’s freeze tolerance is sustained. Moreover, studying Lemna major which has high potential for wastewater filtration and biofuel production would aid the goal for using biotechnology to make wastewater treatment more sustainable.

CONCLUSION

6.1 3 common species of green microalgae were used in this investigation to determine if supplementing glycerol could increase and sustain their freeze tolerance. As global warming, climate change and sustainability become more prevalent issues for countries across the globe, new technologies are required to resolve them. Already in place is a form of wastewater treatment or bioremediation which is highly popular, within tropical climates such as Australia and Malaysia. However this new system becomes highly ineffective when it is applied to cooler climates such as the UK. From this research, the freeze tolerance has been enhanced. Using Chlorella vulgaris as the most commonly researched specimen for bioremediation led to the following outcomes: cell count and biovolume increased by 96.57%, chlorophyll a content increased by 72.90% and finally the chlorophyll a to phaeophytin a ratio was 36:11 which was over 3 times more, when compared with the control.

6.2 One concern which was highlighted by Scarella et al’s. (2010) research was the rapid inflation of the algae’s volume, but under further analysis, this study saw a rapid increase in both cell counts and chlorophyll a determinations. This demonstrated that the samples were able to cope with the osmotic pressure caused by the surplus glycerol (for more information refer to Chapter 5, Page 5.4). Referring back to the main aim of this research, the UK horticultural industry could benefit from this new understanding. Not only UK horticulture but all horticultural industries in temperate climates could benefit, as over 84% of wastewater produced by horticulture is released back into rivers and seas without correct treatment (Driscoll et al., 2013; Boland et al., 2006; Mapanda et al., 2005; Radcliffe, 2004; Brown and Cooper, 2005). Increasing the freeze tolerance of these algae, wastewater could be treated all year around by natural means. Another advantage of using this system would be that after scheduled cycles algae could then be harvested. This harvest could then be used for biofuels, thus increasing sustainability of water treatment, becoming more environmentally friendly and providing a source of renewable energy.

6.3 One improvement which could have been implemented in this experiment could have been to focus on one genus, particularly Chlorella. This focus on one genus could have allowed more detailed study of the mechanisms involved, the filtration efficiency and particular adaptations of them. Furthermore this investigation could have extended the times of the trials, more specifically between the temperature reductions. Instead of allowing 2 weeks per 5ºC decline, a 4 week period between these declines could have allowed. This may have had less of a sudden impact upon the algae cells.

6.4 More research is required to determine what concentrations of glycerol could be used to sustain this new freeze resistance. 20% glycerol was utilised in this experiment but reducing the amount of lipids used, could decrease the industrial cost of application. Another area for research could include what other species could be used for bioremediation. Such species as Lemna major have a high potential for wastewater filtration but share the same characteristics in regards to freeze resistance to microalgae. A possible solution could be to enhance the thickness of the cuticle by means of genetic modification or by selective breeding.

REFERENCES

4^th Algae World Europe Conference. 2012. Are algae players geared for new levels of complexities? [On-line]. A.W.E.C. http://www.cmtevents.com/aboutevent.aspx?ev=120526 [29 September 2012].

Acreman, J. 2010. Modified Chu-10 Medium for algal cultures. [On-line]. www.phycol.ca/system/files/CHU_10.pdf [Accessed 27 September 2012].

Adams, D. 2003. Lab Math: A Handbook of Measurements, Calculations and Other Quantitative Skills for Use at the Bench. New York: Cold Spring Harbour Laboratory Press.

Allaby, M. 2008. A Dictionary of Plant Sciences. 2^nd ed. Oxford: OUP Oxford

Allen, F. 2012. Protein phosphoration in regulation of photosynthesis. Biochimica Biophysica Acta, 1098 (43), 275-335.

Bajguz, A. 2012. Suppression of Chlorella vulgaris growth by cadmium, lead, and copper stress and its restoration by endogenous brassinolide. Environmental Contamination and Toxicology, 60 (3), 406-416.

Barbar, J. and Anderson, B. 2004. Revealing the blueprint of photosynthesis. Nature, 460 (9), 31-34.

Bayer-Giraldi, M. 2010. Antifreeze proteins in polar sea ice diatoms: diversity and gene expression in the genus Fragilariopsis. Environmental Microbiology, 12 (4), 141-152.

BBC. 2011. Panorama - Algae: Fuel of the future. UK: BBC 1, 15^th July, 20:30.

Ben-Amotz, A. and Avron, M. 1973. The role of glycerol in the osmotic regulation of the halophilic alga Dunaliella parva. Plant Physiology, 51(9), 875-878.

Bhatnagar, A. Bhatnagar, M. Chinnasamy, S. and Das, K. 2010. Chlorella --a promising fuel alga for cultivation in municipal wastewaters. Applied Biochemistry and Technology, 161 (8), 523-536.

Bhatnagar, A. Bhatnagar, M. Chinnasay, S and Das, K. 2009. Chlorella – a promising fuel alga for cultivation in municipal wastewaters. Applied Biochemistry and Biotechnology, 16 (8), 523-536.

Blankenship, R. 2001. Molecular Mechanisms of Photosynthesis. In: GBJ Armstrong. Ed. Mechanisms in Photophosphorylation and Carboxylation. Oxford: John Wiley & Sons. pp. 67-86.

Boland, A. Hamilton, A. Stevens, D. and Ziehrl, A. 2006 Opportunities for reclaimed water use in Australian agriculture: In growing food crops with reclaimed wastewater. Melbourne: CSIRO Publishing.

Borowitzka, M. and Moheimani, N. 2012. Algae for Biofuels and Energy (Developments in Applied Phycology). New York: Springer

Brown C. and Cooper, E. 2005. Bioenergetics: An practical approach. Oxford: Oxford OUP.

Brown, P. 2009. The cream of low-carbon ideas, the future wastewater management system. Guardian, 24 February, 6.

Carpentier, R. 2004. Photosynthesis Research Protocols (Methods in Molecular Biology). New York: Humana Press. P. 49, 113.

Carrington, D. 2010. Innovation award for ‘bubble-maker’ that boosts algae growth. Guardian, 11 September, 27.

Chitnis, V. 1996. Concepts in photobiology: photosynthesis and photomorphogensis. Oxford: OUP Oxford.

Clewer, A. and Scarisbrick, D. 2001. Practical statistics and experimental design for plant and crop science. New York: Wiley.

Cooper, J. 2009. Photosynthesis and Productivity in Different Environments (International Biological Programme Synthesis Series). Cambridge: Cambridge University Press.

Daigger, G. and Eckenfelder, W. 1998. Upgrading Wastewater Treatment Plants (Water Quality Management Library). 2^nd ed. Boca Raton: CRC Press

Dainty, A. Goulding, K. Robinson, P. Simpkins, I. and Trevan, M. 1986. Stability of alginate-immobilized algal cells. Biotechnology and Bioengineering, 28 (2), 210-216.

Daligault, F. Reed, D. Savile, C. Nugier-Chauvin, C. Patin, H. Covellao, P. and Buist, P. 2003. Mechanistic characterisation of ώ-3 desaturation in green alga Chlorella vulgaris. Phytochemistry, 63 (5), 739-744.

Deutsch, C. 2008. A single source for clean water and fuel. New Scientist, 2807 (5), 345-351.

Driscoll, W. Espinosa, N. Eldakar, O. Hackett, J. 2013. Allelopathy as an emergent, exploitable public good in the blood-forming microalgarymnesium parvum. Evolution, 10 (11), 120-128.

Etnier, C. and Guterstam, B. 1996. Ecological Engineering for Wastewater Treatment. 2^nd ed. Boca Raton: CRC Press.

Friday, E. Micheal, A. Ayodele, S. 2012. Mixed Cultivation of Euglena Gracilis and Chlorella Sorokiniana: A method of production of single cell protein/vitamins rich algae biomass in large scale. Washington: LAP LAMBERT Academic Publishing.

Furuya, M. 2003. Phytochromes: their monocular species, gene families and functions. Annual Review of Plant Physiology and Plant Molecular Biology, 44 (7), 617-645.

Gacheva, G and Polarski, P. 2008. The Resistance of a New Strain Chlorella SP. R-06/2 Isolated From an Extreme Habitat to Environment Stress Factors. Plant Physiology, 35 (28), 347-352.

Gonzalez-Bashan, L. Lebsky, V. Hernandez, J. Bustillos, J. and Bashan, Y. 2000. Changes in the metabolism of the microalgae Chlorella vulgaris when coimmobilized in alginate with the nitrogen-fixing Phyllobacterium myrsinacearum. Journal of Microbiology, 46 (7), 653-659.

Gorman, D. and Levine, R. 2005. Gorman and Levine modified media. Practical National Academic of Science, 54 (8), 1665-1669.

Gotingen, S. 2008a. Culture Collection of Algae: Medium Recipes. Methods in Cell Physiology, 8 (1), 152-187.

Gottingen, S. 2008b. Culture collection of algae: Bold’s Basal Medium. [On-line]. E.P.S.A.G. Available from: www.epsag.uni-goettingen.de [Accessed 28 August 2012].

Graham, J. Wilcox, L. and Graham, L. 2008. Algae. 2^nd ed. San Francisco: Benjamin Cummings.

Gray, G. Chavin, L. Sarhan, F. and Huner, N. 1997. Cold Acclimation and Freezing Tolerance (A Complex Interaction of Light and Temperature). Plant Physiology, 114 (2), 467-474.

Gray, N. 2010. Water Technology: An Introduction for Environmental Scientists and Engineers. 3^rd ed. London: Butterworth-Heinemann.

Great Britain. House of Parliament. 2011. Parliamentary Office of Science and Technology. Biofuels and wastewater treatment from algae. London: The Parliamentary Office of Science and Technology. (HOPST 2010-2011(15)).

Gronlund, E. 2002a. Microalgae at wastewater treatment in cold climate: A thesis submitted to the University of Lulea for the award of Doctor of Phycology. Norrbotten, Lulea: Lulea University of Technology.

Gronlund, E. 2002b. European Parliamentary Report: Microalgae at wastewater treatment in cold climate. Lucea University Press: Lelea.

Hall, D. and Rao, K. 1999. Photosynthesis (Studies in Biology). In Institute of Biology. Ed. Light absorption and the two photosystems. 6^th ed. Cambridge: Cambridge University Press. pp. 58-75.

Hannan, J., Shutler, L. and Patouillet, C. 2003. A Study of the Feasibility of Oxygen Produced by Algae in Nuclear Submarines. Organic and Biological Chemistry, 17 (4), 239-245.

Hegger, K. 2010. Wastewater treatment by novel hybrid biological – Ion exchange processes. University of Illinois: Illinois.

Hegger, K. 2010. Wastewater treatment by novel hybrid biology – ion exchange process: A thesis submitted to the University of Illinois for the award of Master of Agriculture and Biological engineering. Urbana, Illinois: the University of Illinois.

Henrikson, R. and Edwards, M. 2012. Imagine Our Algae Future: Visionary Algae Architecture and Landscape Design. Washington: CreateSpace Independent Publishing Platform.

Hoek, C. Mann, D. and Jahns, H. 1996. Algae: An Introduction to Phycology. Cambridge: Cambridge University Press.

Humana, R. 2004. Photosynthesis research protocols. New Jersey: Carpentier Press.

ICIS Chemicals. 2013. Current global prices of glycerol. [On-line]. I.C.I.S. http://www.icis.com/search-results/?key=glycerol&filter=16&page=1&searchType=Free Text Search [Accessed 11 February 2013].

Kaiser, M. Attrill, M. Jennings, S. Thomas, D. Barnes, D. Brierley, A. Hiddink, J. Kaartokallio, H. Polunin, N. and Raffaelli, D. 2011. Marine Ecology: Processes, Systems, and Impacts. 2^nd ed. Oxford: Oxford University Press.

Kanno, T. 2005. Chlorella Vulgaris and Chlorella Vulgaris Extract. Salt Lake City: Woodland Publishing Inc.

Kay, W. 2010. Top guns fly on algae in the US battle for fuel. The Times, 21, December, 13.

Ke, B. 2001. Photosynthesis Photobiochemistry and Photobiophysics (Advances in Photosynthesis and Respiration). New York: Springer.

Kim, S. 2011. Handbook of Marine Macroalgae: Biotechnology and Applied Phycology. Oxford: Wiley-Blackwell.

Kirk, O. 2004. Light and photosynthesis in aquatic species. 2^nd ed. Cambridge: Cambridge University Press.

Kirke, B. 2006. Growing microalgae for C0₂ sequestration, wastewater remediation, fuel and other valuable products: A thesis submitted to The University of South Australia for the award of Doctor of Biological engineering. Mawson Lakes, South Australia: The University of South Australia.

Laginestra, M. and Van-oorschot, R. 2011. Wastewater treatment pond systems – an Australian experience. In Australian Wastewater Organisation. Annual report 2010-2011. New South Wales. pp. 34-38.

Lavoie, D. 2005. Modelling ice algae growth and decline in seasonally ice-covered region of the arctic. Journal of Geophysical Research, 110 (10), 29-34.

Lawlor, D. 2000. Photosynthesis. 3^rd ed. Oxford: Garland Science.

Lee, K. and Lee, C. 2001. Effect of light/dark cycles on wastewater treatments by microalgae. Biotechnology, 18 (6), 194-199.

Leegood, R. and Lea, P. 1998. Plant Biochemistry and Molecular Biology. 2^nd ed. Oxford: John Wiley & Sons.

Li, H. Liu, X. Wang, Y. Hu, H. and Xu, X. 2009. Enhanced expression of antifreeze protein genes drives the development of freeze tolerance in an Antarctica isolate of Chlorella vulgaris. Natural Science, 19 (6), 1059-1062.

Lighton, A. 2012. The Big Picture: Algae, curse or cure? The Times, 23 April, 11.

Lim, S. Chu, W. and Phang, S. 2010. Use of Chlorella vulgaris for bioremediation of textile wastewater. Bioresource Technology, 101 (12), 714-722.

Liu, D. and Liptak, B. 1999. Wastewater Treatment. Boca Raton: CRC Press.

Lobban, C. 1996. Seaweed Ecology and Physiology. Cambridge: Cambridge University Press.

Luz, G. L. Lebsky, V. Hernandez, J. Bustillios, J. and Bashan, Y. 2000. Changes in the metabolism of the microalga Chlorella vulgaris and when coimmobilized in alginate with the nitrogen-fixating Phyllobacterium myrsinacearum. Microbiology, 46 (23), 653-659.

Makareviciene, V. Andrulevicute, V. Skorupskaite, V. and Kasperoviciene, J. 2011. Cultivation of microalgae Chlorella sp. and Scenedesmus sp. as a potential biofuel feedstock. Environmental Engineering and Management, 3 (57), 21-27.

Mapanda, F. Mangwayana, E. Nyamangara, J. and Giller, K. 2005. The effect of long-term irrigation using wastewater on heavy metal contents of soils under vegetables in Harare, Zimbabwe. Agriculture, Ecosystems & Environment, 107 (2), 151-165.

Meimers, K. Papadimitriou, S. Thomas, D. Norman, L. and Dieckmann. 2009. Biogeochemical conditions and ice algal photosynthetic parameters in Weddell sea ice during early spring. Polar Biology, 10 (7), 107-113.

Melnikov, A. 1999. Arctic Sea Ice Ecosystem. Boca Raton: CRC Press.

Metcalf, E. and Tchobanoglous, G. 2002. Wastewater Engineering: Treatment and Reuse. 4^th ed. New York: McGraw-Hill Higher Education.

Met Office. 2012. Frost and cold climates statistics of the U.K. [On-line]. M.O. Available from: http://www.metoffice.gov.uk/climate/uk/ [Accessed 26 September 2012].

NASA. 2012. The OMEGA Project: How algae can be used to produce energy and to purify wastewater. [On-line]. NASA. Available from: http://www.nasa.gov/centers/ames/research/OMEGA/index.html [Accessed 17 September 2012].

Newcombe, G. House, J. Ho, L. Baker, P. and Burch, M. 2010. Water Quality Research Australia: Management Strategies for Cyanobacteria (blue-green algae) – a guide for water utilities. Adelaide: CRC for Water Quality and Treatment.

Northumbria Water Group PLC. 2009. The cost of maintaining and developing wastewater treatment systems 2009. Durham: Northumbria Water.

Obata, M. and Taguchi, S. 2009. Photoadaptation of an ice algal community in thin sea ice, Saroma-Ko Lagoon, Hokkaido, Japan. Polar Biology, 32 (9), 1127-1135.

Oilgae. 2010. Oilgae guide to future algae-based water treatment. [PDF]. O.A. Available from: http://www.fao.org/uploads/media/0911_Oilgae_-_Wastewater_Treatment_Using_Algae_Report_Preview.pdf [Accessed 20 September 2012].

Osterhout, W. 2005. The Temperature Coefficient of Photosynthesis. The Journal of General Physiology, 13 (6), 295.

Papageorgiou, G. and Govindjee, G. 2009. Chlorophyll Fluorescence: A Signature of Photosynthesis (Advances in Photosynthesis and Respiration). New York: Springer.

Pentecost, A. 1984. Introduction to Freshwater Algae. Slough: Richmond Publishing Ltd.

Pyle, D. Garica, R. and Wen, Z. 2008. Producing docosahexaenoic acid (DHA)-rich algae from biodiesel-derived crude: effects of impurities on DHA production and algal biomass composition. Journal of Agricultural and Food Chemistry. 56 (11), 3933-3939.

Radcliffe, J. 2004. Water Recycling in Australia. Victoria: Australian Academy of Technological Sciences and Engineering.

Reza, N. 2005. The culture of coccolithophorid algae for carbon dioxide bioremediation: A thesis submitted to Murdoch University for the award of Doctor of Biotechnologies. South Australia: Murdoch University.

Richmond, A. 2003. Handbook of Microalgal Culture. Oxford: Wiley-Blackwell.

Robinson, D. Kolber, Z. and Sullivan, C. 1997. Photophysiology and photoaclimation in surface sea ice from McMurdo Sound, Antarctica. Marine Ecology Progress Series,147 (6), 243-256.

Russel, N. 2000. Psychrophily and resistance to low temperature. Extremophiles, 12 (5), 532-545.

Saha, L. 2007. A Textbook of Algae. New Delhi: CBS Publishers & Distributors.

Sanghera, G. Wani, S. Hussain, W. and Singh, N. 2011. Engineering cold stress tolerance in crop plants. Current Genomics, 12 (1), 30-43.

Scarella, M. Belotti, G. De Fillippis, and P. Bravi, M. 2010. Study on the optimal growing conditions of Chlorella vulgaris in bubble column photobioreactors. Eudossiana, 18 (10), 100-118.

Schmidt, M. 2010. Algae are doing the dirty work at the wastewater facility. [On-line]. P.I.N. Available from: http://progress-index.com/news/algae-is-doing-the-dirty-work-at-wastewater-facility-1.540325 [Accessed 20 September 2012].

Seckbach, J. and Gordon, R. 2012. The Science of Algal Fuels: Phycology, Geology, Biophotonics, Genomics and Nanotechnology (Cellular Origin, Life in Extreme Habitats and Astrobiology). New York: Springer.

Sharma, J. 2009. Dictionary of Microbiology. New Delhi: CBS Publishers & Distributors.

Sharma, R. Singh, G. and Sharma, V. 2011. Comparison of different media formulations on growth. morphology and chlorophyll content of green alga, Chlorella vulgaris. International Journal of Pharma and Bio Sciences, 2 (2), 492-509.

Stolz, J. 2010. Structure of phototropic prokaryotes. Oxford: Oxford OUP.

Sugiyama, J. Vuong, R. and Chanzy, H. 1991. Electron diffraction study on the two crystalline phases occurring in native cellulose from an algal cell wall. Macromolecules, 24 (14), 4168-4175.

Tiquia-Arashiro, S. 2012. Molecular Biological Technologies for Ocean Sensing. New York: Humana Press.

UK National Culture Collection. 2001. Catalogue of the UK national culture collection: list of algae and protozoa. Ambleside: UKNCC Publishing.

Uttley, P. Wilkinson, S. and Plamer, S. 2010. Integration of biological wastewater treatment and algae growth for biofuel. Future, 8 (2), 5.

Wang, B. and Lan, C. 2011. Biomass production and nitrogen and phosphorus removal by the green algae Neochloris oleoabundans in simulated wastewater and secondary municipal wastewater effluent. Bioresources and Technology, 102 (10), 539-544.

Warr, S. Reed, R. and Stewart, W. 1985. Carbohydrate accumulation in osmotically stressed cyanobacteria (blue-green algae): interactions of temperature and salinity. New Phytology. 100 (14), 285-292.

Watanabe, M. 2001. Can bioremediation bounce back? Nature Biotechnology, 19 (7), 1111-1115.

Watanbe, Y. Yamada, N. Machida, T. Honjoh, K. and Kuwano, E. 2011. Influence of cold hardening on Chlorophyll and carotenoid in Chlorella vulgaris. Plant Physiology, 22 (8), 455-462.

Wegmann, K. Ben-amotz, A. and Avron, M. 1980. Effect of temperature on glycerol retention in the halotolerant alga Dunaliella and Asteromonas. Plant Physiology, 66 (7), 1196-1197.

Whitton, B. and Brook, A. 2011. The Freshwater Algal Flora of the British Isles: An Identification Guide to Freshwater and Terrestrial Algae. 2^nd ed. Cambridge: Cambridge University Press.

Wiencke, C. and Gruyter, D. 2010. Biology of Polar Benthic Algae: Marine and Freshwater Botany. Boca Raton: CRC Press.

Wiessner, W. Robinson, D. and Starr, R. 2012. Cell Walls and Surfaces, Reproduction, Photosynthesis (Experimental Phycology). 3^rd ed. New York: Springer.

Wong, S. Nakamoto, L. and Wainwright, J. 1994. Identification of toxic metals in affected algal cells in assays of wastewaters. Journal of Applied Phycology, 6 (4), 405-414.

Met Office. 2012. Frost and cold climates statistics of the U.K. [On-line]. M.O. Available from: http://www.metoffice.gov.uk/climate/uk/ [Accessed 26 September 2012].

ACRONYMS USED

~ - Approximation

µl - Microlitre

0_{2 -} Oxygen

Abs - Absorption bandwidth spectrum

C – Carbon

C0_{2 -} Carbon dioxide

C₆H₈O₇ - Citric acid

Ca(NO₃)2 - Calcium nitrate

CaCI₂ (2H₂0) - Calcium chloride

CoCI₂ (6H₂0) - Cobalt chloride

CuCI₂ - Cupric chloride

CuSO₄ 5H₂0 - Calcium sulphate

dH₂0 - Distilled water

EDTA - Ethylenediamine tetra-acetic acid

Fe(C₆H₅O₇)₂ - Ferric Ammonium citrate

FeCI₂ (7H₂0) - Ferric chloride

GF/C - Borosilicate glass structure

(NH₃) 6Mo₇0₂₄ - Ammonium heptamolybdate

H – Hydrogen

H_{0 -} Null hypothesis

H_{1 –} Hypothesis

H₃BO_{3 -} Boric acid

HCI - Hydrogen chloride

K₂HPO₄ - Dipotassium phosphate

MgC0_{3 -} Magnesium carbonate

MgSO₄ (7H₂0) - Magnesium sulphate

MnCI₂ (4H₂0) - Manganese chloride

MnCI₂ - Manganese chloride

Na₂CO₃ - Sodium carbonate

Na₂SiO₃ 9H₂0 - Sodium silicate

NaNO₃ - Sodium nitrate

NH₄CI - Ammonium chloride

N – Nitrogen

nm – Nanometres

P – Phosphorous

rpm - Revolutions per minute

ZnCI₂ - Zinc chloride

ZnSO₄ - Zinc sulphate

Back to top