INTRODUCTION

1.0 Lemna major has been identified by several major papers within the field of bioremediation as a promising means of water filtration. This simple free-floating plant has attracted the attention of governments around the world including: Bangladesh, Israel and the US; who have heavily invested in this new avenue of research as a possible means of grey-water reconditioning (Jenssen et al., 2004). A litre sample of grey-water contains 57.2g of Kjeldahl nitrogen and 63.5g phosphorous alongside trace amounts of: S, Mg₂⁺, K⁺, Fe₂^- and Ca₂⁺ (Gunther, 2000). Alaert et al. (1996) demonstrated that L. major can remove 74% of nitrogen and 77% of phosphorous from a Bangladesh sewage lagoon with a hydraulic retention time of 21 days. Furthermore, Culley et al. (1981) stipulated that a mixture of Lemna species could remove 1,378kg of nitrogen, 347kg of phosphorous and 441kg of potassium from a hectare of wastewater in a year under temperate conditions.

1.1 As a plant for wastewater treatment, Lemna major has several advantages over other macrophytes including Eichhornia crassipes (water hyacinth) and microphytes including Chlorella vulgaris (microalgae). Lemna produces 50% more 0₂, capturing 40% more C0₂ as other studied genus and has a high rate of nutrient uptake, especially NH₄⁺ (Jefferson et al., 2004). NH₄⁺ as waste organic sources of nitrogen can encourage excess blooms of microalgae and phytoplankton. Once these blooms begin to decay at the sea or lake bed they promote excess growth of anaerobic bacteria. As a result of these bacteria feeding off these blooms they begin to cause hypoxia in the surrounding water, inducing reductions in plant and animal populations. This process is known as eutrophication. Eutrophication is said to cause over 35% of species extinctions within hydroecological systems across the globe and this figure is set to increase to 54% in 10 years (Alvaradoa et al., 2008; Volnlanthen et al., 2012; Harnik et al., 2012).

1.2 Biotechnologists have stipulated that L. major could be used for wastewater whilst being harvested for biofuels. Potentially this could mean that Lemna used in bioremediation could help to reclaim land which has been used for ‘energy crops’ (Daigger and Eckenfelder, 1998; Hegger, 2010). Energy crops have been controversial as they take up land which could be used for food. Additionally, it has been reported that the lipid content of energy crops is 34% lower than in Lemna major. As a result the EU has placed a cap on a member state’s output of energy crops to 5% of total crop turnover (Deutsch, 2008; Lawlor, 2000). Lemna have also been demonstrated to be a viable source of organic fertiliser for land-based industries. Together the above factors, demonstrates the economic ability of Lemna as a source of grey-water reconditioning, fertiliser and fuel.

1.3 Although the application of Lemna as a source of industrial wastewater treatment has been established as effective method of pollution control, what about domestic treatment? Little research has actually been carried out to see if Lemna major could be used as a source of domestic grey-water reconditioning. Though up to 80% of agricultural wastewater is return to lakes and seas without sufficient treatment; this only accounts for an estimated 45% of global eutrophication causes (Vogt et al., 2013; Cusell et al., 2013). An estimated 40% of eutrophication pollutants are as a result of domestic sources according to a UN report on water contamination (Begum, 2009). Together with the rising cost of water this treatment, this could bring about a low cost and environmental friendly method of domestic recovering grey-water.

HYPOTHESES

1.4 The aim of this study is to examine the effectiveness of using Lemna major in the process of water filtration. If proven to be effective, these simple plants could then be used within the domestic sector as a natural and sustainable source of wastewater treatment. The following are to be investigated throughout this study:

Alternative Hypothesis

Lemna major will alter in frequency and mass whistle altering the levels of contamination in the medium.

Null Hypothesis

Lemna major will not alter in frequency and mass whistle altering the levels of contamination in the medium.

LITERATURE REVIEW

Membrane interactions

2.0 Since the early 1970s, considerable research has been done on the use of Lemna as a means of treating wastewater of both agriculture and domestic origin. In order for Lemna major to proceed with bioremediation the organic pollutants must first, cross Lemna’s membranous matrix. Membranes are composed of a mixture of positive phospholipids and negative glycolipids; forming a self-sealing bilipid layer that forms a semipermeable barrier (Alberts et al., 2012; Wiessner et al., 2012; Robinson et al., 1997). This allows the transport of ions by means of facilitated diffusion and osmosis. This bilipid barrier is strengthened by their hydrophilic head which is attracted to the surface and their hydrophobic tails which are attracted towards the intracellular matrix (Mikandawire and Dudel, 2005; Leegood and Lea, 1998). Ions including NH₃^- and P0₄ are unable to pass through the membranes without assistance, unlike water molecules which is controlled by the laws of osmosis (Prassad et al., 2011).

2.1 The assistance needed for ion transportation comes in the form of specialised ion channels which are embedded into the membrane. This is the first stage of facilitated diffusion. These channels are controlled by transmembrane proteins that bind to their specific ion; changing their conformation to deposit those ions across the membranes into the cell’s cytosol, where it is broken down by enzymatic degradation (Oron et al., 2007; Popa et al., 2006). Once this transaction has occurred the protein returns to its original conformation to continue this process (Prytz et al., 2003).

2.2 With the above process occurring many thousands of times a minute, it creates a membrane potential. Plasma membrane function is similar to a combined resistor and capacitor. Resistance arises from the fact that the membrane impedes the movement of charges across it (Hegger, 2010; Laginestra and Van-oorschot, 2011; Wang, 2012). Capacitance arises from the fact that the mosaic bilayer is so thin, that an accumulation of charged particles on one side gives rise to an electrical force. This force pulls oppositely charged particles toward the other side most notably: CI^-, Na⁺ and K⁺ are exchanged (Alberts et al., 2012; Mikandawire et al., 2005). The membrane uses this to create a potential which is curial for providing power to operate a variety of molecular devices such as H⁺ -ATPase; that are embedded in the membrane, effectively powering and depowering a battery (Ullich et al., 1991).

Bioaccumulation capacity

2.3 When L. major cells are exposed to nitrates, sulphates or phosphates the major sources of nutrition, an initial depolarization of the membrane is induced rendering the cytoplastic matrix more positive. Depolarization is followed by repolarization of the membrane, which enhances H⁺-ATPase activity. Another characteristic feature of anion uptake is that it is regulated (Lockhhart et al., 1989; Mann et al., 2008). In the case of phosphate and sulphate, high-affinity transporter activities are derepressed when the plants are deprived of the nutrient. In the case of nitrate, high-affinity transporters are induced by nitrate and are feedback inhibited by reduced and organic forms of nitrogen (Landolt, 1980; Les et al., 2002; Zimmo et al., 2000). These responses allow plants to adjust anion uptake to environmental conditions and internal metabolism.

2.4 Assimilation of nitrate, the primary source of nitrogen for plants involves the reduction by the enzyme nitrate reductase (Miranda and IIangovan, 2006). The resulting ammonia is then incorporated into organic form which can then be used for the construction of molecules required for a cell’s structure. Species of Lemna tend to convert organic nitrogen like NH₃ into carbon at an average rate of 15 g m^–2 day^–1, followed by a conversion rate of 30% proteins in the plant’s dry weight (Hammounda et al., 1995; Valderrama et al., 2002). Lemna has also got the ability to bioabsorb inorganic forms of nitrogen and other elements. The plant does not have the capability to deconstruct inorganic forms into smaller molecules to construct organic substances (Sooknah and Wilkie, 2004). The cells accumulate and store these inorganic elements into their cell wall and vacuole which can then be used for their progeny’s infrastructure. This process is repeated until all the inorganic elements have been exhausted (Korner et al., 2003; Korner et al., 2008).

2.5 Comparable to organic and inorganic nitrates, organic phosphates such as P0₄ can be converted into available molecules which is then metabolised to produce nucleic acids, phospholipids and ATP (Mikandawire et al., 2004; Cecal et al., 2002). However the maintenance of stable cytoplasmic Pi concentrations is essential for many enzyme reactions unlike inorganic nitrogen which can only be accumulated or stored (Obek and Hassar, 2002).

Review

2.60 Cheng et al. (2002) conducted a study into the potential use of Lemna minor 8627 as a source of bioremediation for the removal of nitrogen and phosphorous waste in swine waste lagoons. Cheng et al.’s (2002) investigation was first carried out in a laboratory and then in the field. Within the laboratory, sterilise cultures of L. minor were grown within a growth chamber with 3% sucrose 2 weeks prior to synthetic lagoon media. Light flux density was constantly kept at 40µmol m^-1 s-¹ for a 16 period. Temperature, light levels and initial biomass were kept constant. An artificial mixture of nutrition content was derived from Al-Nozality et al.’s (2000) study which determined the macronutrient content to be NH4 336mg/L, K 483.6mg/L and P 23.80mg/L. The L. minor was culture in 300ml boxes which had a surface area of 25.8cm². These boxes were then placed into a second growth chamber. Test dilutions of the synthetic combination were as flows: 25%, 50%, 75% and 100%.

2.61 Each batch test consisted of 52 vessels containing the dilutions. Of these vessels, 39 contained L. minor cultures corresponding to the triplicate samples for 13 times points, while 13 control boxes did not have any Lemna. Each vessel was grown until the L. minor covered the surface. The control did not contain any Lemna. The samples were cultured at 23°C and 1 sample was taken for destructive sampling every 48 hours to monitor nutrient fluctuations in contaminated water. For the field experiments, samples were grown in swine lagoon with added 3% sucrose. Cheng et al. (2002) set up 2 concrete tanks were constructed and divided into 8 cells; these cells were 91cm deep and topped up with tap water during periods of higher temperatures. The biomass production was monitored by harvesting 20% of the floating plants every alternative day.

2.62 Cheng et al. (2002) found that Lemna minor could effectively remove most of the NH₄ and PO₄ from the artificial mixtures. These results were reflected in the field experiments. From the initial weeks, nutrient uptake was slow but then was followed by a rapid intake. Beyond 336 hours most of the organic nitrogen and phosphorus were removed. Within the field experiment, nitrogen uptake was slower especially within their higher dilutions. Finally, the trial grown Lemna minor was found to have grown 78% more efficiently when compared to the control.

6.70 Mikandawire and Dudel (2007) carried out a met-analysis examining several species of Lemna to carry out phytoremediation. They analysed over 50 studies globally, bringing together the world’s leading authorities of pytoremediation. Their meta-analysis revealed that Lemna major could sustain a growth rate of 0.2-0.3g d^-1, with a doubling time between 0.7 to 2days under control conditions. However when they are subject to high NH₄ and PO₄, they could sustain a growth rate between 0.6g d^-1, with a doubling time of 0.7 to 1 day. In comparison, microalgae Chlorella vulgaris was found to have a sustainable growth rate of 0.37g d^-1, with a doubling time of 1.26 to 2.65 days within effluent.

2.71 Bonomo et al. (1997) and Steen et al. (2008) stipulated that an annual mean yield for Lemna species of 73mT ha^-1 yr^-1 under controlled conditions and 180mT ha^-1 yr^-1. In comparison to C. vulgaris, Lemna has a mean yield of 45% under control conditions and 56% within effluent conditions. Furthermore, Mikandawire and Dudel (2007) demonstrated Lemna major ability to have 41% to 75% of biomass-related extraction potential of heavy metals. Lemna gibba was found to have an extraction rate of 751.9 ± 250 and 662.7 ± 203kg ha^-1 yr^-1 when filtering both arsenic and uranium.

2.72 Mikandawire and Dudel’s (2007) study concluded that Lemna minor, major and gibba have an elimination potential of 6,000kg ha^-1 yr^-1 for N, between 560 and 400kg ha^-1 yr^-1 for P and 4,00kg ha^-1 yr^-1 for K. They stipulated that apart from removing containments from the water, the major species of Lemna bioabsorption shielded toxicity by fixing containments in dead biomass. A few physiology studies have found that metals and inorganic minerals in the vacuole and cellulose of the cell walls. Once these minerals and toxic metals are incorporated into the Lemna cells, they are compartmentalised through enzymatic mediated sequestration Kormer and Vermaat (2008).

2.80 Valderrama et al. (2002) carried out an investigation into developing procedures for biological treatment of anaerobic industrial effluence by means of Chlorella vulgaris and Lemna minusula. Their study occurred within laboratory conditions with the wastewater being synthesized to reflect sugar mill wastewater. The mineral composition was as follows (mg/L): 5 NaCI, 7 CaCI2, 4 MgS0₄, 7 H₂0, 8.5 KH₂PO₄, 33.4 NaHPO₄, 10 NH₄CI. The samples also contained 180mg of lipids, 190mg of proteins and 200mg of aliphatic hydrocarbon. The control media occurred with a hydroponic solution specialised for each species.

2.81 Valderrama et al. (2002) collected species of microalgae and Lemna minusula which were processed from a local natural pond. Batches of the experiment were carried out within an 18L bioreactor. Samples were taken every 48 hours to examine the levels of ammonium ions and phosphorous concentrations. The floating layer of biomass was collected and analysed after lyophilisation treatment (freeze drying) by 2 independent laboratories. C. vulgaris proliferation was measured by using a haemocytometer every 48 hours to measure numbers of cell. The Lemna minusula proliferation was examined by measuring the fresh weight and relative growth rate.

2.82 This research uncovered that when subject to anaerobic conditions L. minusula growth was limited in wastewater compared to growth in hydroponic solution. Lemna grew 12g biomass per bioreactor after 20 days. Lemna had a relative growth rate of 0.03g/g which was 0.05g/g lower than the control. Nevertheless when combined with the microalgae, Lemna doubling time increased by 78% to 8 days. This was achieved by having developed a symbiotic relationship with the C. vulgaris. Within the same conditions as above, the microalgae was found to have a relative growth rate of 0.51g/g within the bioreactor. Lemna minusula was found to have removed 85% of NH₄ and 82% of PO₄. Moreover when mixed with microalgae Lemna removed a further 10% of NH₄ and 11% of PO₄. This symbiotic relationship was also found by the following Kormer et al. (2008), Saygidger et al. (2004) and Susaria et al. (2002).

2.90 A study conducted by Iram et al. (2012) tested Lemna minor potential of bio-treatment of heavy water contamination. The L. minor samples were harvested from bioremediation treatment plants in South-western Pakistan. This field experiment constructed 9 concerted ponds with see-through PVC coverings, having a combined storage of 26,487 L of industrial wastewater. Each trial consisted of 3 months followed by a control which was carried out in a hydroponic pool; situated inside a polytunnel. Iram et al. (2012) carried out daily temperature, light level, humidity and rainfall readings. Each week a total of 100ml from each pond was collected and analysed for its pH, turbidity, NH₄ and PO₄ make up and heavy mental content including: Zn, Cu, Cd, Ni, Mn and Pb. Metal content was determined by using a spectrometer of the dry weight after biomass readings had occurred. Lemna growth was determined by biomass measurements.

2.91 Iram et al.’s (2012) study uncovered that Lemna altered the pH of the ponds between 6-6.5 which is reported as being a favourite pH for its own development. They reported that sewage temperature is one of the most curial design parameters of duckweed reconditioning; as it can promote or inhibit additional growth. In the experiment, the temperature range was between 21.7°C and 23.3°C. According to Culley et al. (2009) the upper temperature limit for Lemna minor to proliferate was 34.5°C. Nevertheless, Lemna showed a 24% decrease in biomass when the temperature reached 10°C after 5 consecutive days. L. minor was found to have removed between 65% and 77% of Zn, Cu, Ni and Cd. whereas it only managed to remove 34% of Pd. Furthermore 92% of both organic and inorganic N and P were removed and incorporated into their vacuoles and cell walls as further suggested by Zimmo et al. (2005) and Omezuruikel et al. (2008). There was a 67% improvement in turbidly after the L. minor had filtered the wastewater.

2.10 El-Kheir et al. (2007) performed an investigation to determine the effectiveness of Lemna minor and L. gibba potential to remove inorganic nitrates and phosphate and heavy metals (500mg) such as Cu, Pb and Zn and uranium. El-Kheir et al. (2007) primary treated sewage water was transferred to the laboratory from the tertiary sewage water treatment plant. The study occurred by using 5 concrete tanks filled with the above solution. The solution was tested prior to the investigation. The control was taken from a small artificial pond, filled with tap water. Heavy metals mentioned in the above were added to the trial tanks. The trials occurred for 5 month period alongside the control. Daily measures of pH, turbidly and physicochemical analyses occurred.

2.101 The growth of the Lemma was determined by their dry and weight weights by using filtrating techniques. Protein content of the duckweeds were used to determined how much nitrogen was be incorporated into the plant’s intracellular structures, the Micro-Kjeldahl method was used.

2.102 El-Kheir et al.’s. (2007) study uncovered that both species of Lemna grew effectively at pH 7.5 with a maximum tolerance of 4 and 8. They also found similar results in relation to Iram et al. (2012) who revealed the sewage temperature yields effect on Lemna minor. Total suspend solids with the media had decrease by 76% reaching 14ml/L. After 2 days of contact with the Lemna gibba and minor the wastewater saw reductions of 7% in N and 6.5% in P. After the end of the investigation 90.6% of P was removed and an increase of 350ml/L in oxygen occurred. The uptake of nitrates varied between 45 and 1,670mg m^-2 d^-2 depending on the light levels. This study concluded that L. minor and L. gibba could be used as an effective fodder for livestock due to its high protein content cause by the bioabsorption of the nitrates within the wastewater. Duckweed could also be employed to reduce soluble salt levels in irrigation water, where the only alterative is to demineralise the water by revise osmosis treatment.

2.111 Ozengin and Elmaci (2007) performed an investigation to verify Lemna minor maximum tolerance levels of grey-water that could filtered. This experimented used 2 tanks with a surface area of 800cm² per tank. The water temperature was monitored every hour and adjusted to ensure a constant level. White LEDs were utilised to ensure a contestant 12:12 hour cycle. The Lemna minor were collected from a local river and then processed. Total nitrogen, COD (Chemical oxygen demand) and phosphate levels were tested weekly. Samples were taken every 3 days and analysed for their chemical content, turbidly, pH and oxygen levels. Interval samples were taken throughout a 48 hour period to determine the best harvesting time. Biomass and leaf counts were used to determine the condition of Lemna minor. The trial occurred over a month which was repeated a further 3 times. There was no control.

2.112 Ozengin and Elmaci (2007) found that municipal and industrial wastewater showed an increase in overall pH from 5.8 to 6.5. An optimal temperature range of 17°C to 35°C; determined which differed from Iram et al. (2012) research. 294mg/L of nitrogen were removed followed by 544mg/L of phosphates. COD removal was 85.6% and finally there was a 298mg/L increase in oxygen levels within the sewage.

METHODOLGY

3.0 Many strains of Lemna can be used within grey-water reconditioning. However without confident verification of what the optimal concentration of grey-water that Lemna major can be cultured in, it is uncertain how effective L. major at water filtration.

Samples

3.1 Lemna major was chosen from previous studies which had been verified effective at grey-water reconditioning and bioremediation (Movafeghi et al., 2012; Parra et al., 2012). A 100ml sample of L. major species was obtained from the researcher’s pond 8 weeks prior to this study. After collection the samples were cultured in an artificial pool under a glasshouse within a day night cycle of 12 hours. It was then further processed by filtering and hand picking each plant until all containments were removed including undesirable species. To ensure validity weakened species were also removed. This experiment occurred within the researcher’s own glasshouse, Newcastle upon Tyne due to the limitations of resources. To gain viable samples for analysis, stainless steel tweezers was used to pick individual plant from the culturing pods.

Procedure

3.2 2 weeks prior to this experiment the glasshouse was deep cleaned by using bleach and environmental conditions monitored to examine the environmental variances. As a note the glasshouse had a backup power source in case power cuts. There was a 12:12 day and night cycle followed. The control comprised of 3 groups that ran alongside the trials with a relatively stable temperature of 15˚C during the day and 10̊C during the night. The control examined the effects without the supplement of the grey-water; this acted as a comparison to the trials. Day and night daily temperature, light level and humidity were recorded. An automated heating system was in place to ensure that the temperature did not vary more than 10% out of environmental specifications. The grey-water was derived from a mixture of machine discharge as recommended by Engina et al. (2011). This mixture was then filtered through a 1mm sieve to remove the solid particles. During the trials, 10ml concentrations of L. major was pipetted into a 200ml sample of grey-water within a 500ml see-through culturing container. This was repeated a further 9 times to complete each trial. This procedure was repeatedly carried out under a plastic hood with sterilised gloves to prevent contamination. To further reduce chances of contamination 70% ethanol solution was used to sterilise work surfaces, materials and equipment and a cover was place over the top of the containers with small vents to allow for gas exchange. See Appendix 1 for Trial Design.

3.3 80mm x 80mm see-through containers were filled with 200ml of grey-water. Within each trial the media had 3 varying concentrations: 40%, 60% and 80% which were recommended by Hocaoglu et al. (2010) Liua et al. (2010) and had been repeated twice, resulting in 6 groups plus 3 control groups. The concentrations were measured by adding the percentage to a 100ml measuring cylinder which was topped up by adding rainwater to make 100ml solution. The control media were brought to pH 7.0 at the start of week 1 to reduce confounding variables. To ensure that the minerals in the media were still present, L. major were injected after filtration and pH checks. To reduce chances of evapotranspiration within the containers, lids were closed throughout the experiment and thin shading covered the samples to reduce chances of scorching.

Apparatus

3.4 The containers were inoculated within under a plastic hood to prevent contamination. For each concentration 2 containers were required. A total of 9 containers was utilised for each trial and 3 containers for the control, totalling 27. Each trial was cultured for 4 weeks. An artificial pool filled with 500ml of concentrated L. major was required for a continuous supply of 10ml L. major throughout the investigation (Rosenkrantz et al., 2013). The artificial pool was located in the glasshouse and the environment continually monitored. When transferring the L. major from the artificial pool to a disposable 5ml pipette was used to safeguard against wastage.

3.5 During testing 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 pods 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 the behaviour of L. major. See Appendix 2 for Environmental Specifications. As a summary there were 3 trials each consisting of 6 different concentrations of grey-water and 3 control groups. This was to test to see what the optimal and maximum capacities of the Lemna to filter them as shown in Table 1, below.

3.6 To determine the effects of grey-water concentrations, weakly leaf counts per cm² and biomass were used as a measure of growth. Weekly pH levels and turbidity determinations were used as an indication of water quality (Singh et al.; 2009; Hawkins et al., 2010). The 4 measurements were recorded weekly alongside observations by using a Nikon camera and magnifying glass. The experimenter was the only one who carried out each of the measurements. This was to reduce chances of human error. The leaf count was determined by dividing the pods in cm² grids and was calculated using the following:

Average count per cm² = (G₁ + G₂ + G₃ + G₄) x T

Where:

T = Total number of grids

G_{1 2 3 4} = Grids (cm²)

3.8 To gain biomass, a random 5ml sample was removed from the media and briefly washed. Plants were then blotted to remove any free surface moisture. Specimens were placed into a drying oven at 100°C for 48 hours. Plants were left to cool for 3 hours and weighed on a scale which measures in milligrams (Reed et al, 2007). To gain the pH results litmus paper was used following the standard protocol of pH testing. The turbidity of the samples was determined by using a Eutech TN100 portable nephelometer. The Nephelometer was calibrated by using dH₂0 weekly to ensure accurate read outs. 1ml sample of medium were removed weekly and placed into the testing tubes of the nephelometer. When turbidity read outs reached over 100 NTU (Nephelometer Turbidity Units) the sample was diluted with dH₂0 until it fell below 100 NTU and the following calculation was used:

Where:

A = NTU found in diluted sample

B = Volume of dilution water in mL

C = Sample volume taken for dilution in mL

Statistical Analysis

3.8 The data from the container studies at weeks 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.

RESULTS

Leaf Count

4.0 From the descriptive statistics it was found when Lemna major was subject to 40% concentration of grey-water it decrease in leaf count by 3.61% when compared with the control. When the 60% concentration of grey-water was tested it too was found to have decrease by 2.42% when compared with the control. When L. major was subject to 80% concentration the leaf count decreased by 0.04% when compared with the control, as demonstrated in Figure 1.

4.1 Two inferential analyses occurred to determine normality and equal variance. The Anderson-Darling test show there was normality within the trial data: 40% P= 0.815, 60% P=0.297 and 80% P=0.390. Furthermore it revealed there was normality within the control as demonstrated by P=0.739. The Levene’s test returned a P-value of 0.103 which showed there was equal variance within the control and within the trial (P=0.270). A One-way independent ANOVA test occurred due to both data sets being parametric. The test was to determine if there was a significant difference between the trial and control:

One-way independent ANOVA test F= 0.78, P 0.519, n= 2,2,2,3

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

Tukey 95% Simultaneous Confidence Intervals

All Pairwise Comparisons among Levels of Container

Individual confidence level = 98.90%

Container = 40% subtracted from:

Container Lower Center Upper ------+---------+---------+---------+---

60% -0.5445 0.0867 0.7178 (------------*-----------)

80% -0.3595 0.2717 0.9028 (-----------*------------)

CON -0.3078 0.2683 0.8445 (----------*-----------)

------+---------+---------+---------+---

-0.50 0.00 0.50 1.00

Container = 60% subtracted from:

Container Lower Center Upper ------+---------+---------+---------+---

80% -0.4462 0.1850 0.8162 (------------*-----------)

CON -0.3945 0.1817 0.7578 (-----------*----------)

------+---------+---------+---------+---

-0.50 0.00 0.50 1.00

Container = 80% subtracted from:

Container Lower Center Upper ------+---------+---------+---------+---

CON -0.5795 -0.0033 0.5728 (-----------*----------)

------+---------+---------+---------+---

-0.50 0.00 0.50 1.00

From this test there was no significant differences between the trials and the control data which is highlighted by the fact the data sets all crossed within the their confidence intervals.

Turbidity

4.3 There was a 1.12 difference in NTU when 40% concentration was compared with the control at week 4. Likewise there was a 1.01 difference when 60% concentration and a 1.16 difference in 80% concentration when they were compared with the control. 40% had an 88.59% decrease in turbidity when weeks 1 and 4 were compared. Furthermore there was a 95.14% decrease in turbidity in the 60% and a 95.50% decrease in the 80% when weeks 1 and 4 were compared.

4.4 Two inferential analyses were carried out to determine normality and equal variance. The Anderson-Darling test show there was normality within the trial data: 40% P= 0.112, 60% P=0.468 and 80% P=0.902. Furthermore it revealed there was normality within the control as demonstrated by P=0.117. The Levene’s test returned a P-value of 0.238 which showed there was equal variance within the control and within the trial (P=0.842). A One-way independent ANOVA test occurred to determine if there was a significant difference between the groups:

One-way independent ANOVA test F= 16.70, P>0.001, n= 2,2,2,3

4.5 This established a significant difference within the leaf counts. To further investigate, a Tukey’s Post-Hoc test occurred to determine where those differences lay:

Tukey 95% Simultaneous Confidence Intervals

All Pairwise Comparisons among Levels of Containers

Individual confidence level = 98.90%

Container = 40% subtracted from:

Container Lower Center Upper -------+---------+---------+---------+--

60% -0.7158 -0.1083 0.4992 (-----*-----)

80% -0.5708 0.0367 0.6442 (-----*-----)

CON -1.6729 -1.1183 -0.5638 (-----*----)

-------+---------+---------+---------+--

-1.0 0.0 1.0 2.0

Container = 60% subtracted from:

Container Lower Center Upper -------+---------+---------+---------+--

80% -0.4625 0.1450 0.7525 (-----*------)

CON -1.5646 -1.0100 -0.4554 (-----*----)

-------+---------+---------+---------+--

-1.0 0.0 1.0 2.0

Container = 80% subtracted from:

Container Lower Center Upper -------+---------+---------+---------+--

CON -1.7096 -1.1550 -0.6004 (----*-----)

-------+---------+---------+---------+--

-1.0 0.0 1.0 2.0

From this test there was a significant difference between the control and 80% concentration. However, there was no significance difference between the other groups and the control.

Biomass

4.6 From the descriptive statistics it was found that when comparing the control to the 40% concentration, there was a 7.07% decreased in biomass. Furthermore the 60% concentration saw a 1.45% decrease in biomass. However, when the 80% concentration was compared with the control there was a 3.70% increase in biomass, as demonstrated in Figure 2.

4.7 Two inferential analyses were carried out to determine normality and equal variance. The Anderson-Darling test show there was normality within the trial data: 40% P= 0.948, 60% P=0.710 and 80% P=0.114. Furthermore it revealed there was normality within the control as demonstrated by P=0.597. The Levene’s test returned a P-value of 0.001 which showed there was no equal variance within the control and within the trial (P=0.003). A Kruskal-Wallace test occurred due to both data sets being nonparametric:

Kruskal-Wallace test H= 5.71, P 0.058, n= 2,2,2,3

4.8 This established a significant difference within the biomass readings. To further investigate, Mann-Whitney U tests occurred to determine where those differences lay:

Mann-Whitney u test W= 54.0, P>0.05, n= 2,2 when comparing the control with 40%

Mann-Whitney u test W= 45.0, p 0.037, n= 2,2 when comparing 60% with 40%

Mann-Whitney u test W= 40.0, p 0.036, n= 2,2 when comparing the control with 80%

Mann-Whitney u test W= 52.0, p 0.053, n= 2,2 when comparing 80% with 40%

From theses tests there were significant differences between 40% and the control, 80% and the control, 60% and 40% and 80% and 40%.

Water pH

4.9 From the descriptive statistics it was found that when Lemna major was subject to 40% concentration, water pH increased in by 8.11% when compared with the control. When the 60% concentration was tested it too was found to have increase by 9.52% when compared with the control. When L. major was subject to 80% concentration the water pH increased by 8.05% when compared with the control, as demonstrated in Figure 3.

4.10 Two inferential analyses were carried out to determine normality and equal variance. The Anderson-Darling test show there was normality within the trial data: 40% P= 0.197, 60% P=0.204 and 80% P=0.689. Furthermore it revealed there was normality within the control as demonstrated by P=0.571. The Levene’s test returned a P-value of 0.342 which showed there was equal variance within the control and within the trial (P=0.072). A One-way independent ANOVA test occurred to determine if there was a significant difference between the groups:

One-way independent ANOVA test F= 0.73, P0.499, n= 2,2,2,3

This established that there was no significant difference of water pH in the samples.

Refer to Appendix 3 for Raw Data.

Refer to Appendix 4 for Observations and Photographic Plates.

4.11 Figure 4 compares the leaf counts and biomass readings of the control and trials. As illustrated, biomass readings peak at 1.17g when L. major was subject to 80% grey-water concentration. Additionally the leaf count peak at 7.71cm², also within the 80% concentration. However biomass readings and leaf counts both consistently were at their lowest within the 40% concentration averaging 1.05g and 7.44cm².

4.12 Figure 5 illustrates the fluctuations in water pH and turbidity. As shown, turbidity readings peak at 2.72 when L. major was subject to 80% grey-water concentration. Additionally the water pH peak at 7.74 within the 60% concentration. However turbidity readings were consistently the lowest within the 60% group which averaged at 2.58. Water pH was at its lowest within the 80% concentration averaging 7.57.

4.13 Below are Figures 6 and 7 which demonstrate the progressive growth of the leaf count and the progressive decline of water turbidly throughout the trials.

DISCUSSION

5.0 There was no significant difference found between the different concentrations of grey-water and the control. There was a large standard error within the 80% concentration demonstrating that there was a lot of variation in the data; refer to Chapter 4, page 4.1 for further information. From the P-value of 0.519 the null hypothesis can be accepted.

5.01 This is supported by Cheng et al.’s (2002) investigation as their laboratory test they did not find a significant difference in leaf count when they tested: 25%, 50%, 75% and 100% dilutions of wastewater. Within Cheng et al.’s (2002) 25% and 50% concentrations they found that the leaf count increased by 78% when compared to their control. Under further scrutiny, as Lemna major absorbs organic waste they cannot convert it directly into building products. A plant’s main source of structural material originates from C0₂ which is absorbed through their stomata. Nutrients such as NH₄ and PO₄ are taken in and are used for fundamental biological processes like cell signalling and behaviour; they cannot be directly used like carbon. As stipulated by Al-Nozality et al.’s (2000) study which verified when the environment of L. major is filled with these organic waste products they can absorb and store them within their vacuoles. When their vacuoles are full, the cell’s plasma membranes reduce or stop their ion channels from absorbing more. Thus water filtration yield is reduced until the Lemna minor can a copulate; this process is repeated from generation to generation as supported by Hell and Hell (2001).

5.02 Mikandawire and Dudel’s (2007) meta-analysis verified that Lemna major increases growth when subject to high levels of NH₄ and PO₄. Though the ability of Lemna growth was limited to the levels of C0₂ within their environment, they were able to integrate them within their parenchyma cells. Although this may seem to contradict 5.1 it stipulates that when subject to high levels of nutrients and a constant supply of C0₂, Lemna can integrate their stored ions into their mechanical components. When comparable invasive species; Eichhornia crassipes and Chlorella vulgaris were subject to high organic wastes, they were found to integrate them into their cell walls or plasma membranes. Bonomo et al. (1997) and Steen et al. (2008) demonstrated that Lemna minor could also act like a hyperaccumulator; having a biomass consisting between 40 and 75% heavy metal within their mechanical constituents. However, Radic et al. (2011) and Suhag et al. (2011) found that this ability to use ions in structural support was limited by the C0₂ and light levels. If a favourable environment persisted for more than 2 days the Lemna would start to replicate at a heightened rate, from having a doubling time of 5 days to 2. As they increased in numbers they began to decrease their surrounding C0₂. They then began to supplement their new growth with the only structural materials that were left within their surroundings.

5.03 As this experiment progressed there was an obvious limitation of the Lemna ability to replicate. From week 3 a 2.5 cm biofilm was produced due to the plants competing for space. Within a commercial tank the competition would be closely regulated as the plants that succumb to the competition would die and begin to decompose at the bottom. This then would begin to realise the captured nutrients back into the substrate. Together with the increase in competition and the energy requirement needed to capture the free ions, the Lemna major would be encouraged to reduce their water filtration activities. Additionally they would begin to store the organic waste until a more favourable environment. This may have occurred within this investigation as the leaf counts were the same as the control. Nonetheless, the leaf counts within the different concentration were not reduced when compared with the control resulting in a strong indication that the L. major were able to survive the extreme differences as stipulated by Christenson and Sims (2011) and Zimmo et al. (2004).

5.1 This investigation established that there was a significant difference (P>0.58) in turbidity within the 80% concentration and the control with no other significant differences. On further scrutiny, the 40% group was found to have removed 88.59% of the organic waste, 60% removed 95.14% and 80% removed 95.50%, refer to Chapter 4, page 4.1. This verifies that the null hypothesis can be rejected due to the P-value of >0.05. This is a strong indication that the organic ions NH₄ and PO₄ were absorbed.

5.12 Rendering Mikandawire and Dudel’s (2007) study, Lemna minor and major have an elimination potential of 6,000kg of N per ha per year, between 560 and 400kg for P and 4,00kg for K. Their study also found that L. minor had a higher biomass-related intake extraction potential than other species. On average Mikandawire and Dudel (2007) uncovered a removal rate of 85% of NH₄ and 82% of PO₄ over a 20 day period with the 25 and 50% groups. These finding reflect the ones found in this investigation, though the concentrations were higher. Moreover Mikandawire and Dudel (2007) revealed when concentrations of grey-water there higher than 75% the filtration rate decreased by 10%, at 100% the Lemna began to decay. The results of this investigation when testing 80% concentration seem to contradict the findings of the above study. As the concentration of grey-water were increased the more effective the filtration rate was. A possible explanation other than environmental variation could be that the experimenters did not report any symbiotic relationship with algae unlike this investigation.

5.13 Valderrama et al. (2002) found that many species of Lemna are capable of developing a symbiotic relationship with many other forms of photoautotrophs including green microalgae. They found that when Lemna had developed this association their water filtration ability increase by 11%. This symbiotic relationship is based within the root layer of a Lemna biofilm. This was apparent in this experiment due to the slim covered roots as shown in Figures 1.0 to 1.15 in Appendix 4 Photographic Plates. This reflects the behaviour that higher plants exhibit as they form many vital associations with mycorrhizal fungi. This fungus expands a plants root network by increasing the area of nutrient and water absorption. Mandyam et al. (2013) and Veiga et al. (2012) found when Arabidopsis thaliana was subject to a mycorrhizal association the plants grew 67% more when compared without. When this idea is extrapolated, it results in the increase in area size for the root system of Lemna, could give the plants an increase ability to filter organic wastes.

5.14 Contrast to Mikandawire and Dudel’s (2007) study Cheng et al. (2002) recorded the rate of nutrient capture. They uncovered that beyond 336 hours, 95% of organic waste was removed when Lemna major was subject to a controlled environment with regular removal of excess growth. When subject to a field trial, L. major removed 84% of the waste in the same amount of time. When the trial specimens were compared with the control they had grown 78% more. From this, it can be concluded that there is a key correlation and trade-off between Lemna growth and its ability to process waste ions.

5.2 This study established that there was a significant difference (P=0.058) in biomass of the concentrations of grey-water when compared with the control. Under further study there was a significant difference between all four groups as demonstrated by the average P-value of 0.05, see Chapter 4, page 4.4 for more information. From this the null hypothesis can be rejected.

5.21 Ozengin and Elmaci’s (2007) study verified when the concentration of grey-water was increased, the biomass of Lemna major decreased. They concluded that the optimum performance of mineral absorption and biomass gain was 65%. In this investigation it was found that the peak gain in biomass was within the 60% group; however there was only a 0.2 g difference with the other groups. In Cheng et al.’s (2002) investigation found that there was an initial reduced uptake of nutrients for up to a week, before a rapid uptake ensued. Beyond 336 hours the researchers uncovered that on average 95.6% of the NH₄ and PO₄ were removed. However, the biomass of L. major was not found to have increase as expected. This unexpected constant mass may have been owed to lower levels of C0₂. As previously discussed, if there was a low availability of C0₂ plant biomass cannot rapidly increase. The cells require more energy to enable to engage in nutrient structural fixation, thus producing a constant mass within the specimens. Nonetheless within this investigation there were no constant mass or sudden increases in biomass.

5.22 During Mikandawire and Dudel’s (2007) trials they uncovered that the biomass of their specimens progressively increased at 0.2-0.3g a day however, when in the presence of heavy metals their biomass seemed to be more irregular; 0.01-0.04g week 1 to 0.1-0.3g at week 2 to 0.6-0.9g at week 3. This irregular pattern of biomass progression related to the fact that the L. major samples take in heavier minerals slower than smaller ones, because the membrane potential does not allow for quick transport of heavier elements through their ion channels. This results in a back log of heavier elements on the extracellular pathways. Steen et al. (2008) also found this effect when testing for heavy metal absorption however; they managed to reduce the effect by increasing the pH of the substrate. The increase in pH was found to balance out the mixture of cations and anions which helped to diminish any back logs as stipulated by Ge et al. (2012) and Cheng and Stomp (2009).

5.3 This study revealed that there was no significant difference (P=0.499) in the pH of water samples within the trials and the control. Though there was no significant difference, this also means that that the water’s pH was no difference when compared with the control at week 4. At week 4 the control was measured to be pH 7.0 however in comparison to week 1 the trial groups were at pH 11.0. From this the null hypothesis can be accepted, see Chapter 4, page 4.5 for more information.

5.31 Iram et al. (2012) verified that Lemna minor and major altered the pH of their environment to become more favourable for its own development. This alteration was found to have been enabled within the range between pH 6.0 to 11.0; if the pH stray out of this range the Lemna were unable to adapt. While the pH was undergoing change the Lemna were unable to increase in mass as this change was unenergetically favourable. Once the pH was stabilised the biomass increased at a rapid rate, this may have been illustrated in Cheng et al.’s (2002) and Steen et al.’s (2008) research.

5.32 El-Kheir et al. (2007) exposed when Lemna minor, gibba and minor were subject to pH 7.5 their biomass increased rapidly: when subject to higher or lower pH the effect described by Iram et al. (2012) was confirmed. Furthermore, since pH is the logarithmic scale of contractions of H⁺ this can be extrapolated to the nutrients within the substrates. The higher the concentrations of certain ions also increased the pH, resulting in the increase uptake of these ions to stabilise the pH. Nevertheless this can also be correlated to the fluctuations seen within the leaf count as verified by Ozengin and Elmaci (2007).

5.4 One improvement which could have been implemented to this study was the use of a more controlled environment. Studies from Cheng et al. (2002), Iram et al. (2012) and Valderrama et al. (2002) used controlled conditions, as they initially found that the slightest increase in C0₂ or light levels could influence the growth of L. major. Since Lemna species are highly adaptable they can utilise any increase in the above to increase their mass or numbers which as a result creates a confounding variable. However, due to the limitations of funding for this paper a semi-controlled glasshouse had to be used. Though most days had been within the environmental specifications some were over by 25%, out of the 10% allowance variance. This may have in turn influenced L. major development. To gain a more accurate outcome a photobioreactor or growth cabinet could have been used to ensure more environmental consistency. Nonetheless this would have increased the cost of this experiment.

5.5 Most of the papers reviewed for this investigation included a field trial to test the ability of Lemna to process wastewater in practice. One of the major differences between this investigation and the reviewed papers is that this study is reviewing the potential for domestic grey-water treatment. This experiment could have tested the Lemna within a photobioreactor to gain a precise measure of the rated influences of filtration. Subsequently, it could have moved into a field trial to test the actual effectiveness of using Lemna. As recommended in Appendix 5: Wider Implication, a tank specialised for this treatment could have been constructed and attached to a grey-water disposal pipe. This could have increased the viability of the practical application of this new technique of grey-water disposal.

5.6 Another improvement that could have been made would have been to precisely monitor the fundamental nutrients within the substrate. As in Ozengin and Elmaci’s (2007) study, all of the major and a chosen few micro organic minerals were recorded throughout their research. With this in mind, this investigation could have precisely monitored how much and at what rate each mineral was being absorbed, refer to 5.7 for further details.

5.7 Future research is required to further understanding of each organic mineral’s rate of uptake, when the more favourable conditions are for each mineral uptake and how much can be absorbed before the saturation point is reached. This would have wider implications not just for the domestic sector but also the commercial sector of grey-water and wastewater treatment. A precise understanding is necessary to be able to improve Lemna water filtration by means of genetic manipulation or by hybridisation. Once this understanding is met, researchers could then move onto other species to see which Lemna species has the better absorption to a particular organic mineral. If this was to occur, a mixture of different species could be used to further improve the species ability to process wastewater with the aim of modifying the process.

5.8 Valderrama et al. (2002) established that a symbiotic relationship between Lemna species and various algae species increased the rate of mineral absorption. This symbiotic relationship requires more study to see if and how it can be manipulated to improve this wastewater treatment. There generally seems to be a lack of understanding of this relationship that various aquatic plants form with protists (Grossart et al., 2013). Comparable to this symbiosis, the manipulation of mycorrhizal fungi has improved the yield of many crops including Zea mays. Though there is a better understanding of mycorrhizal relationships, there also needs to be more research to determine how it can be engineered to increase Valderrama et al.’s (2002) symbiosis.

5.9 In summary, leaf counts, biomass, turbidity and water pH were used to determine the effectiveness of using L. major as a source of grey-water reconditioning. Turbidity was the only variable that was proven to have significant difference between the groups. Though the other measures had no significant difference it does verifies the ability of L. major to absorb and process the waste organic minerals. The lack of a significant difference between the concentration and the control illustrates that the plants were not badly affected by the substrate. If there was a negative impact upon the growth of the Lemna, inevitably the leaf counts and biomass readings would be greatly lower when compared with the control. By using a photobioreactor or growth cabinet this experiment could have improved its repeatability. More research is required to understand the precise mineral take up of various species of Lemna with and without this symbiosis. By increasing this understanding, researchers would be able to modify Lemna to increase its water filtration capacity.

CONCLUSION

6.0 Many species of Lemna including L. major where identified from previous studies as having a potential for water filtration. This simple free-floating plant has a high tolerance to pollution including: heavy metals, high NH₄ and elevated levels of COD. Alaert et al. (1996) initially demonstrated that L. major could remove up to 74% of NH₄ and 77% of PO₄ from a wastewater lagoon. As a simple plant, Lemna has several advantages over the use many microphytes and microphytes species; 50% more oxygen production in and outside of their substrate, capturing 40% more carbon, high nutrient uptake versatility and ease of harvesting. Culley et al. (1981) first uncovered that Lemna species could absorb and capture over 1,300kg of NH₄, 347kg of PO₄ and 441kg of K₂SO₄ per hectare per year under temperate conditions. It is for these reasons that governments around the world including the US, Israel and Bangladesh have begun to invest into bioremediation; focusing on Lemna major, L. minor, L. gibba and several species of green microalgae. By culturing a mixture of Lemna species or by encouraging the development of a symbiosis with green algae, organic mineral uptake increased by 82% and yield increased by 56%.

6.1 From this research, the growth or Lemna major was found not to have been adversely affected by grey-water. Furthermore, it was found that Lemna significantly changed the pH and turbidity which at week 4 was comparable to the control. Using L. minor as the most frequently researched specimen for grey-water filtration led to the following outcomes: at 40% concentration pH was found to have decreased by 47.44%, at 60% pH decreased by 54.44% and at 80% pH decreased by 60.26%. Water turbidity was found to decrease by 88.65% at 40% concentration, at 60% a 95.09% decrease and at 80% 95.61% decrease. Additionally, from the statistical analyses there were no significant differences in the above. From this, it is clear that Lemna major has great potential of grey-water filtration both on a domestic and an industrial scale. Referring back to the aim of this investigation, it is clear that Lemna major has a high capability of bioremediation. By using this plant for wastewater treatment an individual or organisation could greatly increase their sustainability and environmental credibility. Moreover by applying this new technique, one could in fact reduce their costs of treating their grey-water and their tertiary stage wastewater.

6.2 One improvement which could have been implemented to this study was the use of a more controlled environment. Studies from Cheng et al. (2002), Iram et al. (2012) and Valderrama et al. (2002) used controlled conditions, as they initially found that the slightest increase in C0₂ or light levels could influence the growth of L. major. This experiment could have tested the Lemna within a photobioreactor to gain a precise measure of the rated influences of filtration. Subsequently, it could have moved into a field trial to test the actual effectiveness of using Lemna. As recommended in Appendix 5: Wider Implication, a tank specialised for this treatment could have been constructed and attached to a grey-water disposal pipe.

6.3 Future research is required to further understanding of each organic mineral’s rate of uptake, when the more favourable conditions are for each mineral uptake and how much can be absorbed before the saturation point is reached. This would have wider implications not just for the domestic sector but also the commercial sector of grey-water and wastewater treatment. A precise understanding is necessary to be able to improve Lemna water filtration by means of genetic manipulation or by hybridisation.

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