Statistical analysis

After subtraction of absorption due to the blank, linear regressions on the standard curves were generated using MS Office Excel 2010 and used to

calculate protein concentration in µg ml^-1. Using Mintab (V 16.1.0), statistical analysis of protein recovery, expressed as a percentage of the air-dried or lyophilised mass, withthe explanatory model of pre extraction soaking solutions and soaking times was done using ANOVA and a general linear model. Post hoc analysis was done using the Tukey method and 95.0% confidence intervals for the effect of pre-soaking method. The Tukey method conducts multiple comparisons of the differing parameters (pre extraction soaking solutions and soaking times) and its wider confidence intervals provide less precise estimates of the measured parameters but limit the probability that one or more of the confidence intervals does not contain the true difference to a maximum of 5 %.

In trial 2, the results from the Laemmli buffer and sodium hydroxide were not included in the statistical results due to their low levels of protein recovery.

3.3.2 Trial 1

Figure 3.1 shows the results of trial 1 and indicates that sodium

hydroxide, Tris buffer at pH 7.4 and pH 6.8 and Laemmli buffer are not suitable pre-extraction soaking solutions. Of the other pre-treatments, the second extraction into chloroform and methanol has recovered only 0.4 - 0.7 % of protein from the samples and the actual quantities of protein in ug ml^-1 in the samples were at the limits of detection for the Bio-Rad assay used. The 1^st and 3^rd extractions have recovered maximums of 2.4 – 1.7 % protein compared with

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the initial sample mass. The Tukey analysis indicates that the overall protein percentages recovered from pre-soaking treatments; MilliQ water, MilliQ water adjusted to pH 2.02, Tris buffer adjusted to pH 2.02, perchloric acid (HClO4) 0.2 N (pH 0.62) are not statistically different from each other.

Figure 3.1 The effect of pre-soaking solutions and 2 sequential extractions (1^st and 2^nd %) using chloroform and methanol and final extraction of cell debris (3^rd

%) heated to 90 ^oC with 0.1 N NaOH, on protein recovery (percentage of the dry mass) from air dried U. lactuca.

3.3.3 Trial 2

In the ANOVA (table 3.2) the larger Adj SS (Adjusted sum of squares) of 10.0683 of the soaking pre-treatment compared to the Adj SS of 0.515 of the time treatment shows that the majority of the variability is being generated by the soaking pre-treatment and the soaking time 1 hr or overnight.

The Tukey analysis (table 3.3) has ranked the results and indicates that, MilliQ water +HCl to pH 2.02 with 1 h soak, Tris pH 2.02 with overnight soak, Tris pH 7.4 with overnight soak, Tris pH 2.02 with 1 h soak, MilliQ water with 1 h

0.0 1.0 2.0 3.0 4.0 5.0 6.0

MilliQ MilliQpH2 TrispH2 TrispH6.8 TrispH7.4 PerAcidpH0.62 SodHydr Laemmli 1hr Laemmli 0hr

Percentage Protein Recovery

Presoaking Treatment

1st%

2nd%

3rd%

Total%

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soak (all grouping A) had the highest percentage recoveries of protein (2.42 – 2.09 %) and were not statistically different.

Table 3.2 Analysis of variance of soaking pre-treatments on the recovery of protein from Ulva lactuca.

Analysis of Variance (ANOVA)

Source DF Seq SS Adj SS Adj MS F P

Pre-soak treatment 5 10.0826 10.0683 2.0137 3.26 0.012 Soaking time treatment 1 0.0515 0.0515 0.0515 0.08 0.774

Error 58 35.7882 35.7882 0.617

Total 64 45.9223

Table 3.3. Tukey 95% simultaneous confidence intervals for protein removal after soaking pre-treatments of 1 hour (1 h) and overnight (O/N). Means that do not share a letter with other groupings are significantly different.

Grouping Information Using Tukey Method N Mean % Grouping MilliQ water +HCl to pH 2.02 1 h 5 2.42 A

Tris pH2.02 O/N 5 2.28 A

Tris pH7.4 O/N 5 2.12 A

Tris pH2.02 1 h 5 2.10 A

MilliQ water 1 h 5 2.09 A

MilliQ water O/N 5 2.05 A B

Perchloric Acid 0.2 N pH 0.62 1 h 5 1.95 A B

Tris pH6.8 O/N 5 1.84 A B

Tris pH6.8 O/N 5 1.77 A B

MilliQ water +HCl to pH 2.02 O/N 5 1.76 A B

Tris pH6.8 1 h 5 1.48 A B C

Tris pH7.4 1 h 5 0.65 B C

Perchloric Acid 0.2 N pH 0.62 O/N 5 0.20 C

A graphical representation of the results is shown in figure 3.2 where the lower levels of protein recovery from Laemmli buffer (Laemmli) and sodium

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hydroxide (SodHydr) can be seen in comparison the other soaking treatment. Also apparent is a large differential between the soaking pre-treatments 1 hr and overnight (O/N) using Tris buffer adjusted with HCl to pH 7.4.

Figure 3.2 The effect of 1 hour (1 hr) or overnight (O/N) soaking pre-treatments on mean percentage protein extraction with (standard error) from U. lactuca 3.3.4 Trial 3

The mean total protein extracted expressed as a percentage of the dry weight of sample is shown in figure 3.3 and ranged from 5.6 – 10.7 %. In figure 3.3, it can be seen that soaking with perchloric acid 0.2 N does not result large protein losses throughout the extraction process. Protein extracted in portions A, B and C equalled 5.3% of the total extracted compared with the 94.7 % extracted in part D. This is in comparison to Tris buffer pH 2.02

0.00

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Figure 3.3 Percentage protein recovered from different stages in protein extraction protocol after samples were pre-soaked with soaking solution, Tris pH 7.5 extraction, chloroform / methanol extraction and NaOH extraction respectively before extraction where and O/N is overnight pre-extraction soak and 1 h is one hour pre-extraction soak. Percentages of protein portions recovered at different stages of the extraction process are, A%, B%, C% and D%.

0.00 2.00 4.00 6.00 8.00 10.00 12.00

O/N 1Hr O/N 1Hr O/N 1Hr O/N 1Hr

MilliQ MilliQ MilliQ pH2 MilliQ pH2 Tris pH2 Tris pH2 PerAcid pH0.62

PerAcid pH0.62

no pretreat SodHydr

Percentage Protein Recovered

Soaking Pretreatment andTime

A%

B%

C%

D%

Total%

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used as the initial soaking solution where protein extracted in portions A+

B + C equalled 37.9 % of the total extracted compared with the 62.1 % extracted in part D. The extracted portion A + B +C was designated the unbound portion and portion D was designated the bound portion.

The results from the ANOVA below (table 3.4) indicate that the majority of variation i.e. the 119340 value in the Adj SS is being generated by the

extraction set A, B, C or D (P < 0.01). There is no effect of the pre-soaking time 1 hour or overnight (P = 0.552) but there is an effect from the pre-treatment soaking solution (P = 0.028). The Tukey analysis (table 3.5) indicates that this variability is driven by set D extracting a mean of 70.1 µg g^-1 protein which is significantly different from A, B, C (P < 0.05). There is no significant difference between set A, B or C (7.9, 6.7, 6.4 mg g^-1 respectively).

Table 3.4 Analysis of variance of the percentage protein extracted from Ulva lactuca at different stages of the extraction process.

ANOVA of extraction set, soaking time and pre-treatment Source DF Seq SS Adj SS Adj MS F P Extraction Set 3 119340 119340 39780 700.67 <0.01

Soak Time 1 20 20 20 0.36 0.552

Pre- treatment 3 532 532 177 3.13 0.028

Error 152 8630 8630 57

Total 159 128523

Table 3.5 Tukey analysis of protein extracted at different stages of the analysis and with different soaking pre-treatments. Means that do not share a letter with other groupings are significantly different.

Extraction Set N Mean Grouping

D 40 70.1 A

A 40 7.9 B

B 40 6.7 B

C 40 6.4 B

When comparing overall means of the pre-treatment soaking solution, including the results using only the final NaOH stage of the extraction process

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with no pre-soaking treatment, there are significant differences between the total percentage protein recovered (P = 0.003). The Tukey analysis (table 3.6) indicates that of the soaking pre-treatments, Tris buffer at pH 2.02 recovers the highest quantity of protein (8%) and no pre-treatment and heating with NaOH the least (5.6 %).

Table 3.6 Tukey analysis of protein extracted with different soaking pre-treatments. Means that do not share a letter with other groupings are significantly different.

Pre-treatment

Tris pH 2 10 8 A

MilliQ 10 6.7 A B

Perchloric 0.2N 10 6.5 A B

MilliQ pH2 10 6.3 B

no pre treatment NaOH only 10 5.6 B

3.3.5 Observations

Subsamples were examined using light and oil immersion microscopy to determine if the soaking solutions had any easily identifiable effects on the gross cell structure of U. lactuca.

After soaking in MilliQ water and under x 100 and x 400 magnification, the U. lactuca cells looked bleached with no real internal structure visible. The bacteria and flagellates carried in on the surface of the U. lactuca were still active. At x 1000 magnification using an oil-immersion lens, there was evidence of loose cells but the U. lactuca external structure was intact. Internally the structures looked indeterminate. The surface diatoms of the U. lactuca were detached and free-floating.

Under x 100 and x 400 magnification, after soaking in MilliQ water adjusted to pH 2.0 with HCl, the cells looked bleached and although the cell walls looked intact, the internal structures were indeterminate. The liquor round the cells had unidentifiable particles within it. The diatoms were still attached to the U. lactuca external cell surface. Bacteria and flagellates were still active in the surrounding liquor.

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Using Tris buffer adjusted to pH 2.0 with HCl as the soaking solution and at x 100 magnification, the cells were clear and appeared empty. An unidentified nematode was active in the surrounding liquor. Increasing the magnification to x 400, the cells appeared empty but the cell walls were still well formed. There were some conglomerations of debris outside the cells. At x 1000 oil-immersion magnification there appeared to be debris from the internal structures within the cells. The external cell walls were intact but internally no structures could be seen. There were no chloroplasts visible.

Using Perchloric acid as the soaking solution, portions of bleached empty cells could be seen at x 100 magnification along with a brownish indeterminate material. At x 400 magnification, the cell walls looked less well defined than in previous solutions and there was an indeterminate particulate matter in the surrounding liquid. Increasing the magnification to x 1000 oil-immersion, the cells were still in regular arrangements but appeared empty. There was no bacterial or flagellate activity and conglomerates of indeterminate debris were floating free of the cells. These observations are in concordance with Zemke-White et al. (2000) who found the effect of low pH on macroalgae was to lyse the plasma membrane and increase cell porosity but did not affect the overall structure of the cell walls.