Results

Feed stocks were analysed prior to the digestion experiments to ensure optimal conditions in terms of carbon, nitrogen, TS and VS content. Substrates used in this study include Chlorella vulgaris from three different sources name C1, C2 and C3. All Chlorella sources had VS content between 83.77 and 93.95%. Carbon content of these substrates was in the range of 39-45% TS (Table 6.1). The substrates and the inoculum used in this study contained high alkalinity and adequate VFA which project its amenability to anaerobic digestion. Based on substrate characterization and VS content, respective digestions for this study were undertaken.

Table 6.1 Summary of Substrate Characterisation

Parameters pH TS (%) VS (%) Alkalinity (mgCaCO3/ L) C (%T S) N (%T S) H (%TS) O (%T S) C:N Cultivated Chlorella vulgaris (C1) 7.0 7.1 92.75 380 42.8 6.9 5.7 24.2 6.2

Holand & Barrett Chlorella (C2)

7.0 7.16 93.95 375 39.1 8.2 5.7 29.2 4.8

Oneon Uk, Chlorella (C3)

7.0 7.11 83.77 436 45.1 7.4 6.6 27.3 6.1

6.2.1 Methane yield obtained in the study

The methane yield from the comparative experiment of the several Chlorella studied ranged between 150 and 264N mL CH4/g VSadded (Table 6.2). While the lowest methane yield was observed in the Holland and Barrett Chlorella (C2), the cultivated Chlorella and the Oneon Chlorella demonstrated similar yield of 264 and 256N mL CH4/g VSadded respectively. While this values fall within the achievable methane production from a wide range of microalgae species studied in literatures, it also agrees with values obtained from Chlorella when cultivated in nutrient rich environment (Sánchez Hernández and Travieso Córdoba, 1993).

Methane yield of the three Chlorella sources studied were compared in terms of cumulative yield, per g VS added and per g VS destroyed. Results showed consistency under each criterion as cultivated Chlorella (C1) and Oneon Chlorella (C3) portrayed similar/higher yield compared to the Holland & Barrett Chlorella.

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Table 6.2 Achievable methane production from the digested substrates Cumulative methane yield (N ml) Methane Yield (N mL CH4/g VSadded) Methane Yield (N mL CH4/g VSdestroyed) % of VS destroyed Cultivated Chlorella vulgaris (C1) 443.99 264.30 331.22 45

Holand & Barrett Chlorella (C2)

252.28 150.17 180.53 36

Oneon UK, Chlorella (C3)

430.75 256.40 329.50 45

A noticeable lag phase up to three days (Figure 6.1) was observed in Holland & Barrett Chlorella (C2) and this was coupled with the lowest methane yield. This delay could be attributed to improper acclimatization of the feed with the inoculum, presence of resistive compounds to degradation or the presence of a thicker cell wall thus making the cell wall more recalcitrant amongst other possibilities including S:I ratio.

Results (Table 6.2) showed similarities between the cultivated Chlorella (C1) and the Oneon Chlorella (C3). C2 Chlorella demonstrated the least performance under all comparative criteria studied in regards to methane yield. VS destruction from the comparative study also showed similar percentage destruction of 45% for the cultivated Chlorella and the Oneon Chlorella. C2 on the other hand demonstrated a very low VS destruction up to 25% less than the cultivated and the Oneon Chlorella.

6.2.2 Process parameters

The process parameters below (Table 6.3) give an indication of the anaerobic processes as well as the reactor health during digestion.

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Table 6.3 Mean digester parameter

pH VFA (mg/L) NH3 (mg/L) Alkalinity (mg

CaCO3/L) Cultivated Chlorella

vulgaris (C1) 7.4± 0.1 1479± 25 1560± 39 3312± 125

Holand & Barrett

Chlorella (C2) 7.6± 0.1 840± 25 2740± 41 1805± 107

Oneon UK,

Chlorella (C3) 7.6± 0.1 1436± 25 1620± 35 3295± 105

Alkalinity levels in the reactors showed concentration high enough to enhance methanogenesis. While the highest alkalinity was observed in the cultivated Chlorella (C1) 3295 mg CaCO3/L, no obvious difference was observed when compared to the Oneon Chlorella with alkalinity of 3312 mg CaCO3/L. C2 Chlorella however demonstrated the weakest alkalinity of 1805 mg CaCO3/L. Nevertheless, all reactors demonstrated sufficient buffering capacity to maintain a stable pH which averaged between 7.4 and 7.6 for all Chlorella tested.

6.2.3 Comparison of biodegradability rate and methane production

The BMP data obtained in this study was analysed using a first order kinetic model which allows the estimation of the apparent degradability ƒd [L CH4 gVS-1] and the apparent hydrolysis rate, Khyd [day-1]. This was achieved by fitting the data into the first order equation provided by Batstone et al., (2009) and Keymer et al., (2013).

The model was implemented in aquasim 2.1d. The objective function used was the sum of squared errors (χ2). Average values from the triplicates were used and the uncertainty was assessed as described by Keymer et al., (2013) via parameter uncertainty analysis.

Results of the BMP tests with model simulations are shown below (Figure 6.4). The error bars indicate the standard errors from triplicate tests while the model lines shown are based on the best fit of fd and khyd with standard errors which are shown in Table 6.4.

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Figure 6.1 Cumulative methane yield from respective digesters. Error bars indicate

standard error in triplicate tests while the lines show the predicted model trend.

Table 6.4 parameter estimation obtained for degradation ( ) and first order hydrolysis

rate (Khyd) showing standard errors of predictions.

Substrate ƒd [L CH4 gVS-1] Khyd [day-1] χ2

Cultivated Chlorella (C1)

0.26 ± 0.01 0.11 ± 0.003 0.001

Holland & Barrett Chlorella (C2)

0.15 ± 0.01 0.08 ± 0.002 0.001

Oneon Chlorella (C3) 0.26 ± 0.01 0.11 ± 0.003 0.001

Findings once again proved the similarities between the Oneon Chlorella and the cultivated Chlorella which both demonstrated the same hydrolysis rate 0.11 day-1, while the least hydrolysis rate was obtained from C2 Chlorella with a corresponding degradation rate of 0.08 day-1. In view of this C3 Chlorella was picked over C2 as it portrayed the most similar characteristics in terms of methane yield and identical process stability to the cultivated Chlorella in the study.

6.2.4 Oneon Chlorella (C3) Review/Cell wall Integrity

The methane yield of the Chlorella adopted for the research (0.26 L CH4 gVS-1) falls within the literature values ranging between 0.1 and 0.39. This shows the Chlorella

0 0.05 0.1 0.15 0.2 0.25 0.3 0 5 10 15 20 25 30 C u m m u lati v e Me th an e Yield ( L C H4 /g VS a d d ed ) Time (Days)

93 adopted for the research is suitable for further experiments. Nevertheless, the potential of these substrates (microalgae) for anaerobic digestion could be improved as the theoretical methane potential suggests.

This may only be achieved with a complete degradation of the substrate, thus there is need to look into possibility of enhancing microalgae degradation bearing in mind that the cell wall of these microalgae has been identified to be resistive to bacterial attack (Golueke et al., 1957; Sánchez Hernández and Travieso Córdoba, 1993). With the enhancement of the cell wall, the possible methane yield from these substrates of interest stands a chance of competing with high potential substrates, like food waste.

Having identified the C3 (Oneon Chlorella source) to possess the most identical properties and methane yield with that of cultivated Chlorella, another paramount requirement was to confirm the cell wall structure of this Chlorella source to be intact. This step was taken for two reasons:

1) It has been reported that the harvesting and processing of microalgae can cause damages to its cell wall.

2) Considering the fact that one of the research objectives was to see the effect of pre-treatment on cell wall destruction, solubility and overall methane yield, an already disrupted cell wall as a result of cultivation or any other reason would have nullified the use of the Chlorella source.

94 Finally, cell wall integrity of the preferred Chlorella source (C3) was confirmed using the SEM microscope (Figure 6.2). This confirmation seals any other doubts about the source of Chlorella adopted for the research. Also it gives a firm ground for evaluating the effects of pre-treatment on increasing the methane yield achievable from microalgae with intact cell wall. The SEM results visually proved the intactness of the Chlorella (C3) cell wall thus confirming its suitability for further experiments in the research.