Tukey Post Hoc Statistical Analysis of Clay-PEM Microbial Fuel Cell Operation for Improved Process Performance

1.0 INTRODUCTION:

A novel approach to wastewater treatment which represents paradigm shift from the conventional but capital intensive chemical treatment is currently trending (Gude, 2016; He et al., 2017)1). This process is essentially based on the use of renewable energy as a suitable alternative in the treatment and disposal of wastewater with concurrent energy recovery. (Liu et al., 2005; Majumder et al., 2014). This new frontier in wastewater treatment creates a sustainable approach to optimize solution to environmental management in environmentally friendly manner. Microbial Fuel Cells (MFCs) have successfully presented a leeway for efficient involvement of science and technology to exploit biological degradation of wastewater for bioelectricity generation (Gboreyshi et al., 2011; Akujobi et al. 2017).

MFC process has been adjudged flexible in the sense that the output has been variously changed by varying the process inputs such as: the substrates (glucose, acetate, cow dung, domestic wastewater, industrial wastewaters (brewery (Yujie et al., 2008), food processing, starch (Obasi et al, 2012; Lu et al., 2009), soil treated with human urine (Simeon et al., 2020), diary, pharmaceutical industries etc., the proton exchange membrane type and condition of preparation (Ghasemi et al, 2015), and catholytic fluid (Liu et al., 2004; Feng et al., 2018; ). Increase in both internal and external environmental temperatures has been found to impart negatively on cell performance (Calicioglu et al, 2018; Gadkari, 2020). Also, variation in temperature could equally affect proton exchange mechanism (Casciola et al., 2006; Mohammed et al., 2021), and anodic biofilm formation (Min et al., 2008). This sale-up possibility happens to be the basis for MFC proposed application in not just laboratory but industrial processes. Biodegradation of organics in anodic fluids (wastewaters) is also a prominent feature of MFC operation depending on the presence or absence of chemical mediators (Jang et al., 2004, Kim et al., 2020).

The overall effects of MFC process are water pollutant removal (Zhang et al, 2010; Butler et al., 2011; Safwat, 2019), and power generation which could further mitigates the effect of climate change (Mende & Misra, 2020). The power generation output of MFC depends also on the quality of the wastewater in terms of its organic matter content, biodegradability and COD level. Certain electrochemically active bacteria have been found to possess the potential to activate the MFC process through microbial decomposition of organic substrates (Prasertsung & Ratanatamskul, 2013). Certain chemically active microorganisms play active role in bio-electron transfer: Shewanella putrefaciens (Kim et al., 2002) and Oneidesis as bacteria which are associated with MFC. In another study of MFC microorganism, Geobacteraceae sulferreducens were identified (Reguera et al., 2006). Further research by Chaudhuri and Lovely (2008) observed that bacteria identified to possess outer membrane cytochrome such as Geobacter metallireducens and Rhodoferax ferrireducens are able to form biofilm on electrode pores spaces. These microbes are able to shuttle charged ions from the bulk fluids to the anode and cathode conductors in MFCs.

The PEM material type and preparation method as in zirconium phosphate-ionic liquids membrane (Al-Othman et al., 2021), electrode surface area (Lorenzo, 2010), type and nature of participating microorganisms effecting remediation (Gadd, 2010), including mode of feeding and operating pH, anolyte medium temperature (Larrosa-Guerrero et al., 2010) are some of the effective factors that affect the performance of microbial fuel cell (Obasi & Onukwuli, 2019). Materials such as clay has been identified to possess good proton exchange characteristics in MFCs (Neethu et al, 2019). This biological factor has to do with the effect of the type and nature of microbes, the growth and the effect of the synergistic interaction in the community (mixed culture) with the anode surface and the mode of electron transfer to the anode. Different types and species of microbe possess different degrees of electrochemical activity and capacity to form biofilm at the anode surface and such has direct effect on the electrogenesis and electron transfer (Aghababaie et al., 2015). Generally, anolyte medium pH and temperature affect the growth and activity of microbes. The effect of pH categorized bacterial into acidophilic, neutrophilic, and alkaliphilic depending on the pH suitable for the optimum performance. Equally, the hydraulic stability of membrane material such as clay as a function of temperature (Chen et al., 2017, Al-Soudany et al., 2018).), and compounding with doping materials to form a composite (Smitha et al., 2005), play major roles in determining the proton conductivity of a given material. The proton conductivity of coconut shell was improved by compounding with activated carbon (Kammoun et al., 2014).

Certain mathematical models have been also proposed for the modelling of MFC performance with respect to bioenergy production and wastewater clean-up (Kumar et al., 2019). Such models such as response surface methodology (RSM) prediction modeling (Sugumar et al., 2022) and ANN validation are able to consider a number of controllable critical factors which determine MFC performance. Despite numerous efforts, drawbacks are still experienced in MFC modeling as MFC is a multifactorial process. The use of simple and synthetic wastewater is not representative of more complex substrates, and a large number of models are focused on reactions happening in a single chamber (e.g. the anode), neglecting the limitations linked to the presence and importance of the other chamber (e.g. the cathode). In addition, more attention is now focused on Tukey’s post hoc test to establish the operating variables relevant to ensure maximum output by assessing the significance of difference between pairs of group means (Agu et al., 2017).

This work is therefore aimed at evaluating the efficiency of MFC with respect to wastewater treatment and bioelectricity generation using sanitary wastewater and varying critical input operational variables such as PPT, anolyte pH and concentration. The multivariate statistical approach was aimed at assessing the possibility of reducing the process model, and coming up with a better statistical framework that further optimizes the process parameters with a view to finding a leeway to increase the power and wastewater treatment performance of MFC. One way Turkey post hoc statistical analysis is suitable for the study of process variables involving wastewater treatment as it generally simplifies process assessment (Bourget, 2023).

2.0 MATERIALS AND METHODS

2.1 Design and operation of the Microbial Fuel Cell

The dual chamber MFC used in this study was constructed and operated as described in Table 1.

It consists of two polyvinyl chloride (PVC) cylindrical chambers with each of 0.00192m³ internal volume. The chambers were designated anode and cathode chambers. The anode chamber was filled with sanitary waste water (SWW) enriched with 10% glucose solution. 5ml each of di-potassium hydrogen phosphate and potassium di-hydrogen phosphate buffered saline (4.26g/L M K₂HPO₄and 2.76g/L KH₂PO_4,0.1M KCl) (analytical grade) was added in order to stabilize the operating pH between 7.2 and 7.5 of the medium. Also added were 0.2g NaCl and 3.5g NaHCO₃ salts in order to increase the ionic strength of the medium. Excess salinity was avoided as such could impose threat to the life, growth and activity of anaerobes. The cathode chamber was filled with potassium ferricyanide solution, 0.1M K₃Fe(CN)₆(Wei et al., 2012). Graphite rod was selected as the electrode material on account of its good conductivity, large specific surface area and relative inertness for attachment and survival of microorganism and support for biofilm formation and growth. The two reacting chambers were separated by an 0.8m length pipe containing Ekowe clay functioning as proton exchange membrane. The graphite rod was inserted into each of the chambers for electron conduction. A 100Ω external resistor was connected between the electrodes to complete the circuit and equally increase the cell capacity for electron recovery. The anode was inoculated using activated sludge from Imiringi oil spill site in Bayelsa State. The anodic chamber was fed with sanitary wastewater, collected from male hostel at Federal Polytechnic Ekowe, Bayelsa State and stored at 4^oC until use.

4.2 Tukey post-hoc HDS statistical test for process variables effect on MFC performance

4.2.1 One-way AVOVA for effect of PPT

Analysis of variance was carried out to examine the effect and hence validate that thermal degradation of the kaolinite-rich Akaso clay functioning as a medium of proton exchange on the performance of MFC with respect to COD removal efficiency and electrical energy recovery was not by chance. Table 4 shows the ANOVA for variables effects on the performance of MFC with respect to power generation and wastewater clean-up. Furthermore, as shown in Table 5, the temperatures effect on PEM preparation was subjected to Tukey post hoc HSD (honestly significant difference) test in order to validate that varying the PPT had significant effect on the overall performance of MFCs. This analysis indicated which possible comparisons between group means performance that were actually statistically significant.

The use of one-way ANOVA and Tukey post hoc HSD analyses at P<0.05 showed that a statistical significance existed between and within the group means, which is an indication of a possible rejection of the null hypothesis. This is evident in the existence of statistical difference between the various temperatures to which the samples were subjected in the study.

The analysis examines the possible statistical mean difference and the particular PPT (PEM preparation temperature) that was indeed significant. The post hoc result in Table 5 shows that the PPT in the range 100 °C to 450°C were statistically significant at p-value (p<0.05) with mean differences distinguished asterisks (*). This validates the statistical significance of clay PPT as indicated in Table 4. Conversely, the PPT in the rage above 450^oC whose mean differences are without asterisks are considered insignificant (p>0.05). This is unlike nafion-117 and ZrP with limited PPT of <100^oC and <200^oC (Mohammed et al., 2021). Clay shows characteristic increase in swelling pressure while the suction pressure decreases when wet (Wang et al, 2012). Specifically, at temperatures > 300^oC, kaolinite shows characteristic reduced swelling behavior with improved stabilizing properties (Yilmaz, 2011; Trusilewicz et al., 2012).). These properties could have affected the performance of kaolinite clay as a proton exchange membrane in MFC (Liu et al., 2018). The performance of the cell was optimal at PPT of 300^oC beyond which a decline in performance sets in.

4.2.2 One-way AVOVA for effect of anolyte concentration

Similarly, the effect of change in anolyte concentration on MFC performance was examined using the one-way ANOVA as presented in Table 4. The one-way ANOVA result for the effect of concentration with p-value of 0.003 indicated the statistical significance of concentration (p<0.05). Accordingly, the null hypothesis was rejected, and this gave rise to the existence of statistical differences between the anolyte concentrations studied.

Furthermore, the %COD removal and power generation were also subjected to honest significant difference (HSD) Tukey post analysis in order to establish whether or not anolyte concentration had significant effect on MFC performance. Close examinations between and within the group means were performed as shown in Table 4. The post hoc analysis in Table 5 shows the multiple comparisons of the effect of concentration on energy recovery from sanitary wastewater. From the analysis, the mean differences shown with asterisks (*) were used to denote the pairs that were indeed statistically significant. The analysis showed that the mean pairs within the concentrations of 70 and 80 (v/v) were statistically significant as indicated by the p-values (p<0.05). Increase in anolyte concentration effects a corresponding increase in microbial activity with its attendant wastewater parameter removal and equally induce particle electrocoagulation (Garg, & Prasad, 2019). The significant effect of concentration was also in accordance with the works of Feng et al. (2008) that reported that the COD removal efficiency increases with increase in concentration from 84 to 1600mg/l, and 800mg/l to 2500mg/l (Ullah, & Zeshan, 2020). Ni et al., 2020).

4.2.3 One-way AVOVA for effect of anolyte pH

This is to ascertain the extent to which operational parameters and solution chemistry affect the performance of the cell (Yujie et al, 2008). The effect of anolyte pH on the performance of MFC with respect to sanitary wastewater treatment and energy recovery was investigated using the one-way ANOVA and Tukey’s post analysis as presented in Tables 4 and 5 respectively. From the one-way ANOVA result in Table 4, the existence of a statistical significance between and within the group means (<0.05) clearly indicates that pH has a significant effect on the performance of MFC. The statistical significance was evident as the p-value when the effect of pH was 0.000 (Table 4). However, according to the statistical significant effect of pH on sanitary waste water treatment and energy recovery, the null hypothesis was rejected.

Also, the Tukey post hoc analysis in Table 5 was used to investigate the specific group means that were indeed statistically significant (<0.05). The post hoc analysis shows that the development and operation of microbial fuel cell for waste water treatment and energy recovery was statistically significant at pH 7.5. pH has an obvious consequence on the performance of MFC due to its effect of the microbial activity and influence on power generation. Hence it is important to control the anodic pH around point of neutrality for optimal performance (Zhang et al, 2012). Tremouli et al., (2017) reported that a 35 % increase in power density was achieved as the anolyte pH was increased from 6 to 9 while optimum coulombic efficiency (CE) was recorded at pH 7.

Furthermore, the asterisks (*) in Table 5 were used to clarify the possible comparison of pH that were indeed statistically significant (Agu & Menkiti, 2017). The Tukey post hoc analysis in table 5 indicates that the mean comparison within the pH of 7.5 were statistically significant as shown by their p-values (p < 0.05).

5. CONCLUSION

Within the limit of the experimental conditions, MFC using sanitary wastewater as fuel and thermally modified clay as PEM showed great potential for wastewater treatment. This is evident in the reduction of specific pollution indicators such as COD, BOD, phosphate, conductivity, salinity, TSS, TDS. The study shows maximum cell efficiency at PPT between 100 and 540^oC after a 30-day operational period. This suggests greater potential for proton transfer at temperatures in the range 100^oC<PPT<540^oC as against nafion-117 and imidazolium based ionic liquids incorporated into ZrP which show less tendency to improve their proton conductivity functions at PPT above 100^oC and 200^oC respectively. Nevertheless, the trio show potentials for better performance at elevated temperatures of preparation when applied as PEM in MFC. From the FTIR results, the presence of inner surface hydroxyl (OH) and Al-Al-OH functional groups in both the clay at room and elevated temperatures justifies improved hopping of protons across the sites. This by extension enhances the biodegradability of the wastewater (fuel) driving the cell.

The ANOVA and HDS Turkey’s post Hoc analysis showed that the effects PEM preparation temperature, anolyte concentration and pH of the medium on the performance of MFC are statistically significant.

Therefore, it can be concluded that modifying the physicochemical properties of the anolyte and PEM base substance before compounding with other doping substances could, in addition to opening a leeway for scale-up, improve the capability of MFC in wastewater water degradation before disposal into the receiving human environment (Pandit et al., 2020).

Acknowledgements

The authors acknowledge the support of Analytical Concept Limited, Elelenwo, Port Harcourt Nigeria for providing the Laboratory quality assistance for the analyses. We also appreciate the contributions from the academic resources of the Department of Chemical Engineering, Nnamdi Azikiwe University Awka, Anambra State.

Conflicting interest statement

The authors declare that there is no known conflicting interests or personal relationship that could have influence on the work presented in this paper.

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Cite this Article: Obasi, LA; Nevo, CO (2023). Tukey Post Hoc Statistical Analysis of Clay-Pem Microbial Fuel Cell Operation for Improved Process Performance. Greener Journal of Biological Sciences, 13(1): 30-39.

ARTICLE INFO	ABSTRACT
*Article No.:* 111123128 *Type: Research* *Full Text:* PDF, PHP, HTML, EPUB, MP3	The aim of this work is to analyze the performance of microbial fuel cell (MFC) using clay-proton exchange membrane (PEM) and running on sanitary wastewater SWW as substrate with Tukey’s multivariate statistical approach. Tukey’s statistical method was adopted to analyze Microbial Fuel Cell performance statistically as a function of three variables such as: PEM preparation temperatures (PPT), anolyte concentration, and pH with respect to power generation and water treatment (COD removal). The dual MFC was operated for 30 days using sanitary wastewater (SWW) anolyte enriched with 10% glucose solution influent with varying internal conditions. The results showed that three factors considered where statistically significant in determining the functionality of MFC. While the first variable (PPT) was directly related to the proton conductivity, anolyte concentration and pH were determining performance factors for ion (proton and electron) exchange and transfer within the anolyte medium. The results describe the relationship between cell operational variables and the two main explanatory response variables that would directly link improved process optimization and to further MFC process modifications. The three factors considered together results in the overall cell output with respect to power density generation and wastewater clean-up.
*Accepted:* 13/11/2023 *Published:* 30/11/2023
Corresponding Author Dr.* Obasi, Livinus A. *E-mail:* engrlaobasi@gmail.com
*Keywords: bioelectrochemical systems, performance factor analysis, wastewater treatment, sanitary wastewater, Tukey’s statistical method.*

MFC type	Dual chamber H-type
Chamber (Reactor) Volume	0.00192 m³
Electrode type	Carbon (graphite) rod
Surface area of projected electrode	0.001183 m²
Electrode volume	2.002x10^-6 m³
External resistor applied	100 Ω
Clay-based PEM length	0.08 m
Concentration of catholyte	0.1MK₃Fe(CN)₆
Anodic Substrate	Municipal, Brewery and Sanitary wastewaters
Bacteria (Inoculum)	Mixed microbial consortium (community)
Mediator used	Mediator-less
Operational Condition	Batch

Functional group	Frequency (cm^-1)			Intensity
OH stretching		3697.5		strong
OH stretching			3652.8	strong
OH stretching		3623.0		strong
C=C=C stretching		1982.9		medium
C= N stretching		1640.0		medium
C-F stretching		1114.5		strong
C-N stretching		1028.7		medium
Si-O stretching		1002.7		strong
Al-Al-OH stretching		909.5		strong
C=C bending		790.2		medium
C=C bending		749.2		medium
C-Br stretching		670.9		medium

Table 4: One way ANOVA for variable effect on MFC performance using sanitary wastewater as substrate.
	PEM preparation temperature (⁰C)					Anolyte concentration (v/v)					Anolyte pH
	Sum of squares	df	Mean square	f-value	p-value	Sum of squares	df	Mean square	f-value	p-value	Sum of squares	df	Mean square	f-value	p-value
Between groups	1099	4	477.5	75.29	0.001	1009	3	537.5	64.5	0.003	1575	1	845.5	90.59	0.000
Within groups	30	3	6.343			80	3	8.333			65	3	9.333
Total	1129	7				1089	6				1640	4