OTRAS RESERVAS

Table 5-1 outlines the abundances of bacteria as detected in EBPR systems by other researchers, but the results of this study do not agree well with these findings. The difference in results could be attributed to the negative effect of α-Proteobacteria on β-Proteobacteria and Actinobacteria during EBPR upset (Okunuki et al., 2004). β-proteobacteria and Actinobacteria are reported to contribute to good EBPR performance, while α-proteobacteria is suggested to reduce EBPR performance at laboratory scale (Okunuki et al., 2004; Bond et al., 1999). In this system α-Proteobacteria had a greater effect on Actinobacteria than β-Proteobacteria with r=-0.71 and r=-0.51, respectively, explaining the low abundance of β-Proteobacteria. Figure A-7 shows graphically the dramatic decline in β-Proteobacteria and Actinobacteria as α-Proteobacteria increases to a maximum of 86% abundance. Figure A-7 also shows the concurrent increase of Bacilli with α-Proteobacteria, showing Bacilli are potentially resistant to and synchronise with α-Proteobacteria. Bacilli and Clostridia are sister classes, both of the phylum Firmicutes, that share phylogenetic properties which supports their mutual existence with α- Proteobacteria from days 189 to 239 (see Figure 5-2) (Berman, 2012).The high abundance of Firmicutes in Slough WWTP is attributed to the high presence of food, beverage, and pharmaceutical related industries in the catchment area (Haakensen et al., 2008; Meng et al., 2015).

Table 5-1: Abundance of microorganism order found in EBPR systems as reported in literature Muszynski et al. (2015) Lee et al. (2003) Juretschko et al. (2002) Kong et al. (2007) Current study Actinobacteria 15% <10% 13% 26% Acidobacteria <1% <1% 6% α-Proteobacteria <10% 16% 30% Bacteroidetes 17% 2% β-Proteobacteria 23% 16-47% 47% 10% Chloroflexi 12% 8% 8% Firmicutes <10% <1% 11% γ-Proteobacteria <10% <1% 6% Planctomycetes <1% Proteobacteria 56%

Figure 5-2 shows the opposing relationships between Firmicutes and Actinobacteria as seen on days 189-415. The substitution of Firmicutes and Actinobacteria bacteria between days 189-239 and days 349-415 is likely due to the fact that these bacteria contain a family of proteins functionally equivalent to each other (Ravagnani et al., 2005). The decrease in α-Proteobacteria from day 349-415 allowed a rise in Actinobacteria and β-Proteobacteria abundances. Due to the increase of Actinobacteria, Firmicutes were no longer required due to their shared functions.

5.5.3 Diversity

Figure 5-3a shows the dramatic decrease in diversity which is followed by a recovery of diversity and mutual improvement of EBPR performance. The recovery of diversity may be due to the mutation and adaptation of new dominant bacteria strains (Li & Jin, 2009). Flowers et al. (2013) suggest diversity in their samples increased due to the washing effect of heavy rainfall transporting non-activated sludge bacteria into the EBPR process. This is a possible explanation for the peak in diversity seen on day 217 following the heaviest rainfall event as seen in Figure A-8 of Supplementary Data. However, for this EBPR system an increase in total rainfall and therefore washing effect caused a decrease in diversity as seen in the negative correlation r=-0.35. From October to December, total rainfall averaged 3.4mm/day with diversity averaging 2.82; excluding these months total rainfall averaged 1.3mm/day with diversity averaging 5.5. Therefore, no distinct conclusion can be drawn on the effect of rainfall and it’s washing effect on EBPR microorganisms.

As shown in Figure 5-3b and through correlation analysis there was no significant relationship found

between diversity and specific PO4-P release/uptake (r=0.19 and r=0.26). In this system, a more diverse

community does not equate to improved EBPR performance. Much of the literature suggests that increased diversity provides improved process stability and reduces risk of EBPR failure (Loy et al., 2002;

Curtis et al., 2003; Mielczarek et al., 2013; Wagner & Loy 2002; Saikaly et al., 2005; Wilmes et al., 2008). It is thought that an increase in diversity may improve the chance of obtaining species with complementary physiological traits that can better handle changes to their environment (Saikaly & Oether, 2004). The results of this research may differ from others because of excess chemical dosing onsite.

Figure 5-3c shows the undulating effect of Fe concentration on diversity, a weak positive correlation of r=0.25 was calculated between Fe concentration and diversity. To withstand the effects of FeCl2 solution

dosing, diversity increased to select for bacteria better able to deal with these conditions. The fact that only Proteobacteria and Firmicutes correlated negatively with diversity is attributed to their synchronised relationship, as described previously. This relationship is underlined by the strong correlation r=0.60 between Lactobacillus and Ochrobactrum and their very strong negative correlations with diversity (r=-0.78 and r=-0.81, respectively). The heat map in Figure A-3 displays the opposite behaviour of Actinobacteria and Firmicutes, substituting from low-medium-high diversity, reinforcing the fact that these bacteria have similar functions and are interchangeable.

Lactobacillus competes with other microorganisms due to their fast growth and tolerance to low pH, which may explain their presence at low diversities (Gois et al., 2013). However, as diversity increases Lactobacillus loses its ability to compete with a wide range of microorganisms and eventually withdraws, to be replaced by Ochrobactrum with which it has a strong positive correlational relationship.

5.5.4 Low & high iron concentrations

Much of the literature on chemical dosing and heavy metals in EBPR suggests that metals inhibit

microorganism growth and diversity. De Gregorio et al. (2010) suggest that the addition of FeCl2 solution

dosing creates stress conditions which reduces the number of organisms present and promotes the development of organisms more tolerant to chemical than others. Motlagh et al. (2015) state heavy metals can cause PAO infection and death through intra-cellular phage induction. However, viewing Figure 5-4 and Figure A-5 it is clear that the presence of Fe did not reduce organisms present; rather it appeared to increase organism counts, which does not support the hypothesis of this research. A positive correlation exists between Fe concentration and diversity (r=0.25), and Fe and genus count (r=0.44).

According to Britton, (2011) some bacteria are acidophilic, deriving energy from oxidation of Fe2+ to Fe3+.

A strong correlation of r=0.60 was measured between Bacteroidetes and Fe concentration. Bacteroidetes are very resistant versatile microorganisms which can degrade complex organic compounds and biopolymers (Wan et al., 2013; Naumoff & Dedysh, 2012). In terms of genera, Terrimonas measured the highest correlation with Fe concentration at r=0.60, but little is known of the environmental significance of Terrimonas (Zhang et al., 2011). Terrimonas are a strictly aerobic non-

motile genus, dominant genus in aerobic, anoxic, and anaerobic sludge (Jin et al., 2013; Zhang et al., 2015). Terrimonas pekingensis, the species of Terrimonas in these samples, grow between 10-37°C (optimum 30°C) and pH 5.0-8.0 (optimum pH 7.0) (Jin et al., 2013). Figure A-4 and Figure A-5 show Terrimonas has higher abundance at increased Fe concentrations and diversity, displaying it is a resilient and competitive genus. From this research Aestuariimicrobium and Dokdonella are also considered resistant to Fe, because they were present in all five highest Fe samples, as seen in Figure 5-4.

Little is known of Aestuariimicrobium; indeed the only relevant information available is the species Aestuariimicrobium kwangyangens present in these samples. Aestuariimicrobium kwangyangens is aerobic, with growth occurring between 4-40°C (30°C optimum) and pH range 7.5-8.5 (Jung et al., 2007). Through this research the authors suggest Aestuariimicrobium is a resilient microorganism able to withstand high concentrations of Fe, a correlation of r=0.04 was measured showing Fe has little impact on Aestuariimicrobium.

Much of the biological function of Dokdonella is still unknown; its ferrous-oxidising ability has yet to be proven (Wang et al., 2015). Dokdonella is strictly aerobic and heterotrophic and has been recovered from activated sludge in anaerobic/aerobic reactors (Liu et al., 2013). Dokdonella growth occurs between 16-37°C (optimum 25°C) and pH 5.0-8.5 (optimum between 6.5 and 7.0) (Liu et al., 2013). Similar to Aestuariimicrobium, this analysis shows Dokdonella are resistant to high concentrations of Fe dosing. Due to the higher correlation (r=0.25) between Fe concentration and Dokdonella, it is thought that Dokdonella may be more resistant to Fe than Aestuariimicrobium. A positive correlation of r=0.47 between Dokdonella and diversity was found, suggesting this genus out-competes other genera present at higher diversities.