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Aggregation and surface hydrophobicity of selected microorganism due to the effect of substrate, pH and temperature
International Biodeterioration & Biodegradation 93 (2014) 202e209
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Aggregation and surface hydrophobicity of selected microorganism due to the effect of substrate, pH and temperature Khalida Muda a, b, *, Azmi Aris a, b, Mohd Razman Salim a, b, Zaharah Ibrahim c, Mark C.M. van Loosdrecht d, Mohd Zaini Nawahwi c, Augustine Chioma Affam a, b a
Faculty of Civil Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia Institute of Environmental and Water Resource Management, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia d Department of Biotechnology, Delft University of Technology, Julianalaan 67,2628BC Delft, The Netherlands b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2014 Received in revised form 26 May 2014 Accepted 27 May 2014 Available online
Aggregation and surface hydrophobicity are two important indicators used to evaluate the successful development of biogranules. The effect of substrate concentration, pH, and temperature on these properties of selected bacteria that are isolated from biogranules used in treating textile wastewater was investigated. Statistical response surface methodology was used to quantitatively determine the effects and to determine the relationship between the responses and variables. Thirty experimental runs were performed at substrate concentrations of 500e3000 mg L1, pH of 5e9, and temperatures of 20e40 C. The results show that all variables investigated were either positively or negatively correlated with aggregation and surface hydrophobicity and induced significant linear responses in these indicators. Interaction effects were also observed for some of the variables. An acceptable statistical model describing the relationship between aggregation and surface hydrophobicity of the selected bacteria and substrate concentration, pH, and temperature has been developed for the stated experimental conditions. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Biogranules Aggregation pH Substrate Surface hydrophobicity Temperature
1. Introduction The development of granules is a complex process and is initiated by the formation of small microbial aggregates (Adav et al., 2008). The aggregation of microbial cells can be formed by either cell-to-cell interaction or by combination with other particulates, which eventually lead to the development of granules of stable multicellular associated bioflocs. This initial cell interaction is affected by many factors, including the type and concentration of substrate, nature of the seed sludge, availability of essential nutrients, presence of exopolymeric protein and the ratio between exopolymeric protein and polysaccharides in the extracellular polymeric substrates (EPS), composition of the media, pH, temperature, and the operational set-up of the reactor system (Dignac et al., 1998; Wang et al., 2009; Zhu et al., 2012).
* Corresponding author. Faculty of Civil Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. E-mail address: [email protected] (K. Muda). http://dx.doi.org/10.1016/j.ibiod.2014.05.023 0964-8305/© 2014 Elsevier Ltd. All rights reserved.
Aggregation ability and surface hydrophobicity of the bacteria are two independent traits that have been used to indirectly evaluate the adhesion capacity of the bacteria (Marin et al., 1997; Ibrahim et al., 2005; Rahman et al., 2008; Wan et al., 2011). Since the adhesion ability is important in the granulation process, several studies have been conducted to investigate factors that can affect this, measured in terms of aggregation and surface hydrophobicity (Wang et al., 2005; Adav and Lee, 2008). The ability of the Bifidobacterium spp. to form aggregates was studied against the effect of pH, type of culture media, and temperature, while variations in the surface hydrophobicity were investigated under the influence of superficial velocity and organic loading rate. Nevertheless, most previous studies of this initial granulation stage focused only on the effect of individual factors, without looking into the effects of the interaction of these factors that may cause different outcomes. In reality, there will be more than one factor that can affect the process and there is the possibility of an interaction between these factors. Moreover, most studies that used the traditional one factor at a time approach did not fully explore all phenomena that could take place, and therefore, could result in wrong conclusions.
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In this study, the effects of substrate concentration, pH, and temperature on the adhesion ability, in terms of cell aggregation and surface hydrophobicity, of isolated bacteria from biogranules were explored. Statistical experimental design was used to provide a quantitative understanding of the main and interaction effects of the variables and to develop the correlation between the variables and the responses. 2. Materials and methods 2.1. Wastewater composition Synthetic textile wastewater as described by Muda et al. (2010) was used in the study. Glucose (0.5 g L1), ethanol (0.125 g L1), and sodium acetate (0.5 g L1) were used as the mixed carbon sources. The trace elements used were based on the composition recommended by Smolders et al. (1995). Mixed dyes, consisting of Sumifix Black EXA, Sumifix Navy Blue EXF, and Synozol Red K-4B, with a total concentration of 10 mg L1 were used as the dye model compounds. 2.2. Source of biogranules The mature biogranules with an average size on 0.45 mm used in this experiment were developed in sequential batch reactor (SBR) as reported in our previous study (Muda et al., 2010).
%Ag ¼
Ago Agf 100 Ago
The synthetic textile dyeing wastewater was sterilized by autoclaving the media for 20 min at 121 C and was used as the growth medium. The mixed dyes, glucose, and minerals were filtered through a 0.2-mm membrane filter for sterilization purposes. These materials were then added to the autoclaved medium. Using an aseptic technique, a small amount of mature granules were added to synthetic dyeing medium (15 mL) and mixed in a sterilized beaker in order to dissolve the granules. After going through serial dilution, about 1 mL of liquid sample was spread onto nutrient agar using a glass spreader. The isolation of microorganisms was carried out by the spread plate method (Madigan et al., 2000). The plates were inverted and incubated at room temperature and were monitored over several weeks. Pure bacterial cultures were obtained by repeatedly sub-culturing onto new nutrient agar plates until single pure colonies were obtained. 2.4. Aggregation assay The aggregation assay was conducted based on a modification of the procedure presented by Rahman et al. (2008). Each sample was aerated using an air diffuser at a flow rate of 5 L/min until the growth of the bacterial culture reached a stationary phase. The cell growth was monitored by optical density (OD) measurements with an optical density meter (100 VIS Spectrophometer) at a wavelength of 600 nm. After the stationary phase had been attained, 15 mL of each bottle was taken for the aggregation assay. Turbidity was measured using a turbidity meter (H 93703 Microprocessor). The initial turbidity reading (Ago) of the samples was recorded and followed by centrifugation at a speed of 650g for 2 min, as described by Malik et al. (2003). Subsequently, the turbidity of the samples was measured again (Agf). The aggregation ability is expressed as the percentage aggregation (%Ag) and calculated using Eqn. (1).
(1)
2.5. Surface hydrophobicity assay The surface hydrophobicity (SH) of the bacterial strains as mixed cultures was based on the microbial adhesion to hydrocarbon assay. This assay, originally described by Rosenberg et al. (1980) and Canzi et al. (2005), was modified to suit the experimental conditions and the focus of this study. Fifteen milliliters of the sample were taken from the sample bottle and used for the surface hydrophobicity assay. The bacterial cells from the samples were harvested by centrifugation at 14,000g for 5 min. The supernatant was discarded, while the pellets were washed twice with 50 mM K2HPO4 (pH 7.0). The pellets were then resuspended in the same buffer to obtain an absorbance of about 0.5 at 660 nm. Five milliliters of the bacterial suspension were mixed with 1 mL of xylene (C6H4(CH3)2) by vortexing for 120 s and then allowed to stand for 1 h at room temperature. The absorbance of the bacterial suspension in the aqueous phase after extraction with xylene (Ai) was compared against the absorbance of sample before mixing with xylene (Ao). The absorbance was measured at 660 nm using an optical density meter (Jenwai6300 Spectrophometer). The surface hydrophobicity is expressed as the percentage surface hydrophobicity (%SHb) and calculated using Eqn. (2).
%SHb ¼ 2.3. Bacteria isolation
203
Ao Ai 100 Ao
(2)
2.6. Experimental procedures Bacteria isolated from the granules, as previously explained, were used in this study. Each bacterial cell was cultured separately in nutrient broth until the OD was close to 1. The cell cultures were harvested and resuspended in saline water. Thereafter, the bacteria were mixed together and about 25 mL (10% v/v) of the sample culture were inoculated in a separate 250-mL Schott bottle containing synthetic textile wastewater with a concentration of 10 mg L1. The inoculated synthetic textile sample was allowed to decolorize before the aggregation and surface hydrophobicity assay were carried out. Color was measured in terms of the American Dye Manufacturing Index (ADMI) using a HACH Spectrophotometer (DR/4000U) (APHA, 2005). After 90% decolorization, the synthetic wastewater was aerated at a flow rate of 5 L/min for 5 h. Instead of using physical shaking, as commonly reported in previous aggregation works (Nishiyama et al., 2007; Rahman et al., 2008; Adav and Lee, 2009), aeration was used to provide a close resemblance of the techniques used in the granulation process. Throughout the aeration period, 10 mL samples were taken hourly and used for the aggregation and surface hydrophobicity assays. To determine the effect of pH, temperature, and substrate on aggregation and surface hydrophobicity of the mixed culture, a central composite rotatable experimental design was used. Using this approach, the effect of each factor or variable separately (termed as the main effect) and the effect of the interaction between the variables (termed as the interaction effect) on the responses (i.e., aggregation and surface hydrophobicity) can be determined quantitatively instead of qualitatively. The central composite rotatable experimental design consisted of a 2-level had a matrix factorial design (coded as þ1 and 1) with the addition of star (coded as þa and -a) and center points (coded as 0). The factorial design comprised of eight runs, which were duplicated, the star points comprised of six runs, while the center point comprised of one run that was replicated six times. The
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Table 1 The variables and their values used in the experiments. Variables
Factorial
A: Substrate (mg/L) B: pH C: Temperature ( C)
Star points
Center point
Lower value (1)
Upper value (þ1)
Lower value (-a)
Upper value (þa)
(0)
500 5 20
3000 9 40
1007 6 24
2493 8 36
1750 7 30
design and analysis were carried out using Minitab® (Version 13.32) and Design Expert® (Version 7.0). The substrate, pH and temperature range of values that were used in the experiments are shown in Table 1. The responses measured were percentage aggregation (%Ag) and percentage surface hydrophobicity (%SHb) of the mixed bacteria. 3. Results and discussion 3.1. Isolated bacteria A total of 12 bacteria were successfully isolated as pure cultured bacteria from the granular sludge developed in the synthetic textile wastewater using the SBR system. All of the bacteria were characterized in terms of form, shape, edges, and colony surface for their colony characteristics. A gram staining procedure was carried out for each bacterium in order to characterize the cellular morphology of the isolated bacteria. Among the 12 pure bacterial cultures, six bacteria were selected and used in this study. The selection of the six bacteria was based on the percentage chemical oxygen demand (COD) and color removal, aggregation, and surface hydrophobicity of each isolated pure culture (Muda, 2010). 3.2. Factorial analysis The results of the factorial experiments for aggregation and surface hydrophobicity are shown in Table 2. Depending on the experimental conditions, the %Ag and %SHb after 6 h of aeration varied between 39% to 85% and 19%e54%, respectively. A summary of the factorial analyses is shown in Table 3. The significance of the variables tested, i.e., substrate concentration, pH, and temperature, are based on p-values evaluated at a confidence level above 95% (pvalue less than 0.05). Based on the experimental conditions used in the study, the p-value indicates that all factors, i.e., substrate
Table 2 Experimental results for 2-level factorial design analysis. Run no.
Substrate concentration
pH
Temperature
%Ag
%SHb
RUN01 RUN02 RUN03 RUN04 RUN05 RUN06 RUN07 RUN08 RUN09 RUN10 RUN11 RUN12 RUN13 RUN14 RUN15 RUN16
1 1 þ1 þ1 1 1 þ1 þ1 1 1 þ1 þ1 1 1 þ1 þ1
1 1 1 1 þ1 þ1 þ1 þ1 1 1 1 1 þ1 þ1 þ1 þ1
1 1 1 1 1 1 1 1 þ1 þ1 þ1 þ1 þ1 þ1 þ1 þ1
49.6 44.7 67.2 64.1 46.8 44.1 65.6 62.9 65.2 61.9 84.5 85.4 38.8 38.7 57.4 57.6
19.0 21.7 33.8 29.4 37.3 41.2 39.6 36.9 32.1 31.5 53.9 52.0 20.4 18.2 8.6 6.8
concentration, pH, and temperature, have a significant effect on aggregation. A significant interaction effect is observed between pH and temperature (pH temperature), while the three-way interaction effect (substrate concentration pH temperature) is not significant. As for surface hydrophobicity, all model terms (main, 2way, and 3-way interaction) are significant except for the 2-way interaction between substrate concentration and temperature (substrate concentration temperature). The significance of the substrate concentration on bacterial aggregation and surface hydrophobicity is shown by very low pvalues of less than 0.05; their estimated positive main effects are 19.36 and 4.95, respectively, which indicates that higher substrate concentrations enhance the aggregation and surface hydrophobicity. This can be explained by the fact that an increase in the concentration of the substrate means more food being supplied to the bacteria, which results in an increase in bacterial growth. The presence of more cell biomass increases the collisions between cells and causes more aggregation to occur. As reported by Liu and Tay (2002), the collision between particles is one of the key factors influencing the formation and stabilization of biofilm, anaerobic, and aerobic granules. An increase in substrate concentration may also enhance the bacterial growth rate, which would also increase the production extracellular polymeric substances, particularly when faced with starvation (Wang et al., 2006). The presence extracellular polymeric substances on the cell surface could alter the physicochemical properties of the bacterial cell surface, which include surface charge and surface hydrophobicity. As reported by Wang et al. (2005), the poorly soluble and non-biodegradable extracellular polymeric substances on the surface of biogranules is responsible for the higher hydrophobicity of the granules, which may facilitate the adhesion or aggregation processes of the granules (Kjelleberg et al., 1987; Jiang et al., 2004). More recently, Lin et al. (2010) found alginate-like exopolysaccharides as one of the dominant components in aerobic granules and that they significantly influence the physical and chemical characteristics of the granules. The effect of pH on %Ag and %SHb was also found to be significant, with a p-value of less than 0.05. However, based on the estimated effects of 13.84 and 8.05, increases in pH are expected to reduce aggregation and surface hydrophobicity of the bacteria. This phenomenon is thought to be related to bacterial surface charge. Under neutral conditions, the bacteria would be negatively charged owing to the ionization of carboxyl, sulfate, and phosphate that act as functional groups on the surface of the cell (Sutherland, 2001). As they have the same charges, there will be a repulsive force between the two cell surfaces, which inhibits cell aggregation. Under acidic conditions, the presence of excess Hþ will neutralize the cell surface charge. This will reduce the repulsion and in turn favors cell-to-cell approach and the formation of cell aggregates can be initiated (Derjaugin and Lanadau, 1941). However, under alkaline conditions, the excess OH would enhance the surface charges on the cells and consequently increase the repulsive forces causing cells to be driven further apart, indicated by the reduction in %Ag and %SHb. Differences in pH of the media can also be associated with the type of substrate used in the growth media. Different types of
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Table 3 The p-values of the estimated main and interaction effects of substrate concentration, pH, and temperature on %Ag and %SHb after 6 h of aeration. Effects
Estimated effect
%Ag
Main Substrate concentration pH Temperature
19.36 13.84 5.56
<0.0001 <0.0001 0.0004
4.95 8.05 4.43
0.001 <0.0001 0.0019
2-way interaction Substrate conc. pH Substrate conc. temperature pH temperature
0.59 0.71 12.29
0.5607 0.4827 <0.0001
11.25 0.18 20.82
<0.0001 0.8624 <0.0001
0.74
0.4679
5.13
0.0008
3-way interaction Substrate conc. pH temperature
growth substrate may result in acidic or alkaline conditions when hydrolyzed. The concentration of Hþ and OH will affect the surface tension of the liquid media and eventually affect the surface hydrophobicity of the bacteria cell (Pasdar and Chan, 2000). The effect of pH on the surface hydrophobicity depends on the net surface charge of the exoprotein of the bacterial cell, which ultimately depends on the type of protein in the bacterial cell wall (Wang et al., 2006). Further investigations are required for a better understanding of the effect of pH on the different proteins in the bacterial cell wall and consequently on the surface hydrophobicity. Temperature was also found to have a significant positive effect on %Ag with a p-value of less than 0.05. However, the estimated effect was only 5.56, suggesting a weaker effect than the other two variables. An increase in temperature within the range of the experimental conditions is expected to increase microbial activity, which includes the metabolic rate and mobility of the microorganisms (Voet and Voet, 2004). In other words, a rise in temperature increases the specific growth rate of the bacteria and hence cell biomass, which increases the microbial aggregation process, as mentioned earlier. Ibrahim et al. (2005) reported the same observation that, as the incubation temperature was increased, the ability of the Bifidobacteria to aggregate also increased. While the effect of temperature on %SHb was significant (pvalue of 0.002), the estimated effect was negative (4.43). This indicates that the surface hydrophobicity of the bacteria decreases with increasing temperature. It is expected that as temperature increases, the liquid surface tension decreased (Moraes et al., 2008), thus allowing the adhesion of hydrophilic cells and resulting in a reduction in the percentage of cell surface hydrophobicity. Blanco et al. (1997) reported that the majority of the 42 strains of Candida albicans were hydrophobic at 20 C and became hydrophilic when the temperature was increased to 37 C. It is interesting to note that while surface hydrophobicity is expected to be the triggering force of cell aggregation (Liu et al., 2004), the effects of temperature on surface hydrophobicity and aggregation are opposing. To explain this observation, Rahman et al. (2008) categorized bacteria (Bifidobacteria) into high, medium, and low aggregator groups. Among the high aggregators, increases in temperature caused a decrease in the percentage aggregation. As for the medium and low aggregators, increases in temperature caused an increase in aggregation. It is likely that in addition to temperature, other factors such as pH of the media, and the types of protein bound on the cell surfaces of different bacterial strains at different temperatures (Maclagan and Old, 1980) may cause different effects on their ability to aggregate. Different types of bacteria may respond differently when exposed to different environmental conditions with the overall effect being influenced by the most dominant species in the group.
Estimated effect
%SHb
Fig. 1. Interaction effect plot between (a) pH and temperature for the aggregation process, (b) substrate and pH, and (c) pH and temperature for the surface hydrophobicity (-temperature ¼ 20 C, þtemperature ¼ 40 C; pH ¼ 5, þpH ¼ 9).
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Table 4 Experimental results for CCRD analysis. Run
Substrate
pH
Temperature
Aggregation (%Ag)
Surface hydrophobicity (%SHb)
RUN01 RUN02 RUN03 RUN04 RUN05 RUN06 RUN07 RUN08 RUN09 RUN10 RUN11 RUN12 RUN13 RUN14 RUN15 RUN16 RUN17 RUN18 RUN19 RUN20
1 þ1 1 þ1 1 þ1 1 þ1 1.682 þ1.682 0 0 0 0 0 0 0 0 0 0
1 1 þ1 þ1 1 1 þ1 þ1 þ1 þ1 1.682 þ1.682 0 0 0 0 0 0 0 0
1 1 1 1 þ1 þ1 þ1 þ1 þ1 þ1 0 0 1.682 þ1.682 0 0 0 0 0 0
49.6 67.2 46.8 65.6 65.2 84.5 38.8 57.4 68.8 73.0 54.2 5.5 29.3 58.3 59.4 56.4 55.1 60.3 57.5 60.2
19.0 33.8 37.3 39.6 32.1 53.9 20.4 8.6 50.9 70.7 62.4 7.8 71.2 83.9 75.2 77.2 72.7 74.0 76.2 72.2
The interaction effects between the factors on the responses were also investigated to provide a better understanding of how the responses behave when changes of more than one variable take place. Fig. 1 shows the significant interaction effect plots between aggregation and surface hydrophobicity. Of the three variables, only the interaction effect between pH and temperature was significant for %Ag with a p-value of less than 0.05 (Fig. 1a). As for %SHb, the interaction effects were significant between pH and temperature and between pH and substrate with p-values less than 0.05. At low temperature, %Ag was almost the same at low and high pH (i.e., pH 5 and 9) at about 53%. However, at high temperature, % Ag under acidic conditions increased up to about 75% while %Ag under alkaline conditions dropped slightly to less than 50%. This suggests that the effect of pH is strongly dependent on the temperature of the solution. The main effect that temperature and pH have on %Ag and their probable causes were discussed earlier. Apparently, under alkaline conditions, the high concentration of OH, which causes the repulsive force between cells, is greater and overshadows the positive effect of high temperature. Under acidic conditions, the synergistic positive effect of both high Hþ concentration and high temperature significantly increase %Ag.
The significant interaction effect of substrate and pH on %SHb is shown in Fig. 1b. At high substrate concentrations, a change in pH from 5 to 9 causes the surface hydrophobicity to decrease, while at low substrate concentrations, the change in pH has an insignificant effect on the surface hydrophobicity. The phenomenon is believed to be caused by the presence of acetate in the synthetic textile dyeing wastewater, which is hydrolyzed to release OH during the aeration phase (Voet and Voet, 2004). This increases the concentration of OH and makes the wastewater more alkaline when the initial pH is 9. As the cell hydrophobicity is inversely correlated to the quantity of surface charge on the bacteria (Liao et al., 2001), such a synergistic condition (i.e., initial pH 9 and OH from acetate hydrolysis) significantly reduces the surface hydrophobicity, as observed in this study. At low pH, the OH released from the hydrolysis of acetate will neutralize the existing Hþ. In this situation, the electrostatic force is reduced and when the substrate concentration is increased, the production of extracellular polymeric substances may contribute to the cell hydrophobicity and make the polymeric interaction force dominant and promote cell adhesion (Tsuneda et al., 2003). The interaction effects between pH and temperature on %SHb are inversely correlated, as shown in Fig. 1c. At lower pH value (pH 5), increasing the temperature (from 20 C to 40 C) increases %SHb (from 26% to 40%). However, at higher pH level (pH 9), increasing temperature (from 20 C to 40 C) reduces %SHb (from 38% to about 14%). Similarly, increasing the pH from 5 to 9 at 20 C, increases % SHb, but the same action at 40 C reduces %SHb. This interaction effect does not agree with the previous discussion. It appears that there are some byproducts from the degradation of the wastewater that are affecting the aggregation and surface hydrophobicity of the bacteria and that further in-depth study is required for a better understanding of this phenomenon. The factorial analyses also identify the significance of the 3-way interaction between substrate concentration, pH, and temperature on %SHb (p-value of 0.001) under the current experimental conditions. 3.3. Response surface analysis Response surface analysis was conducted to model the relationships between the aggregation and surface hydrophobicity and the factors being considered (i.e., substrate concentration, pH, and temperature). In addition, the curvature effect of the significant interactions could be further investigated. The results of the central composite rotatable experimental design experiments are shown
Table 5 Summary of the p-values from the response surface modeling analysis. Term
% Ag Full quadratic terms
% SHb Linear þ square þ pH temperature
The P-valuea Substrate pH Temperature Substrate pH Substrate Temperature pH Temperature Substrate Substrate pH pH Temperature Temperature R-squared value Lack of Fit (LOFT) a
0.0067 0.0022 0.0369 0.6329 0.8147 0.02 0.0042 0.0427 0.2397 86.6% 0.0029
0.0037 0.0009 0.0262 e e 0.0133 0.0016 0.0352 e 84.1% 0.048
0.01e0.04: Highly significant; 0.05e0.1: significant; 0.1e0.2: less significant; <0.2: insignificant (Vecchio, 1997).
0.3582 0.075 0.9194 0.3596 0.8854 0.1221 0.0423 0.0014 0.3393 75.9% <0.0001
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Fig. 2. (i) Contour and (ii) 3D response surface plots representing the relationship between pH, temperature, and %Ag.
in Table 4. The analyses were carried out using full quadratic terms, including linear, square, and interaction terms, with the aid of Design-Expert 7P statistical software (version 7). The summarized results of the ANOVA with respect to %Ag and %SHb are shown in Table 5. As in the factorial analysis, the significance of the terms (i.e., linear, interaction, and curvature (term2)) is based on a 95% confidence limit (i.e., p-value less than 0.05). For aggregation (%Ag), all linear terms, interaction between pH and temperature, and all curvature terms, except that of temperature, are significant. The model is acceptable with an R-squared value of 86.6%. In order to improve the model for %Ag, the previously determined insignificant terms, which were the two interaction terms (substrate pH and substrate temperature) and one square term (temperature temperature), were omitted. Omitting the insignificant terms improved the p-value of all of the included terms (reduced quadratic terms) compared to the previous analysis (full quadratic terms). The R-squared term decreased from 86.6% to 84.1%, which indicates that the omitted terms only cost 2.5% in goodness-of-fit, which is acceptable. As for %SHb, the only significant terms were pH and the squared pH and substrate terms. The R-squared value of this model is slightly lower than that of %Ag, but within an acceptable range with a value of 75.9%. The best statistical models that can be used to represent the relationship between %Ag and %SHb and substrate concentration, temperature, and pH within the range of the experimental conditions are given below:
where: A: Substrate concentration (mg L1) B: pH C: Temperature ( C) The response surface and contour plots, which illustrate the relationship between the %Ag and pH and temperature at a fixed substrate concentration (i.e., 1750 mg L1), are shown in Fig. 2. According to these analyses, the best condition for achieving the maximum %Ag would be a high temperature and low pH. Fig. 3(a)e(c) show the relationships between surface hydrophobicity and the independent variables of substrate, pH, and temperature. The contour plots of the RSM are drawn as a function of two variables, keeping the remaining variables at a fixed value. The surface plots of the interactions between variables for %SHb as the response were observed to have a symmetrical mound shape (Fig. 3a and b). Fig. 3b illustrates %SHb as a response at various substrate concentrations and temperatures at a constant pH of 7. The symmetrical mound shape plot shows that at any temperature level, %SHb increases as the substrate concentration increases from 500 to 1750 mg L1. The relationship between pH and temperature at a constant substrate concentration of 1750 mg L1, as shown in Fig. 3c, appears to have an elliptical shape. The response illustrated in Fig. 3(a)e(c) shows that the maximum predicted % SHb is indicated by the surface confined in the smallest curve of the contour diagram. 4. Conclusions
ðAggregationÞ2 ð%Þ ¼ 30449:2 4:02A þ 7114:5B þ 843:1C 108:1BC þ 1:4 103 A2 329:1B2 (3) Surface Hydrophobicityð%Þ ¼ 1122:2 þ 0:12A þ 239:7B þ 18:1C 6:5 103 AB 2:01 104 AC 1:4BC 1:9 105 A2 13:8B2 0:1 C2 (4)
The investigated variables imposed significant linear effect on the selected bacterial aggregation and surface hydrophobicity. The substrate concentration and temperature are positively correlated with aggregation, while a change from acidic to alkaline conditions reduces aggregation; the only significant interaction effect is that between pH and temperature. Surface hydrophobicity increases with substrate concentration, but decreases with bothda decrease in temperature and a change from acidic to alkaline conditions. The interaction effects between pH and substrate concentration, between pH and temperature, and between all three variables are significant.
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Fig. 3. (i) Contour and (ii) 3D response surface plots representing the relationship between variables (pH, temperature, and substrate concentration) and %SHb.
Acknowledgments The authors wish to thank the Ministry of Science, Technology and Innovation (MOSTI), Ministry of Higher Education (MOHE), and Universiti Teknologi Malaysia for financial support for this research (Grant Nos.: 79137, 78211 and 75221). References Adav, S.S., Lee, D.J., 2008. Single-culture aerobic granules with acinetobacter calcoaceticus. Appl. Microbiol. Biotechnol. 78, 551e557. Adav, S.S., Lee, D.J., 2009. Intrageneric and intergeneric co-aggregation with acinetobacter calcoaceticus I6. J. Taiwan Inst. Chem. Eng. 40, 344e347.
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K. Muda et al. / International Biodeterioration & Biodegradation 93 (2014) 202e209 Ibrahim, S.A., Hassan, O.A., Salameh, M.M., Shahbazi, A., 2005. Effect of media composition and incubation temperatures on auto aggregation behavior of bifidobacteria. Milchwiss. Milk. Sci. Int. 60, 127e129. Jiang, H.L., Tay, J.H., Tay, S.T.L., 2004. Bacterial diversity and function of aerobic granules engineered in a sequencing batch reactor for phenol degradation. Appl. Environ. Microbiol. 70, 6767e6775. Kjelleberg, S., Hermansson, M., Marden, P., Jones, G.W., 1987. The transient phase between growth and nongrowth of heteoprothic bacteria, with emphasis on the marine environment. Annu. Rev. Microbiol. 41, 25e49. Liao, B.Q., Allen, D.G., Droppo, I.G., Leppard, G.G., Liss, S.N., 2001. Surface properties of sludge and their role in bioflocculation and settleability. Water Res. 35, 339e350. Lin, Y., de Kreuk, M., van Loosdrect, M.C.M., Adin, A., 2010. Characterization of alginate like exopolysaccharides isolated from aerobic granular sludge in pilot plant. Water Res. 44, 3355e3364. Liu, Y., Tay, J.H., 2002. The essential role of hydrodynamic shear force in the formation of biofilm and granular sludge. Water Res. 36, 1653e1665. Liu, Y., Yang, S.F., Tay, J.H., Liu, Q.S., Qin, L., Li, Y., 2004. Cell hydrophobicity is a triggering force of biogranulation. Enzyme Microbiol. Technol. 34, 371e379. Maclagan, R.M., Old, D.C., 1980. Heamaglutinins and fimbriae in different serotypes and biotypes of Yersinia enterocolitica. J. Appl. Bacteriol. 49, 353e360. Madigan, J.E., Pusterla, N., Johnson, E., Chae, J.S., Pusterla, J.B., DeRock, E., Lawler, S.P., 2000. Transmission of Ehrlichia risticii, the agent of potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report. Equine. Vet. J. 32, 275e279. Malik, A., Sakamoto, M., Hanazaki, S., Osawa, M., Suzuki, T., Tochigi, M., Kakii, K., 2003. Coaggregation among nonflocculating bacteria isolated from activated sludge. Appl. Environ. Microbiol. 69, 6056e6063. Marin, M.L., Benito, Y., Pin, C., Fernandez, M.F., Garcia, M.L., Selgas, M.D., Casas, C., 1997. Lactic acid bacteria: hydrophobicity and strength of attachment to meat surfaces. Lett. Appl. Microbiol. 24, 14e18. Moraes, M.A., Rosa, G.S., Pinto, L.A.A., 2008. Moisture sorption isotherms and thermodynamic properties of apple fuji and garlic. Int. J. Food Sci. Technol. 43, 1824e1831. Muda, K., 2010. Facultative Granular Sludge for Textile Wastewater Treatment. PhD thesis. Universiti Teknologi Malaysia, Malaysia. Muda, K., Aris, A., Salim, M.R., Ibrahim, Z., Yahya, A., van Loosdrecht, M.C.M., Ahmad, A., Nawahwi, M.Z., 2010. Development of granular sludge for textile wastewater treatment. Water Res. 44, 4143e4150.
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Nishiyama, S., Murakami, Y., Nagata, H., Shizukuishi, S., Kawagishi, I., Yoshimura, F., 2007. Involvement of minor components associated with the fima fimbriae of porphyromonas gingivalis in adhesive functions. Microbiology 153, 1916e1925. Pasdar, N.A., Chan, C.Y.L., 2000. Comparison of protein surface hydrophobicity measured at various ph values using three different fluorescent probes. J. Agr. Food Chem. 48, 328e334. Rahman, M.M., Kim, W.S., Kumura, H., Shimazaki, K.K., 2008. Autoaggregation and surface hydrophobicity of bifidobacteria. World J. Microbiol. Biotechnol. 24, 1593e1598. Rosenberg, M., Gutnick, D., Rosenberg, E., 1980. Adherence of bacteria to hydrocarbons: a simple method for measuring cell surface hydrophobicity. FEMS Microbiol. Lett. 9, 29e33. Smolders, G.J.L., Klop, J., van Loosdrecht, M.C.M., Heijnen, J.J., 1995. A metabolic model of the biological phosphorus removal process: effect of the sludge retention time. Biotechnol. Bioeng. 48, 222e233. Sutherland, I.W., 2001. Biofilm exopolysaccharides: a strong and sticky framework. Microbiology 147, 3e9. Tsuneda, S., Jung, J., Hayashi, H., Aikawa, H., Hirata, A., Sasaki, H., 2003. Influence of extracellular polymers on electrokinetic properties of heterotrophic bacterial cells examined by soft particle electrophoresis theory. Colloids Surf. B. 29, 181e188. Vecchio, R.J.D., 1997. Understanding Design of Experiments, a Primer for Technologists. Hanser/Gardner Publications, Cincinnati, USA. Voet, D., Voet, J.G., 2004. Biochemistry 1. John Wiley & Sons Inc., Hoboken, New Jersey. Wan, J., Mozoa, I., Filalia, A., Line, A., Bessiere, Y., Sperandio, M., 2011. Evolution of bioaggregate strength during aerobic granular sludge formation. Biochem. Eng. J. 58e59, 69e78. Wang, S.G., Gai, L.H., Zhao, L.J., Fan, M.H., Gong, W.X., Gaoa, B.Y., Maa, Y., 2009. Aerobic granules for low strength wastewater treatment: formation, structure and microbial community. J. Chem. Technol. Biotechnol. 84, 1015e1020. Wang, Z.W., Liu, L., Yao, J., Cai, W., 2006. Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors. Chemosphere 63, 1728e1735. Wang, Z.W., Liu, Y., Tay, J.H., 2005. Distribution of EPS and cell surface hydrophobicity in aerobic granules. Appl. Microbiol. Biotechnol. 69, 469e473. Zhu, L., Li, M., Dai, X., Yu, Y.W., Qi, H.Y., Xu, X.Y., 2012. Role and significance of extracellular polymeric substances on the property of aerobic granule. Bioresour. Technol. 107, 46e54.
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Aggregation and surface hydrophobicity of selected microorganism due to the effect of substrate, pH and temperature Khalida Muda a, b, *, Azmi Aris a, b, Mohd Razman Salim a, b, Zaharah Ibrahim c, Mark C.M. van Loosdrecht d, Mohd Zaini Nawahwi c, Augustine Chioma Affam a, b a
Faculty of Civil Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia Institute of Environmental and Water Resource Management, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia d Department of Biotechnology, Delft University of Technology, Julianalaan 67,2628BC Delft, The Netherlands b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2014 Received in revised form 26 May 2014 Accepted 27 May 2014 Available online
Aggregation and surface hydrophobicity are two important indicators used to evaluate the successful development of biogranules. The effect of substrate concentration, pH, and temperature on these properties of selected bacteria that are isolated from biogranules used in treating textile wastewater was investigated. Statistical response surface methodology was used to quantitatively determine the effects and to determine the relationship between the responses and variables. Thirty experimental runs were performed at substrate concentrations of 500e3000 mg L1, pH of 5e9, and temperatures of 20e40 C. The results show that all variables investigated were either positively or negatively correlated with aggregation and surface hydrophobicity and induced significant linear responses in these indicators. Interaction effects were also observed for some of the variables. An acceptable statistical model describing the relationship between aggregation and surface hydrophobicity of the selected bacteria and substrate concentration, pH, and temperature has been developed for the stated experimental conditions. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Biogranules Aggregation pH Substrate Surface hydrophobicity Temperature
1. Introduction The development of granules is a complex process and is initiated by the formation of small microbial aggregates (Adav et al., 2008). The aggregation of microbial cells can be formed by either cell-to-cell interaction or by combination with other particulates, which eventually lead to the development of granules of stable multicellular associated bioflocs. This initial cell interaction is affected by many factors, including the type and concentration of substrate, nature of the seed sludge, availability of essential nutrients, presence of exopolymeric protein and the ratio between exopolymeric protein and polysaccharides in the extracellular polymeric substrates (EPS), composition of the media, pH, temperature, and the operational set-up of the reactor system (Dignac et al., 1998; Wang et al., 2009; Zhu et al., 2012).
* Corresponding author. Faculty of Civil Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. E-mail address: [email protected] (K. Muda). http://dx.doi.org/10.1016/j.ibiod.2014.05.023 0964-8305/© 2014 Elsevier Ltd. All rights reserved.
Aggregation ability and surface hydrophobicity of the bacteria are two independent traits that have been used to indirectly evaluate the adhesion capacity of the bacteria (Marin et al., 1997; Ibrahim et al., 2005; Rahman et al., 2008; Wan et al., 2011). Since the adhesion ability is important in the granulation process, several studies have been conducted to investigate factors that can affect this, measured in terms of aggregation and surface hydrophobicity (Wang et al., 2005; Adav and Lee, 2008). The ability of the Bifidobacterium spp. to form aggregates was studied against the effect of pH, type of culture media, and temperature, while variations in the surface hydrophobicity were investigated under the influence of superficial velocity and organic loading rate. Nevertheless, most previous studies of this initial granulation stage focused only on the effect of individual factors, without looking into the effects of the interaction of these factors that may cause different outcomes. In reality, there will be more than one factor that can affect the process and there is the possibility of an interaction between these factors. Moreover, most studies that used the traditional one factor at a time approach did not fully explore all phenomena that could take place, and therefore, could result in wrong conclusions.
K. Muda et al. / International Biodeterioration & Biodegradation 93 (2014) 202e209
In this study, the effects of substrate concentration, pH, and temperature on the adhesion ability, in terms of cell aggregation and surface hydrophobicity, of isolated bacteria from biogranules were explored. Statistical experimental design was used to provide a quantitative understanding of the main and interaction effects of the variables and to develop the correlation between the variables and the responses. 2. Materials and methods 2.1. Wastewater composition Synthetic textile wastewater as described by Muda et al. (2010) was used in the study. Glucose (0.5 g L1), ethanol (0.125 g L1), and sodium acetate (0.5 g L1) were used as the mixed carbon sources. The trace elements used were based on the composition recommended by Smolders et al. (1995). Mixed dyes, consisting of Sumifix Black EXA, Sumifix Navy Blue EXF, and Synozol Red K-4B, with a total concentration of 10 mg L1 were used as the dye model compounds. 2.2. Source of biogranules The mature biogranules with an average size on 0.45 mm used in this experiment were developed in sequential batch reactor (SBR) as reported in our previous study (Muda et al., 2010).
%Ag ¼
Ago Agf 100 Ago
The synthetic textile dyeing wastewater was sterilized by autoclaving the media for 20 min at 121 C and was used as the growth medium. The mixed dyes, glucose, and minerals were filtered through a 0.2-mm membrane filter for sterilization purposes. These materials were then added to the autoclaved medium. Using an aseptic technique, a small amount of mature granules were added to synthetic dyeing medium (15 mL) and mixed in a sterilized beaker in order to dissolve the granules. After going through serial dilution, about 1 mL of liquid sample was spread onto nutrient agar using a glass spreader. The isolation of microorganisms was carried out by the spread plate method (Madigan et al., 2000). The plates were inverted and incubated at room temperature and were monitored over several weeks. Pure bacterial cultures were obtained by repeatedly sub-culturing onto new nutrient agar plates until single pure colonies were obtained. 2.4. Aggregation assay The aggregation assay was conducted based on a modification of the procedure presented by Rahman et al. (2008). Each sample was aerated using an air diffuser at a flow rate of 5 L/min until the growth of the bacterial culture reached a stationary phase. The cell growth was monitored by optical density (OD) measurements with an optical density meter (100 VIS Spectrophometer) at a wavelength of 600 nm. After the stationary phase had been attained, 15 mL of each bottle was taken for the aggregation assay. Turbidity was measured using a turbidity meter (H 93703 Microprocessor). The initial turbidity reading (Ago) of the samples was recorded and followed by centrifugation at a speed of 650g for 2 min, as described by Malik et al. (2003). Subsequently, the turbidity of the samples was measured again (Agf). The aggregation ability is expressed as the percentage aggregation (%Ag) and calculated using Eqn. (1).
(1)
2.5. Surface hydrophobicity assay The surface hydrophobicity (SH) of the bacterial strains as mixed cultures was based on the microbial adhesion to hydrocarbon assay. This assay, originally described by Rosenberg et al. (1980) and Canzi et al. (2005), was modified to suit the experimental conditions and the focus of this study. Fifteen milliliters of the sample were taken from the sample bottle and used for the surface hydrophobicity assay. The bacterial cells from the samples were harvested by centrifugation at 14,000g for 5 min. The supernatant was discarded, while the pellets were washed twice with 50 mM K2HPO4 (pH 7.0). The pellets were then resuspended in the same buffer to obtain an absorbance of about 0.5 at 660 nm. Five milliliters of the bacterial suspension were mixed with 1 mL of xylene (C6H4(CH3)2) by vortexing for 120 s and then allowed to stand for 1 h at room temperature. The absorbance of the bacterial suspension in the aqueous phase after extraction with xylene (Ai) was compared against the absorbance of sample before mixing with xylene (Ao). The absorbance was measured at 660 nm using an optical density meter (Jenwai6300 Spectrophometer). The surface hydrophobicity is expressed as the percentage surface hydrophobicity (%SHb) and calculated using Eqn. (2).
%SHb ¼ 2.3. Bacteria isolation
203
Ao Ai 100 Ao
(2)
2.6. Experimental procedures Bacteria isolated from the granules, as previously explained, were used in this study. Each bacterial cell was cultured separately in nutrient broth until the OD was close to 1. The cell cultures were harvested and resuspended in saline water. Thereafter, the bacteria were mixed together and about 25 mL (10% v/v) of the sample culture were inoculated in a separate 250-mL Schott bottle containing synthetic textile wastewater with a concentration of 10 mg L1. The inoculated synthetic textile sample was allowed to decolorize before the aggregation and surface hydrophobicity assay were carried out. Color was measured in terms of the American Dye Manufacturing Index (ADMI) using a HACH Spectrophotometer (DR/4000U) (APHA, 2005). After 90% decolorization, the synthetic wastewater was aerated at a flow rate of 5 L/min for 5 h. Instead of using physical shaking, as commonly reported in previous aggregation works (Nishiyama et al., 2007; Rahman et al., 2008; Adav and Lee, 2009), aeration was used to provide a close resemblance of the techniques used in the granulation process. Throughout the aeration period, 10 mL samples were taken hourly and used for the aggregation and surface hydrophobicity assays. To determine the effect of pH, temperature, and substrate on aggregation and surface hydrophobicity of the mixed culture, a central composite rotatable experimental design was used. Using this approach, the effect of each factor or variable separately (termed as the main effect) and the effect of the interaction between the variables (termed as the interaction effect) on the responses (i.e., aggregation and surface hydrophobicity) can be determined quantitatively instead of qualitatively. The central composite rotatable experimental design consisted of a 2-level had a matrix factorial design (coded as þ1 and 1) with the addition of star (coded as þa and -a) and center points (coded as 0). The factorial design comprised of eight runs, which were duplicated, the star points comprised of six runs, while the center point comprised of one run that was replicated six times. The
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Table 1 The variables and their values used in the experiments. Variables
Factorial
A: Substrate (mg/L) B: pH C: Temperature ( C)
Star points
Center point
Lower value (1)
Upper value (þ1)
Lower value (-a)
Upper value (þa)
(0)
500 5 20
3000 9 40
1007 6 24
2493 8 36
1750 7 30
design and analysis were carried out using Minitab® (Version 13.32) and Design Expert® (Version 7.0). The substrate, pH and temperature range of values that were used in the experiments are shown in Table 1. The responses measured were percentage aggregation (%Ag) and percentage surface hydrophobicity (%SHb) of the mixed bacteria. 3. Results and discussion 3.1. Isolated bacteria A total of 12 bacteria were successfully isolated as pure cultured bacteria from the granular sludge developed in the synthetic textile wastewater using the SBR system. All of the bacteria were characterized in terms of form, shape, edges, and colony surface for their colony characteristics. A gram staining procedure was carried out for each bacterium in order to characterize the cellular morphology of the isolated bacteria. Among the 12 pure bacterial cultures, six bacteria were selected and used in this study. The selection of the six bacteria was based on the percentage chemical oxygen demand (COD) and color removal, aggregation, and surface hydrophobicity of each isolated pure culture (Muda, 2010). 3.2. Factorial analysis The results of the factorial experiments for aggregation and surface hydrophobicity are shown in Table 2. Depending on the experimental conditions, the %Ag and %SHb after 6 h of aeration varied between 39% to 85% and 19%e54%, respectively. A summary of the factorial analyses is shown in Table 3. The significance of the variables tested, i.e., substrate concentration, pH, and temperature, are based on p-values evaluated at a confidence level above 95% (pvalue less than 0.05). Based on the experimental conditions used in the study, the p-value indicates that all factors, i.e., substrate
Table 2 Experimental results for 2-level factorial design analysis. Run no.
Substrate concentration
pH
Temperature
%Ag
%SHb
RUN01 RUN02 RUN03 RUN04 RUN05 RUN06 RUN07 RUN08 RUN09 RUN10 RUN11 RUN12 RUN13 RUN14 RUN15 RUN16
1 1 þ1 þ1 1 1 þ1 þ1 1 1 þ1 þ1 1 1 þ1 þ1
1 1 1 1 þ1 þ1 þ1 þ1 1 1 1 1 þ1 þ1 þ1 þ1
1 1 1 1 1 1 1 1 þ1 þ1 þ1 þ1 þ1 þ1 þ1 þ1
49.6 44.7 67.2 64.1 46.8 44.1 65.6 62.9 65.2 61.9 84.5 85.4 38.8 38.7 57.4 57.6
19.0 21.7 33.8 29.4 37.3 41.2 39.6 36.9 32.1 31.5 53.9 52.0 20.4 18.2 8.6 6.8
concentration, pH, and temperature, have a significant effect on aggregation. A significant interaction effect is observed between pH and temperature (pH temperature), while the three-way interaction effect (substrate concentration pH temperature) is not significant. As for surface hydrophobicity, all model terms (main, 2way, and 3-way interaction) are significant except for the 2-way interaction between substrate concentration and temperature (substrate concentration temperature). The significance of the substrate concentration on bacterial aggregation and surface hydrophobicity is shown by very low pvalues of less than 0.05; their estimated positive main effects are 19.36 and 4.95, respectively, which indicates that higher substrate concentrations enhance the aggregation and surface hydrophobicity. This can be explained by the fact that an increase in the concentration of the substrate means more food being supplied to the bacteria, which results in an increase in bacterial growth. The presence of more cell biomass increases the collisions between cells and causes more aggregation to occur. As reported by Liu and Tay (2002), the collision between particles is one of the key factors influencing the formation and stabilization of biofilm, anaerobic, and aerobic granules. An increase in substrate concentration may also enhance the bacterial growth rate, which would also increase the production extracellular polymeric substances, particularly when faced with starvation (Wang et al., 2006). The presence extracellular polymeric substances on the cell surface could alter the physicochemical properties of the bacterial cell surface, which include surface charge and surface hydrophobicity. As reported by Wang et al. (2005), the poorly soluble and non-biodegradable extracellular polymeric substances on the surface of biogranules is responsible for the higher hydrophobicity of the granules, which may facilitate the adhesion or aggregation processes of the granules (Kjelleberg et al., 1987; Jiang et al., 2004). More recently, Lin et al. (2010) found alginate-like exopolysaccharides as one of the dominant components in aerobic granules and that they significantly influence the physical and chemical characteristics of the granules. The effect of pH on %Ag and %SHb was also found to be significant, with a p-value of less than 0.05. However, based on the estimated effects of 13.84 and 8.05, increases in pH are expected to reduce aggregation and surface hydrophobicity of the bacteria. This phenomenon is thought to be related to bacterial surface charge. Under neutral conditions, the bacteria would be negatively charged owing to the ionization of carboxyl, sulfate, and phosphate that act as functional groups on the surface of the cell (Sutherland, 2001). As they have the same charges, there will be a repulsive force between the two cell surfaces, which inhibits cell aggregation. Under acidic conditions, the presence of excess Hþ will neutralize the cell surface charge. This will reduce the repulsion and in turn favors cell-to-cell approach and the formation of cell aggregates can be initiated (Derjaugin and Lanadau, 1941). However, under alkaline conditions, the excess OH would enhance the surface charges on the cells and consequently increase the repulsive forces causing cells to be driven further apart, indicated by the reduction in %Ag and %SHb. Differences in pH of the media can also be associated with the type of substrate used in the growth media. Different types of
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205
Table 3 The p-values of the estimated main and interaction effects of substrate concentration, pH, and temperature on %Ag and %SHb after 6 h of aeration. Effects
Estimated effect
%Ag
Main Substrate concentration pH Temperature
19.36 13.84 5.56
<0.0001 <0.0001 0.0004
4.95 8.05 4.43
0.001 <0.0001 0.0019
2-way interaction Substrate conc. pH Substrate conc. temperature pH temperature
0.59 0.71 12.29
0.5607 0.4827 <0.0001
11.25 0.18 20.82
<0.0001 0.8624 <0.0001
0.74
0.4679
5.13
0.0008
3-way interaction Substrate conc. pH temperature
growth substrate may result in acidic or alkaline conditions when hydrolyzed. The concentration of Hþ and OH will affect the surface tension of the liquid media and eventually affect the surface hydrophobicity of the bacteria cell (Pasdar and Chan, 2000). The effect of pH on the surface hydrophobicity depends on the net surface charge of the exoprotein of the bacterial cell, which ultimately depends on the type of protein in the bacterial cell wall (Wang et al., 2006). Further investigations are required for a better understanding of the effect of pH on the different proteins in the bacterial cell wall and consequently on the surface hydrophobicity. Temperature was also found to have a significant positive effect on %Ag with a p-value of less than 0.05. However, the estimated effect was only 5.56, suggesting a weaker effect than the other two variables. An increase in temperature within the range of the experimental conditions is expected to increase microbial activity, which includes the metabolic rate and mobility of the microorganisms (Voet and Voet, 2004). In other words, a rise in temperature increases the specific growth rate of the bacteria and hence cell biomass, which increases the microbial aggregation process, as mentioned earlier. Ibrahim et al. (2005) reported the same observation that, as the incubation temperature was increased, the ability of the Bifidobacteria to aggregate also increased. While the effect of temperature on %SHb was significant (pvalue of 0.002), the estimated effect was negative (4.43). This indicates that the surface hydrophobicity of the bacteria decreases with increasing temperature. It is expected that as temperature increases, the liquid surface tension decreased (Moraes et al., 2008), thus allowing the adhesion of hydrophilic cells and resulting in a reduction in the percentage of cell surface hydrophobicity. Blanco et al. (1997) reported that the majority of the 42 strains of Candida albicans were hydrophobic at 20 C and became hydrophilic when the temperature was increased to 37 C. It is interesting to note that while surface hydrophobicity is expected to be the triggering force of cell aggregation (Liu et al., 2004), the effects of temperature on surface hydrophobicity and aggregation are opposing. To explain this observation, Rahman et al. (2008) categorized bacteria (Bifidobacteria) into high, medium, and low aggregator groups. Among the high aggregators, increases in temperature caused a decrease in the percentage aggregation. As for the medium and low aggregators, increases in temperature caused an increase in aggregation. It is likely that in addition to temperature, other factors such as pH of the media, and the types of protein bound on the cell surfaces of different bacterial strains at different temperatures (Maclagan and Old, 1980) may cause different effects on their ability to aggregate. Different types of bacteria may respond differently when exposed to different environmental conditions with the overall effect being influenced by the most dominant species in the group.
Estimated effect
%SHb
Fig. 1. Interaction effect plot between (a) pH and temperature for the aggregation process, (b) substrate and pH, and (c) pH and temperature for the surface hydrophobicity (-temperature ¼ 20 C, þtemperature ¼ 40 C; pH ¼ 5, þpH ¼ 9).
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Table 4 Experimental results for CCRD analysis. Run
Substrate
pH
Temperature
Aggregation (%Ag)
Surface hydrophobicity (%SHb)
RUN01 RUN02 RUN03 RUN04 RUN05 RUN06 RUN07 RUN08 RUN09 RUN10 RUN11 RUN12 RUN13 RUN14 RUN15 RUN16 RUN17 RUN18 RUN19 RUN20
1 þ1 1 þ1 1 þ1 1 þ1 1.682 þ1.682 0 0 0 0 0 0 0 0 0 0
1 1 þ1 þ1 1 1 þ1 þ1 þ1 þ1 1.682 þ1.682 0 0 0 0 0 0 0 0
1 1 1 1 þ1 þ1 þ1 þ1 þ1 þ1 0 0 1.682 þ1.682 0 0 0 0 0 0
49.6 67.2 46.8 65.6 65.2 84.5 38.8 57.4 68.8 73.0 54.2 5.5 29.3 58.3 59.4 56.4 55.1 60.3 57.5 60.2
19.0 33.8 37.3 39.6 32.1 53.9 20.4 8.6 50.9 70.7 62.4 7.8 71.2 83.9 75.2 77.2 72.7 74.0 76.2 72.2
The interaction effects between the factors on the responses were also investigated to provide a better understanding of how the responses behave when changes of more than one variable take place. Fig. 1 shows the significant interaction effect plots between aggregation and surface hydrophobicity. Of the three variables, only the interaction effect between pH and temperature was significant for %Ag with a p-value of less than 0.05 (Fig. 1a). As for %SHb, the interaction effects were significant between pH and temperature and between pH and substrate with p-values less than 0.05. At low temperature, %Ag was almost the same at low and high pH (i.e., pH 5 and 9) at about 53%. However, at high temperature, % Ag under acidic conditions increased up to about 75% while %Ag under alkaline conditions dropped slightly to less than 50%. This suggests that the effect of pH is strongly dependent on the temperature of the solution. The main effect that temperature and pH have on %Ag and their probable causes were discussed earlier. Apparently, under alkaline conditions, the high concentration of OH, which causes the repulsive force between cells, is greater and overshadows the positive effect of high temperature. Under acidic conditions, the synergistic positive effect of both high Hþ concentration and high temperature significantly increase %Ag.
The significant interaction effect of substrate and pH on %SHb is shown in Fig. 1b. At high substrate concentrations, a change in pH from 5 to 9 causes the surface hydrophobicity to decrease, while at low substrate concentrations, the change in pH has an insignificant effect on the surface hydrophobicity. The phenomenon is believed to be caused by the presence of acetate in the synthetic textile dyeing wastewater, which is hydrolyzed to release OH during the aeration phase (Voet and Voet, 2004). This increases the concentration of OH and makes the wastewater more alkaline when the initial pH is 9. As the cell hydrophobicity is inversely correlated to the quantity of surface charge on the bacteria (Liao et al., 2001), such a synergistic condition (i.e., initial pH 9 and OH from acetate hydrolysis) significantly reduces the surface hydrophobicity, as observed in this study. At low pH, the OH released from the hydrolysis of acetate will neutralize the existing Hþ. In this situation, the electrostatic force is reduced and when the substrate concentration is increased, the production of extracellular polymeric substances may contribute to the cell hydrophobicity and make the polymeric interaction force dominant and promote cell adhesion (Tsuneda et al., 2003). The interaction effects between pH and temperature on %SHb are inversely correlated, as shown in Fig. 1c. At lower pH value (pH 5), increasing the temperature (from 20 C to 40 C) increases %SHb (from 26% to 40%). However, at higher pH level (pH 9), increasing temperature (from 20 C to 40 C) reduces %SHb (from 38% to about 14%). Similarly, increasing the pH from 5 to 9 at 20 C, increases % SHb, but the same action at 40 C reduces %SHb. This interaction effect does not agree with the previous discussion. It appears that there are some byproducts from the degradation of the wastewater that are affecting the aggregation and surface hydrophobicity of the bacteria and that further in-depth study is required for a better understanding of this phenomenon. The factorial analyses also identify the significance of the 3-way interaction between substrate concentration, pH, and temperature on %SHb (p-value of 0.001) under the current experimental conditions. 3.3. Response surface analysis Response surface analysis was conducted to model the relationships between the aggregation and surface hydrophobicity and the factors being considered (i.e., substrate concentration, pH, and temperature). In addition, the curvature effect of the significant interactions could be further investigated. The results of the central composite rotatable experimental design experiments are shown
Table 5 Summary of the p-values from the response surface modeling analysis. Term
% Ag Full quadratic terms
% SHb Linear þ square þ pH temperature
The P-valuea Substrate pH Temperature Substrate pH Substrate Temperature pH Temperature Substrate Substrate pH pH Temperature Temperature R-squared value Lack of Fit (LOFT) a
0.0067 0.0022 0.0369 0.6329 0.8147 0.02 0.0042 0.0427 0.2397 86.6% 0.0029
0.0037 0.0009 0.0262 e e 0.0133 0.0016 0.0352 e 84.1% 0.048
0.01e0.04: Highly significant; 0.05e0.1: significant; 0.1e0.2: less significant; <0.2: insignificant (Vecchio, 1997).
0.3582 0.075 0.9194 0.3596 0.8854 0.1221 0.0423 0.0014 0.3393 75.9% <0.0001
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Fig. 2. (i) Contour and (ii) 3D response surface plots representing the relationship between pH, temperature, and %Ag.
in Table 4. The analyses were carried out using full quadratic terms, including linear, square, and interaction terms, with the aid of Design-Expert 7P statistical software (version 7). The summarized results of the ANOVA with respect to %Ag and %SHb are shown in Table 5. As in the factorial analysis, the significance of the terms (i.e., linear, interaction, and curvature (term2)) is based on a 95% confidence limit (i.e., p-value less than 0.05). For aggregation (%Ag), all linear terms, interaction between pH and temperature, and all curvature terms, except that of temperature, are significant. The model is acceptable with an R-squared value of 86.6%. In order to improve the model for %Ag, the previously determined insignificant terms, which were the two interaction terms (substrate pH and substrate temperature) and one square term (temperature temperature), were omitted. Omitting the insignificant terms improved the p-value of all of the included terms (reduced quadratic terms) compared to the previous analysis (full quadratic terms). The R-squared term decreased from 86.6% to 84.1%, which indicates that the omitted terms only cost 2.5% in goodness-of-fit, which is acceptable. As for %SHb, the only significant terms were pH and the squared pH and substrate terms. The R-squared value of this model is slightly lower than that of %Ag, but within an acceptable range with a value of 75.9%. The best statistical models that can be used to represent the relationship between %Ag and %SHb and substrate concentration, temperature, and pH within the range of the experimental conditions are given below:
where: A: Substrate concentration (mg L1) B: pH C: Temperature ( C) The response surface and contour plots, which illustrate the relationship between the %Ag and pH and temperature at a fixed substrate concentration (i.e., 1750 mg L1), are shown in Fig. 2. According to these analyses, the best condition for achieving the maximum %Ag would be a high temperature and low pH. Fig. 3(a)e(c) show the relationships between surface hydrophobicity and the independent variables of substrate, pH, and temperature. The contour plots of the RSM are drawn as a function of two variables, keeping the remaining variables at a fixed value. The surface plots of the interactions between variables for %SHb as the response were observed to have a symmetrical mound shape (Fig. 3a and b). Fig. 3b illustrates %SHb as a response at various substrate concentrations and temperatures at a constant pH of 7. The symmetrical mound shape plot shows that at any temperature level, %SHb increases as the substrate concentration increases from 500 to 1750 mg L1. The relationship between pH and temperature at a constant substrate concentration of 1750 mg L1, as shown in Fig. 3c, appears to have an elliptical shape. The response illustrated in Fig. 3(a)e(c) shows that the maximum predicted % SHb is indicated by the surface confined in the smallest curve of the contour diagram. 4. Conclusions
ðAggregationÞ2 ð%Þ ¼ 30449:2 4:02A þ 7114:5B þ 843:1C 108:1BC þ 1:4 103 A2 329:1B2 (3) Surface Hydrophobicityð%Þ ¼ 1122:2 þ 0:12A þ 239:7B þ 18:1C 6:5 103 AB 2:01 104 AC 1:4BC 1:9 105 A2 13:8B2 0:1 C2 (4)
The investigated variables imposed significant linear effect on the selected bacterial aggregation and surface hydrophobicity. The substrate concentration and temperature are positively correlated with aggregation, while a change from acidic to alkaline conditions reduces aggregation; the only significant interaction effect is that between pH and temperature. Surface hydrophobicity increases with substrate concentration, but decreases with bothda decrease in temperature and a change from acidic to alkaline conditions. The interaction effects between pH and substrate concentration, between pH and temperature, and between all three variables are significant.
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Fig. 3. (i) Contour and (ii) 3D response surface plots representing the relationship between variables (pH, temperature, and substrate concentration) and %SHb.
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