#
Using a Statistical Model to Examine the Effect of COD: SO_{4}^{2}^{−} Ratio, HRT and LA Concentration on Sulfate Reduction in an Anaerobic Sequencing Batch Reactor

^{*}

^{†}

^{‡}

## Abstract

**:**

_{4}

^{2−}ratio, hydraulic retention time (HRT) and linoleic acid (LA) concentration on sulfate (SO

_{4}

^{2−}) reduction in an anaerobic sequencing batch reactor using glucose as the electron donor. Based on the OA, optimum condition for maximum SO

_{4}

^{2−}reduction was evaluated. Increasing the COD/SO

_{4}

^{2−}ratio and HRT caused decreasing SO

_{4}

^{2−}reduction while increased SO

_{4}

^{2−}reduction was observed with increasing LA concentration (1 g L

^{−1}). In control (not fed LA) cultures, higher SO

_{4}

^{2−}reduction (87% ± 3%) was observed at a low COD/SO

_{4}

^{2−}ratio of 0.8. This indicates that increasing SO

_{4}

^{2−}reduction was observed at increasing SO

_{4}

^{2−}loading rates. In general, results from this study reveal that limiting the substrate concentration with high SO

_{4}

^{2−}levels (low COD/SO

_{4}

^{2−}ratio) favors high SO

_{4}

^{2−}removal. Surface plots were used to evaluate the significant interactions between the experimental factors. Accuracy of the model was verified using an analysis of residuals. Optimum conditions for maximum SO

_{4}

^{2−}reduction (97.61%) were observed at a COD/SO

_{4}

^{2−}ratio of 0.8 (level 1), 12 h HRT (level 1) together with 1000 mg L

^{−1}LA addition (level 3). In general, the Taguchi OA provided a useful approach for predicting the percent SO

_{4}

^{2−}reduction in inhibited mixed anaerobic cultures within the factor levels investigated.

## 1. Introduction

_{4}

^{2−}) is found in natural environments such as sediments, seawater and areas rich in decaying organic matter. Sulfate is also released in effluents from many industries such as pulp and paper processing, coal powered power plants, edible oil industries, tannery operations, molasses fermentation and mining [1,2]. Effluents generated from these industries also contain other sulfur species, which include thiosulfate, sulfite, sulfide and dithionite [3].

_{4}

^{2−}(Reactions (1) and (2); Table 1). Under acidic conditions, dissolution of heavy metals from metal oxides and carbonates results in the formation of metal and SO

_{4}

^{2−}containing wastewater known as acid mine drainage (AMD) [4,5,6].

_{2}S) in the presence of electron donors such as hydrogen (H

_{2}) or easily degradable organic chemicals [7]. Biological SO

_{4}

^{2−}reduction is a promising methodology to treat AMD due to the combined removal of acidity, SO

_{4}

^{2−}and heavy metals. Sulfate removal is accomplished by SO

_{4}

^{2−}reducing bacteria (SRB). SRBs utilize electron donors, such as volatile fatty acids (VFAs), alcohols and H

_{2}. SRBs often out-compete methane producing bacteria (MPB) for substrates, such as H

_{2}(Reactions (3) and (4); Table 1). When H

_{2}is utilized as an electron donor, SRBs produce H

_{2}S and hydroxide ions (Reaction (5); Table 1).

Reaction No. | Stoichiometric Reaction | ΔG°' (kJ mol^{−1}) |
---|---|---|

(1) | 2FeS_{2} + 7O_{2} + 2H_{2}O→2Fe^{2+} + 4SO_{4}^{2−} + 4H^{+} | −2168.0 |

(2) | ZnS + 2O_{2}→Zn^{2+} + SO_{4}^{2−} | −690.0 |

(3) | 4H_{2} + HCO_{3}^{−} + H^{+}→CH_{4} + 3H_{2}O | −135.6 |

(4) | 4H_{2} + H^{+} + SO_{4}^{2−}→4H_{2}O + HS^{−} | −152.2 |

(5) | 8H_{2} + 2SO_{4}^{2−}→H_{2}S + HS^{−} + 5H_{2}O + 3OH^{−} | −146.9 |

_{4}

^{2−}ratio is large or with decreasing SO

_{4}

^{2−}levels, MPBs out-compete SRB for available electrons. Conversely, SRBs out-compete MPBs if the COD/SO

_{4}

^{2−}ratio is low. A minimum COD/SO

_{4}

^{2−}mol ratio of 0.67 is required for SO

_{4}

^{2−}reduction [9]. The percent SO

_{4}

^{2−}reduction is variable with different COD/SO

_{4}

^{2−}ratios [10,11]. SRB and MPB competition is also dependent on the operational pH with SRB growth favored at high pH [12]. Since the chemical equilibrium of different sulphide species is pH dependent [13,14], pH is a crucial factor affecting the competition between SRBs and MPBs.

_{4}

^{2−}ratio and HRT. Another factor controlling the activity of SRBs and MPBs is inhibitory chemicals. Successful inhibition of MPB growth will favor SRB growth and increase the quantity of SO

_{4}

^{2−}reduced. Diverting the fraction of substrate electron flow from MPBs to SRBs is achievable using different treatment methods, which selectively inhibit methanogenic growth. Among the physical methods, heat treatment is used to inhibit non-spore forming MPB [17]. However, due to the high cost associated with heat treatment, the method is unsuitable for full-scale application. Alternate methods to heat treatment include utilizing chemical inhibitors. Various chemical inhibitors have been used successfully to inhibit MPBs [18]. Inhibitors specific to inhibiting methanogens include 2-bromo ethane sulfonic acid (2-BESA). Other methanogenic and non-methanogenic inhibitors, which have been extensively studied, include saturated long chain fatty acids (SLCFAs) and unsaturated long chain fatty acids (ULCFAs). Lauric acid (C12:0), a SLCFA, and as linoleic acid (LA, C18:2), a ULCFA, are able to suppress gram positive bacteria and methanogens [19,20].

_{4}

^{2−}reduction can be examined using statistical methods, such as the Taguchi design [21]. A significant difference between Taguchi’s optimization technique and other similar methods is the ability to reduce process variability by involving factors that cause variability in the experimental design, modeling and optimization process [22]. The Taguchi method has been used in many biotechnological applications Rao et al. [21]. The method has been used by many researchers to optimize the operation of microbial processes [21,23,24].

_{4}

^{2−}ratio, HRT and LA concentration on mesophilic biological SO

_{4}

^{2−}reduction in anaerobic sequencing batch reactors (ASBRs) using a Taguchi design.

## 2. Materials and Methods

#### 2.1. Inoculum Source

^{−1}, respectively. Cultures A and B were diluted with basal medium to 25 and 12 g VSS L

^{−1}in 9 L reactors, respectively (designated as reactor A and B). The bioreactors were operated in accordance with procedures reported by Ray et al. [27]. Reactors A and B were operated at 37 °C in a sequencing batch mode with a 14 d HRT and a feed concentration of 2000 mg glucose L

^{−1}. The pH of the reactors was maintained at 7.0 ± 0.5. In addition to glucose, reactor B was acclimated incremental to increasing SO

_{4}

^{2−}levels of 250 mg L

^{−1}to 2000 mg L

^{−1}for 2 months. During the acclimation period, the quantity of gas and VFAs were monitored to establish quasi-steady state conditions. Inoculum for the experiments under consideration was combined from reactors A (80%) and B (20%) and diluted with basal medium to 8 g VSS L

^{−1}. The basal medium composition used for dilution and feed was adapted from Wiegant and Lettinga [28]. All the chemicals for basal medium were procured from ACP Chemicals Inc. (Montreal, QC, Canada) and Sigma Aldrich (Oakville, ON, Canada). The feed substrate (glucose) was procured from Spectrum Chemicals, Gardena, CA, USA.

#### 2.2. Sulfate Reduction Studies

^{−1}) and then purged with nitrogen (N

_{2}) (99.99% purity, Praxair, Windsor, ON, Canada) for 5 min to maintain anaerobic conditions. The experimental reactors (R1 and R2) were operated under identical conditions with a feed concentration of 2000 mg glucose L

^{−1}(2.134 g COD L

^{−1}) as a carbon source. The SO

_{4}

^{2−}concentration was varied according to the COD/SO

_{4}

^{2−}ratio shown in Table 2.

_{4}

^{2−}reduction with ±10% variation). Different LA levels (0, 0.5 and 1.0 g L

^{−1}) were fed to cultures according to experimental conditions shown in Table 2. Cultures were incubated with LA for 24 h prior to initiating the experiment (substrate addition).

#### 2.3. Analytical Methods

_{4}and H

_{2}were 0.0032 kPa (0.5 mL/bottle (160 mL)) and H

_{2}S was 0.0315 kPa (5 mL/bottle (160 mL)), respectively. The liquid samples collected at the end of each cycle were analyzed for SO

_{4}

^{2−}using an ion chromatography (IC) [7]. The detection limits for the SO

_{4}

^{2−}was 0.5 mg L

^{−1}. The total suspended solids (TSS) and VSS levels were measured according to Standard Methods [31].

**Table 2.**Design matrix for experimental factors and corresponding response function at different factor levels.

Exp. No. | COD/SO_{4}^{2−} Ratio ^{1} | HRT (h) | LA conc. (mg L^{−1}) | Experimental SO_{4}^{2−} Reduction (%) ^{2} | Predicted SO_{4}^{2−} Reduction (%) | |||
---|---|---|---|---|---|---|---|---|

X_{1} | Level | X_{2} | Level | X_{3} | Level | |||

1 | 0.8 | 1 | 12 | 1 | 0 | 1 | 86.5 ± 2.6 | 83.5 |

2 | 0.8 | 1 | 24 | 2 | 500 | 2 | 65.8 ± 1.9 | 67.8 |

3 | 0.8 | 1 | 36 | 3 | 1000 | 3 | 80.6 ± 0.7 | 81.6 |

4 | 1.6 | 2 | 12 | 1 | 500 | 2 | 75.1 ± 1.9 | 76.1 |

5 | 1.6 | 2 | 24 | 2 | 1000 | 3 | 78.2 ± 3.7 | 75.2 |

6 | 1.6 | 2 | 36 | 3 | 0 | 1 | 58.3 ± 2.7 | 60.3 |

7 | 2.4 | 3 | 12 | 1 | 1000 | 3 | 89.9 ± 6.0 | 91.9 |

8 | 2.4 | 3 | 24 | 2 | 0 | 1 | 61.5 ± 8.6 | 62.5 |

9 | 2.4 | 3 | 36 | 3 | 500 | 2 | 64.5 ± 2.9 | 61.5 |

_{4}

^{2−}= sulfate;

^{1}COD/SO

_{4}

^{2−}ratio of 0.8 denotes a glucose COD concentration of 2.134 g L

^{−1}and SO

_{4}

^{2−}concentration of 2.668 g L

^{−1}. Similarly in order to attain a COD/SO

_{4}

^{2−}ratio of 1.6 and 2.4, SO

_{4}

^{2−}concentration of 1.334 and 0.889 g L

^{−1}were used by keeping a constant glucose COD concentration of 2.134 g L

^{−1};

^{2}values shown in mean ± standard deviation represents the average sulfate reduction from two reactor samples for at least three consecutive cycles;

#### 2.4. Taguchi Design

#### 2.4.1. Fractional Factorial Design of Experiments (FFDOE) (Phase 1)

_{4}

^{2−}removed. The normal practice has been to experiment with a feasible range so that the variation inherent in the process does not mask the factor effect. Factors were selected and the ranges were assigned based on data from work reported by Moon et al. [7,32] and Kaksomen et al. [32]. Three factors (HRT, COD/SO

_{4}

^{2−}ratio, LA concentration) with significant influence on the SO

_{4}

^{2−}removal rate were selected for the Taguchi orthogonal array (OA) study. The levels for the three factors are shown in Table 2. The L9 OA can handle up to four factors at three levels with eight degrees of freedom. Since only three factors were examined in this study, the fourth column in the OA was left empty. Orthogonality is not lost by maintaining one or more columns of an array empty [33]. Taguchi’s OA are used to estimate main effects using only a few experimental runs. An OA (n, q, s, t) is an n × p array with entries from a set of s distinct symbols such that for any collection of t columns of the array, each of the s

^{t}row vectors appears equally often in the matrix [34].

#### 2.4.2. Sulfate Removal ASBR Experiments with Selected Factors and Levels (Phase 2)

_{4}

^{2−}reduction) are outlined in Section 2.3.

#### 2.4.3. Analysis of Experimental Data (AED) and Prediction of Performance (POP) (Phase 3)

_{4}

^{2−}removal rate data obtained from the L-9 experiments were analyzed using the Qualitek-4 software with the “bigger-is-better” quality characteristics selected to determine the optimum conditions (higher SO

_{4}

^{2−}removal rate) and to identify individual factor influence on the SO

_{4}

^{2−}removal rate. In the Taguchi’s method, quality is measured by the deviation of a characteristic from a target value using the loss function (Equation (2)).

_{4}

^{2−}removal. In the optimization, the bigger-is-better quality characteristic for the loss function is represented as Equation (3).

^{2}) can be estimated from a sample of as Equation (5):

## 3. Results and Discussion

#### 3.1. Experimental Design Analysis

_{4}

^{2−}reduction). The residual quantity of SO

_{4}

^{2−}measured at the end of each experimental run in the effluent was used to compute the percent of SO

_{4}

^{2−}removed (Table 2). This response variable was used to predict the optimum response using the three factors and three levels. The regression coefficients computed for the experimental response (%SO

_{4}

^{2−}reduction) were used to derive a model equation involving the three independent factors (Equation (6)).

#### 3.2. Analysis of Variance

_{4}

^{2−}reduction) at different conditions and to determine variation in contribution of each factor to the response variable (Table 3). The Fisher statistic (F-test) was used to establish whether the factors under investigation have any significant effects on the quality characteristic. In particular, the F ratio is used to determine the significance of the different experimental factor. The calculated F ratios indicate all the individual factors are statistically significant at a 95% confidence limit. The p values were used to determine the significance of each factor on SO

_{4}

^{2−}reduction. Based on the p values, the HRT contributed the maximum impact (49.99%) on the overall SO

_{4}

^{2−}reduction followed by LA with 41.42% (Figure 1 and Table 3).

_{4}

^{2−}ratio showed the least impact at the individual level (8.58%). The results from the study indicate that both HRT and LA concentration contributed more than 91% towards SO

_{4}

^{2−}reduction.

Factor | DOF (f) | Sum of Squares (s) | Mean Squares | Variance (v) | F ratio (F) ^{1} | Pure Sum (S’) | Percent p (%) ^{2} |
---|---|---|---|---|---|---|---|

COD/SO_{4}^{2−} | 2 | 84.57 | 42.29 | 42.29 | 845,749.5 ^{3} | 84.57 | 8.58 |

HRT | 2 | 492.67 | 246.33 | 246.33 | 4,926,686.4 ^{3} | 492.67 | 50.00 |

LA | 2 | 408.16 | 204.08 | 204.08 | 4,081,612.0 ^{3} | 408.16 | 41.42 |

Error | 2 | 0.001 | 0.0005 | 0.001 | 0.002 | ||

Total | 8 | 985.40 | 492.70 | 100.00 |

^{1}Critical F

_{0.05, 2, 8}= 4.46;

^{2}Percent p (%) = (Sum of squared deviations/total sum of squared deviations) × 100;

^{3}denotes significant at 95% confidence level.

**Figure 1.**Percent contribution of each variable on the sulfate removal rate. (1. HRT = hydraulic retention time; LA = linoleic acid; 2. Percent contribution of each experimental variable was estimated using ANOVA).

#### 3.3. Effect of Factors on the Response Variables

#### 3.3.1. Main Effects Plot

_{4}

^{2−}reduction.

#### Effect of COD/SO_{4}^{2−} Ratio

_{4}

^{2−}ratio is a major factor affecting SO

_{4}

^{2−}reduction [36]. According to Velasco et al. [36], for a given SO

_{4}

^{2−}concentration, the feed COD/SO

_{4}

^{2−}ratio was used to control H

_{2}S production, which in-turn was used for metal precipitation. Varying quantities of the % SO

_{4}

^{2−}removed was observed in cultures fed different COD/SO

_{4}

^{2−}ratios (Figure 2a). The main effects plot showed a maximum mean SO

_{4}

^{2−}removal of 78% at a COD/SO

_{4}

^{2−}ratio of 0.8. With increasing COD/SO

_{4}

^{2−}ratios of 1.6 and 2.4, the mean SO

_{4}

^{2−}reduction reached approximately 70% irrespective of the experimental HRT and LA concentration. These results indicate that low COD/SO

_{4}

^{2−}ratios are favorable for high SO

_{4}

^{2−}removal. Studies conducted by Choi and Rim [9] reported reduced SRB activity at COD/SO

_{4}

^{2−}ratios exceeding 2.7 for acetate and hydrogen electron donors. They attributed the reduced SRB activity to competition by MPBs. In similar work by El Bayoumy et al. [37], they concluded that SRB growing on lactate and acetate with COD/SO

_{4}

^{2−}ratios between 0.75 and 2.25 was enhanced in comparison to ratios greater than 2.25. Studies by Velasco et al. [36] have indicated that COD/SO

_{4}

^{2−}ratios greater than 1.5 resulted in increasing sulfide levels while at lower COD/SO

_{4}

^{2−}ratios, sulfur species such as H

_{2}S, dissolved sulfide was produced. Higher SO

_{4}

^{2−}reduction at low COD/SO

_{4}

^{2−}ratios is likely attributed to higher SRBs growth rates under these conditions. Evidence by Erdirencelebi et al. [10] also support the argument that at higher COD/SO

_{4}

^{2−}ratios, SRBs are unable to compete with MPBs for electrons derived from substrate oxidation.

**Figure 2.**Impact of selected experimental factors on percent sulfate reduction. (

**a**) Effect of COD/SO

_{4}

^{2−}ratio; (

**b**) Effect of HRT; (

**c**) Effect of LA concentration.

#### Effect of Hydraulic Retention Time

_{4}

^{2−}removal was conducted after optimizing the COD/SO

_{4}

^{2−}ratio. According to Neculita et al. [38], increased treatment efficiency was observed with increasing HRT. Reduced treatment efficiency with decreasing HRT at a constant COD/SO

_{4}

^{2−}ratio was reported by Zhou et al. [39]. These authors reported decreasing SO

_{4}

^{2−}removal efficiencies from 89% to 82% as the HRT was decreased from 24 h to 12 h at a constant COD/SO

_{4}

^{2−}ratio of 4. In this study, a mean experimental response of 84% SO

_{4}

^{2−}reduction was observed at a 12 h HRT (Figure 2b).

_{4}

^{2−}removals of 69% and 68% observed in cultures operating at 24 h and 36 h HRT indicated that long HRT conditions are unfavorable for SO

_{4}

^{2−}reduction. The low SO

_{4}

^{2−}removals might be due to the ability of MPBs competing with SRB for the available substrate. MPBs are able to compete with SRBs for substrates derived electrons and subsequently produce CH

_{4}. Higher SO

_{4}

^{2−}removal at lower HRT (12 h) is likely associated with elevated growth rates of SRB in comparison to MPBs [37]. MPB have longer doubling times and are washed out at lower HRT thus favoring SO

_{4}

^{2−}reduction [16].

#### Effect of Linoleic Acid Concentration

_{4}

^{2−}removal (experimental response) of 69% was observed in control cultures (not fed LA). Cultures fed 0.5 g L

^{−1}LA performed the same as the controls with mean SO

_{4}

^{2−}removal reaching approximately 68% (Figure 2c). However, with 1 g L

^{−1}LA, the SO

_{4}

^{2−}removal was more effective with a 22% increase. This result indicates that adding 1 g L

^{−1}LA was effective in selectively inhibiting MPBs and re-directing the substrate derived electrons to SRBs. In work conducted by Ray et al. [40] and Chowdhury et al. [41], they indicated that methanogenic inhibition by a threshold LA level lead to H

_{2}production. In comparison, Moon et al. [7] concluded no significant difference in SO

_{4}

^{2−}reduction was detected at low (0.5 g L

^{−1}) and high (1.5 g L

^{−1}) LA levels using glucose fed batch cultures maintained at pH 6.0 to 7.5 and COD/SO

_{4}

^{2−}ratios varying from 0.5 to 2.5. This difference in comparison to the work reported herein could be due to no pH control in the batch studies. Additionally, in comparison to the work reported by Moon et al. [7], variation in HRT might have exerted a significant effect at varying LA levels.

#### 3.3.2. Surface Plots

_{4}

^{2−}ratio and HRT (Figure 3a) suggest that maximum SO

_{4}

^{2−}reduction (>80%) was observed at a COD/SO

_{4}

^{2−}ratio of 0.8 and a 12 h HRT. Similar trends were observed with a low COD/SO

_{4}

^{2−}ratio of 0.8 and an elevated LA concentration of 1 g L

^{−1}(Figure 3b). The effect of HRT and LA concentration on SO

_{4}

^{2−}removal is shown in Figure 3c. Lower HRT (12 h) and higher LA concentration (1 g L

^{−1}) resulted in maximum SO

_{4}

^{2−}removal. In general, from these surface plots, a combination of lower HRT and COD/SO

_{4}

^{2−}ratio together with higher LA concentration resulted in maximum SO

_{4}

^{2−}removal.

#### 3.4. Model Verification

^{2}value for predicted versus experimental SO

_{4}

^{2−}reduction (%) was 0.9592 (data not shown). The residuals (model predicted value - experimental value) for the experimental response were used to assess the adequacy of the fit. The Anderson-Darling (AD) plot confirmed a normal distribution of the residuals. The observed AD statistic for the model response was 0.290 (Supplementary Figure A1). This value is smaller than the critical AD value of 0.752 for a sample size of 18 at a 5% significance level. The observed p-value (Supplementary Figure A1) of 0.572 (greater than 0.05) also confirms a normal distribution of residuals. This suggests that the model-predicted response values correlated reasonably well with the experimental response values (Table 2) over the factor space under consideration. The results obtained from this study are comparable with data reported in literature (Table 4). In general, the results (%SO

_{4}

^{2−}reduction) reported in literature for various reactor systems fed with different type of substrates obtained higher SO

_{4}

^{2−}reduction at higher COD/SO

_{4}

^{2−}ratios (>2; Table 4).

**Figure 3.**Surface plots for the experimental response (% sulfate reduction) (

**a**) COD/SO

_{4}

^{2−}ratio versus HRT (at constant LA = 0.5 g L

^{−1}); (

**b**) COD/SO

_{4}

^{2−}ratio versus LA (at constant HRT = 24 h); (

**c**) HRT versus LA (at constant COD/SO

_{4}

^{2−}ratio = 1.6).

COD/SO_{4}^{2−} Ratio | Reactor Type; Mode of Operation | Temp. (°C) | pH | SO_{4}^{2−} Reduction (%) | Substrate | HRT | Ref. |
---|---|---|---|---|---|---|---|

2.5 | UASBR; Continuous | 30 | 7.0 ± 0.5 | 94 ± 1 | Ethanol | 4 d | [36] |

4 | FBR; Continuous | 35 | 7.4 ± 0.2 | 90 | Ethanol | 6.5 h | [32] |

3.2, 4, 5 | UASBR; Continuous | 30–33 | 7.3 ± 0.7 | 70, 81, 74 | Glucose | 24 h | [10] |

3.15, 2.7 | CSTR; Continuous | 30 | NR | 29, 28 | Glucose | NR | [10] |

2.7, 1.23, 0.6 | Serum bottle; Batch | 30 | NR | 9, 4, 4.5 | Acetate | NA | [10] |

0.41, 1.03, 2.07 | Serum bottle; Batch | 35 ± 1 | 7.3 ± 0.1 | 26, 60, 93 | Propionate | NA | [11] |

1, 4 | UASBR; Continuous | 55 | 6.0 | 25–35, 65 | Sucrose | 10 h | [42] |

6.67 | UASBR; Continuous | 35 ± 1 | 7.0–7.5 | 80–86 | Sulfate rich vinasse | 4.86 days | [43] |

4 | AFR; Continuous | 37 ± 0.5 | 9.5 | 97.8 ± 1.1 | Ethanol | 18 h | [39] |

0.8, 1.6, 2.4 | ASBR; Sequencing batch | 37 ± 0.1 | 6.5 ± 0.1 | 87 ± 3, 58 ± 3, 62 ± 9 | Glucose | 12, 36, 24 h | This study * |

_{4}

^{2−}reduction of 81% and 29% in UASBR and CSTR, respectively, using COD/SO

_{4}

^{2−}ratios >3 and mixed anaerobic cultures fed glucose at neutral pH 7.0. High SO

_{4}

^{2−}reduction (86.5% ± 2.6%; Table 2) in control cultures (LA unfed cultures) at a low COD/SO

_{4}

^{2−}ratio of 0.8 and a 12 h HRT indicate that higher SO

_{4}

^{2−}levels is associated with high SO

_{4}

^{2−}removal in comparison to data reported by other researchers (Table 4). In comparison, statistically the same percent SO

_{4}

^{2−}reduction (89.9% ± 6%; Table 2) was observed in the presence of 1000 mg L

^{−1}LA, a 12 h HRT and a COD/SO

_{4}

^{2−}ratio of 2.4. In the control cultures with a low COD/SO

_{4}

^{2−}ratio of 0.8 (i.e., at high SO

_{4}

^{2−}concentration), low methane production (data not shown) was coupled with high SO

_{4}

^{2−}reduction. Since LA is an effective methanogenic inhibitor (at threshold levels), the LA treated cultures with high levels of SO

_{4}

^{2−}reduction at all COD/SO

_{4}

^{2−}ratios indicated that the substrate-derived electrons were utilized for SO

_{4}

^{2−}reduction rather than CH

_{4}formation.

#### 3.5. Factor Interactions and Their Influence on Sulfate Reduction

_{4}

^{2−}reduction is shown in Supplementary Table A1. The difference among the levels (L1−L2, L1−L3, L3−L2, L2−L1, L3−L2, and L3−L1) of each factor indicates the relative influence on the response (Supplementary Table A1). A larger difference is associated with a strong influence on the response variable. The data clearly indicate that HRT showed the greatest influence (83.8%) at level 1 (12 h HRT) when compared to the other factors. The next factors were the LA concentration (level 3) and COD/SO

_{4}

^{2−}ratio (level 1; Supplementary Table A1). Notice the decreasing percent SO

_{4}

^{2−}reduction from 83.8% to 63.8% is associated with increasing HRT from 12 h to 36 h (Supplementary Table A1). Increasing the LA concentration from 0 to 1 g L

^{−1}showed an increase in SO

_{4}

^{2−}reduction from 68.8% to 82.9% (Supplementary Table A1). In comparison, minimum variation in the percent SO

_{4}

^{2−}removed was observed with varying COD/SO

_{4}

^{2−}ratios.

_{4}

^{2−}reduction. In general, interaction effects are studied because of the possibility of one factor interacting with one or all of the other factors. The interaction severity index [SI] was calculated to determine the influence of the experimental factors at varying factor levels (Supplementary Table A2). The analysis indicates that the COD/SO

_{4}

^{2−}ratio and LA concentration had the largest SI (59.33%) followed by the COD/SO

_{4}

^{2−}ratio and HRT (37.65%; Supplementary Table A2). The SI value for the HRT and LA concentration was the lowest (24.84) among the factors investigated. Note that in the interactions, experimental variables with the least impact factor (COD/SO

_{4}

^{2−}ratio (p% = 8.582); Table 3) were associated with a stronger impact factor (HRT (p% = 49.99) and LA concentration (p% = 41.42); Table 3). Data from this study indicate that the influence of selected factors on SO

_{4}

^{2−}reduction was independent of the individual influence.

#### 3.6. Optimum Conditions for Sulfate Reduction

_{4}

^{2−}reduction. The data shows the relative interactions of the parameters on SO

_{4}

^{2−}reduction. The contribution by each individual factor is the key for enforcing control over SO

_{4}

^{2−}reduction. The expected improvement on SO

_{4}

^{2−}reduction in mixed anaerobic culture using the experimental variables is shown in Figure 4.

**Figure 4.**Variation reduction plot showing the performance distribution of sulfate removal under current and improved conditions. (LCL = lower control limit; UCL = upper control limit.)

_{4}

^{2−}removal shown is approximately 73.9% (Figure 4). The improved and current average percent SO

_{4}

^{2−}removal is the same; however, the improved condition frequency is larger when compared to the current condition frequency. A summary of the two conditions is shown in Table 5.

Parameters | Current Condition | New/Improved Condition |
---|---|---|

Mean | 72.49 | 72.49 |

Standard deviation | 11.50 | 7.93 |

C_{p} | 1.00 | 1.45 |

C_{pk} | 1.00 | 1.45 |

Quality characteristic (QC) | Bigger is better | Bigger is better |

Lower control limit (LCL) | 37.99 | 37.99 |

Upper control limit (UCL) | 106.99 | 106.99 |

_{p}represents the capability index expressed in terms of a number (ratio) indicating the narrowness of the population distribution within the LCL and UCL; C

_{pk}represents the capability index is very similar to C

_{p}which captures the position of the mean performance as well as the variation of the data within the specification limits; LCL = Mean − (3 × standard deviation of current condition); UCL = Mean + (3 × standard deviation of current condition).

_{4}

^{2−}reduction) while the improved condition is based on the minimization of the variation in the experimental response. The C

_{p}and C

_{pk}values are designated as capability indices [44]. C

_{p}is a measure of the process capability with respect to the difference between the upper control limit (UCL) and lower control limit (LCL). The C

_{pk}value measures the process variation with respect to the mean. A high the C

_{pk}indicate the capability of the process to meet its requirements. For the improved condition case, the capability index is larger when compared to the current condition. A capability index greater than 1.33 (Table 5) indicate the percent SO

_{4}

^{2−}removals are within the tolerances (LCL and UCL). The optimum conditions for SO

_{4}

^{2−}removal was determined by the Qualitek 4 software based on the results obtained using the Taguchi OA.

_{4}

^{2−}removal were observed at a COD/SO

_{4}

^{2−}ratio of 0.8, a 12 h HRT together with 1 g L

^{−1}LA (Supplementary Table A3). Under the optimum conditions, the maximum SO

_{4}

^{2−}reduction attained was 97.6%. The total contribution from the experimental factors on SO

_{4}

^{2−}reduction was 24.2%. The observed 73.4% average performance of the mixed microbial cultures and 24.2% contribution from all experimental factors revealed the potential of these variables and their interaction on SO

_{4}

^{2−}reduction in the ASBRs.

## 4. Conclusions

_{4}

^{2−}reduction using glucose as substrate under different experimental conditions. The factors investigated in this study included the COD/SO

_{4}

^{2−}ratio, HRT and LA concentration. In general, the percent SO

_{4}

^{2−}removed decreased with increasing COD/SO

_{4}

^{2−}ratio and HRT levels and increased with increasing LA concentration. An analysis of the residuals indicates a normal distribution. The surface plots and ANOVA indicates significant interactions between the experimental factors investigated. The Taguchi model predicted an optimum SO

_{4}

^{2−}removal of 97.6% at a COD/SO

_{4}

^{2−}ratio of 0.8 (level 1), a 12 h HRT (level 1) and 1000 mg L

^{−1}LA (level 3). The maximum SO

_{4}

^{2−}removal of 87% ± 3% was obtained at a lower feed COD/SO

_{4}

^{2−}ratio (high SO

_{4}

^{2−}loading conditions) in combination a lower HRT (12 h) in the control cultures (without LA addition). The results obtained from this current study indicated that higher biological SO

_{4}

^{2−}reduction using anaerobic cultures could be achieved in an ASBR at high SO

_{4}

^{2−}levels.

## Acknowledgments

## Author Contributions

## Conflicts of Interest

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**MDPI and ACS Style**

Singh, R.; Moon, C.; Veeravalli, S.S.; Shanmugam, S.R.; Chaganti, S.R.; Lalman, J.A.
Using a Statistical Model to Examine the Effect of COD: SO_{4}^{2}^{−} Ratio, HRT and LA Concentration on Sulfate Reduction in an Anaerobic Sequencing Batch Reactor. *Water* **2014**, *6*, 3478-3494.
https://doi.org/10.3390/w6113478

**AMA Style**

Singh R, Moon C, Veeravalli SS, Shanmugam SR, Chaganti SR, Lalman JA.
Using a Statistical Model to Examine the Effect of COD: SO_{4}^{2}^{−} Ratio, HRT and LA Concentration on Sulfate Reduction in an Anaerobic Sequencing Batch Reactor. *Water*. 2014; 6(11):3478-3494.
https://doi.org/10.3390/w6113478

**Chicago/Turabian Style**

Singh, Rajesh, Chungman Moon, Sathyanarayan S. Veeravalli, Saravanan R. Shanmugam, Subba Rao Chaganti, and Jerald A. Lalman.
2014. "Using a Statistical Model to Examine the Effect of COD: SO_{4}^{2}^{−} Ratio, HRT and LA Concentration on Sulfate Reduction in an Anaerobic Sequencing Batch Reactor" *Water* 6, no. 11: 3478-3494.
https://doi.org/10.3390/w6113478