Significance of Algae in eliminating and deactivating pathological index Organisms in Wastewater Stabilization Pond Systems

. Wastewater stabilization ponds (WSPs) are an economical alternative to conventional wastewater treatment technologies. They are commonly favored by small and rural communities. Due to long hydraulic retention time required by the treatment in these systems, algal activity is often expected during summer season. This study investigated the effects of algal growth on the removal and inactivation of pathogenic indicator organisms in WSP systems. Two predominant algae species (Mougeotia sp. and Nostochopsis sp) that were found in Iraq (Alkhademiyah sewage station in Baghdad) WSP were able to increase both pH and DO. The highest inactivation rates of both E. coli and total coliforms (TC) were observed at pH 10.6 compared to other pH (5.1, 8.2, 8.4, 10.6) investigated in the bench-scale experiments. Both high (20mg/L) and low DO (1 mg/L) levels can facilitate the removal and inactivation of both E. coli and TC. Enterococci were significantly reduced at both intermediate (8.6 mg/L) and high (20 mg/L) DO concentrations. Therefore, the presence of algae can potentially promote the removal of E. coli, TC and Enterococci. Much higher inactivation of E. coli, TC and Enterococci at a higher temperature (20°C) than at a lower temperature (4°C) indicates temperature is one the most important removal factor. Two potential/additional indicator organisms (Enterococci and Clostridium perfringens (C. perfringens)) exhibited different inactivation trends than the traditional indicators (E. coli and TC) under the same pH and DO conditions. C. perfringens were tolerant to all the tested pH, DO and temperature conditions. Their resistance to environmental stresses may pose potential health risk. Hence, both Enterococci and C. perfringens could be potentially used as indictor organisms to predict the overall level of pathogens in treatedwastewater.


Introduction
Wastewater stabilization ponds (WSPs) are environmentally sustainable ecosystems that require lower energy inputs and maintenance compared to conventional forms of wastewater treatment [1,2]. As such, they have become a popular alternative to conventional technologies, especially in small, rural and remote regions. However, the performance of WSPs is highly dependent on environmental and climatic conditions, hence, the treatment efficiency can be sensitive to both location and season.
Maturation WSPs, with depth of 1-1.4 m, are designed for disinfection and are often implemented as a tertiary polishing step in wastewater treatment. Over the past two decades, one of the research interests in WSPs has been to understand the mechanisms of pathogen removal in these systems. Previous studies have shown that the main factors contributing to disinfection in WSP systems are: sunlight, pH, dissolved oxygen (DO), temperature, nutrient limitation, water depth, hydraulic retention time, attachment, sedimentation and predation [2][3][4][5][6][7][8].
As a result of the long hydraulic retention times associated in these systems, algal activity is often expected during warmer seasons. Consequently, high dissolved oxygen (DO) (~ 15 mg/L) and pH levels (~ 9.5) are often observed in WSPs containing algae [1,3,8,9] . It has been noted in the literature that pH and DO are two important factors that can affect the removal and inactivation of pathogenic microorganisms in WSPs [3,4,6]. The presence of algae may influence the various treatments occurring in WSP systems, such as nutrient removal and pathogen removal [8] . Escherichia coli (E. coli), fecal [10,11] coliforms and total coliforms are common indicator organisms that are used to assess the quality of treated wastewater and determine the presence of pathogenic microorganisms [12,13] Their presence can be a good indication of fecal contamination. However, the limitations associated with the use of traditional fecal indictor organisms have been recognized in the literature; particularly as they can sometimes be limited in their ability to represent overall pathogen levels [14][15][16]. A number of potential indicator organisms have been proposed as representative of pathogenic microorganisms in addition to those traditionally employed. Enterococci are one of the potential indicator organisms that have been reported to have better survival rates than fecal coliforms at high pH. In a bench scale study using grab samples of sewage obtained from the Academic Board of Baghdad University treatment plant, [5] observed that Enterococci could survive at pH levels as high as 11. Therefore, the use of E. coli, fecal coliforms or total coliforms as indicators may under predict the presence or concentration of Enterococci, especially under high pH conditions. Clostridium perfringens (C. perfringens), an anaerobic, spore-forming bacteria found in high concentrations in human and animal feces, has been recently used as an additional indicator. Its spore phase is considered highly resistant to environmental conditions, as well as conventional disinfection processes, whereby, [17] found that the inactivation rate of E.coli was one order of magnitude greater than that of C. perfringens. Therefore, it was suggested that rather than E. coli, this microorganism could be used to predict the presence of resistant protozoan and helminthic pathogens such as Cryptosporidium, Giardia and helminth eggs [18][19][20]; However, a comprehensive understanding of how these indicator organisms would respond to different pH, DO and temperature conditions is missing in the literature.
The main objective of this study was to investigate the indirect effect of algae on the removal and inactivation of traditional (E. coli and total coliforms) and alternative indicator organisms (Enterococcus and C. perfringens).
Bench scale experiments were conducted to examine whether the presence of algae could be responsible for the high pH and DO levels in WSP systems, then to assess the effect of the high pH and DO conditions on the removal and inactivation of both typical and potential indicator organisms.
2 Material and Methods:

Source of wastewater samples Description of the study area
Wastewater samples were collected from Alkhademiyah sewage station in Baghdad. extended from southwest of AlKarkh city with their elongation for more than 30 km passes through vast farmlands, orchards and several residential quarters , followed the figure below ( Figure .1

Algae cultivation experiments
At the Alkhademiyah sewage station in Baghdad , algal blooms are typically observed to start in early June. During the warmer months, from June to September, pH generally ranged from 9.5 to 11 [3]. Two types of algae were found to be predominant . One was Nostochopsis sp ., also known as Water Net algae, and tended to float on the water surface. The other one was filamentous aglae, Mougeotia sp. which preferentially attached to the m-acrophytes in the WSP. A Nikon SMZ25 microscope (Nikon Instruments Inc.) was used to take images of algae. Microscopic images of both algae are shown in Figure .2. These two types of algae were collected on August 17, 2016, and transported to the laboratory in a cooler packed with ice. They were washed five times with autoclaved DI water and cultivated in Bold's Basal Medium (BBM) separately for 6 days at room temperature (~20°C). Three 500-mL Mason jars, each containing 400 mL of BBM and 10 g algae, were prepared for each type of algal. A jar of BBM only (without any algae samples) was also maintained as a control. pH and DO levels were monitored daily using a HQ40d portable multi-parameter (Hach, Loveland, CO).   The purpose of the laboratory pH experiment was to assess the effect of pH alone, on the inactivation of indicator organisms. A grab sample was collected from the treatment plant secondary effluent, which is the influent to the first WSP. Seven Liters of the wastewater grab sample was distributed equally into fifteen 500-mL Mason jars. All Mason jars were autoclaved before use. Five different pH conditions (5.1 , 8.2, 8.4, 9.7 and 10.6) were tested in triplicate. pH was adjusted daily with 0.2N HCl and 0.2N NaOH solutions as required. All the jars were placed in an incubator (Thermo Scientific Refrigerated Incubator) with the temperature of 20 °C and with a light intensity of 3400 Lux, measured with a REED LM-81LX Compact Light Meter (Reed instruments), and DO was kept at 8.5 mg/L by bubbling air using aquarium air diffusers. The concentrations of indicator organisms were measured on days 1, 3, 5, 7 and 9.

pH experiments
The indicator organisms selected for this study were E. coli, total coliforms, Enterococci and C. perfringens. . Plate counting was performed after a 24-hour incubation period at 37°C for E. coli, total coliforms and Enterococci, and a 48-hour anaerobic incubation at 44°C for C. perfringens in an anaerobic jar (Merk, Germany). In this study, the "inactivation" and "removal" of indicator organisms referred to the disappearance of culturable colonies. The detection limit was 1 CFU/100 mL. If no culturable organisms were found in 100 mL sample, the concentration was noted as zero.

DO experiments
Three DO levels of 1, 8.5 and 20 mg/L were investigated to simulate low, intermediate (control) and high DO concentrations. pH and temperature were maintained at 7 and 20°C, respectively. A secondary effluent sample was collected from the wastewater treatment plant. Three replicates of each DO condition were prepared. A sample volume of 400 mL was transferred into each of nine 500-mL Mason jars. Low, intermediate and high DO conditions were maintained by constantly bubbling N 2 , air and O 2 using aquarium air diffusers, respectively. The concentrations of indicator organisms were measured on days 1, 3, 7, 9, 11 and 13.

Temperature experiments
The inactivation and removal of indicator organisms were measured at room temperature (20°C) to simulate warmer season, and at an incubator temperature of 4°C to simulate colder season. A grab sample from Alkhademiyah sewage station in Baghdad effluent was obtained. Sample volume of 400 mL was used in each Mason jar. Both temperature condition experiments were conducted in triplicate. pH and DO were kept at 7 and 8.6 mg/L, respectively. The concentrations of indicator organisms were measured on days 1, 3, 5, 8, 10, 13 and 15.

Statistical analysis
t-test is most commonly applied when the test statistic would follow a normal distribution. The t-test was used to determine if two sets of data are significantly different from each other for different pH, DO and temperature conditions.

Results and Discussion
Over a monitoring period of 6 days, pH and DO were noted to increase from 5.2±0.1 to 8.1±0.3, and from 6.1±0.1 to 9.1±0.2, respectively, in Mougeotia sp. jars ( Figure.3). Similarly, in the Nostochopsis sp . jars, pH increased from 6.0±0.1 to 9.0±0.3, and DO from 6.2±0.1 to 7.5±0.4 ( Figure .4). In the control Mason jar, pH varied between 5.9 and 6.2, while DO fluctuated between 5.8 and 6.2 ( Figure .5). The increases in both pH and DO were considered significant compared to the control (p<0.05). There were significant increases in both pH (p<0.05) and DO (p<0.05) in both Nostochopsis sp. and Mougeotia sp. jars. This experiment confirmed that the two predominant algal species found were likely responsible for the high pH and DO levels observed.
where C 0 is the initial concentration (CFU/100 mL), and C t represents the concentration (CFU/100 mL) at time, t. The inactivation of the selected indicator organisms throughout the 9 days of exposure to different pH levels is compared in Figure .6. Table .1 summarized the inactivation rates of the four indicator organisms under the different pH conditions. First order inactivation rate constants for E. coli ranged from 0.11 day -1 at pH 8 to 0.19 day -1 at pH 10.5. Inactivation rates for total coliforms varied between 0.08 day -1 at pH 8 and pH 8.4 and 0.12 day -1 at pH 10.5. In general, the inactivation rates of both E. coli and total coliforms increased with an increase in pH. However, no significant differences (p>0.05) between the tested pH were found. These observations were consistent with the studies done in the literature. In a study by [6] , it was found that pH of 8 and 8.8 (tested pH include 6.6, 7.3, 8 and 8.8) inhibited the survival of E. coli. [7] found that the inactivation rate of E. coli increased when pH was above 8.5. A study by [8] and [1] also reported that no significant difference in decay rates of fecal coliforms at pH levels of 5, 7, 9 and 10, and under both sunlight and dark conditions. In a previous study, Liu  coli and total coliforms to represent pathogenic Enterococci as these organisms showed different responses to various pH conditions. Inactivation of E. coli increased with increasing pH while in contrast, Enterococci was noted to exhibit similar rates of inactivation at all pH levels [7][8][9] also reported that low pH (pH 4 and 5) had a more bactericidal effect on Enterococci in domestic wastewater than alkaline conditions. Spore forming bacteria, C. perfringens, showed resistance to all the pH levels investigated during the test period. They may require a longer period of time to be inactivated or pH is not a significant removal mechanism for C. perfringens.
The effects of different DO concentrations on the removal and inactivation of indicator organisms were also investigated in this study. Table .2 presents the inactivation rates of the four indicator organisms at low, intermediate and high DO levels. Figure .7 shows the concentrations of E. coli, total coliforms, Enterococci and C. perfringens at different DO levels over the 13-day testing period. The highest inactivation rates for both E. coli and total coliforms were observed at the high DO concentration of 20 mg/L ( Figure .7 (a) and (b)). Among the selected indicator organisms, E. coli, total coliforms and Enterococci are facultative anaerobes, while C. perfringens are obligate anaerobes. Facultative anaerobes can metabolize either aerobically or anaerobically; however these organisms prefer aerobic conditions as aerobic respiration generates more adenosine triphosphate (ATP) than either fermentation or anaerobic respiration. This could explain why comparatively low inactivation rates were generally found under  For the removal and inactivation of Enterococci, a higher inactivation rate was found at high and intermediate DO concentrations. The Enterococci species obtained from the secondary effluent appeared to exhibit a higher growth rate at lower DO concentrations. Moreover, a significant difference was found between low DO and high DO (p=0.004). As such, this would suggest that when DO was enhanced by algal photosynthesis during warm season, it could facilitate the inactivation of Enterococci. Although C. perfringens are obligate anaerobes, and oxygen can be considered toxic to them, they showed a minimum inactivation rate of 0.01 day -1 at high DO concentration over the monitoring period of 13 days. This again shows the resistance to environmental stresses of C. perfringens.  Two temperatures (4°C and 20°C) were selected to simulate the cold and warm seasons, and to assess their effects on the removal and inactivation of indicator organisms. Figure .8 shows the concentrations of E. coli (a), total coliforms (b), Enterococci (c) and C. perfringens (d) under warm (T=20 °C) and cold (T=4 °C) temperatures over a monitoring period of 15 days. The inactivation rates of E. coli were -0.02 day -1 at 4°C and 0.11 day -1 at 20°C, while for total coliforms, the inactivation rates ranged from -0.01 day -1 and 0.03 day -1 , respectively. There was no Enterococci colony detected on day 12 under the 20°C condition and the inactivation rate for the first 10 days was 0.23 day -1 . Meanwhile, the inactivation rate of Enterococci at 4°C was 0.05 day -1 . The concentrations of E. coli, total coliforms and Enterococci were generally significantly reduced at 20°C compared to at 4°C (p<0.05). This finding agrees with observation made by several researchers as they have reported increased removal efficiencies of fecal coliforms at higher temperatures [26]; Pearson [10,25,27] . In a study by [24], fecal coliforms were reduced by 98.95% during the warmer seasons and by 94.91% during the cold seasons in a WSP system, operating in Marrakesh, Morocco. This system consisted of an anaerobic and an aerobic pond in series. They also found that when temperature was increased from 8°C to 23°C, the removal rate of E. coli also increased. For C. perfringens removal in this study, they were generally tolerant to the wide range of temperature during a 15-day test period. Figure .8. Inactivation and removal of E. coli (a), total coliforms (b), Enterococci (c) and C. perfringens (d) in the secondary effluent wastewater collected from the Alkhademiyah sewage station in Baghdad governorate over a 15-day period when they were exposed warm (T=20°C) and cold (T=4°C) temperatures.

Conclusion
This paper investigated the effects of pH (5.1, 8.2, 8.4, 9.7 and 10.6), DO (1, 8.5 and 20 mg/L) and temperature (4 °C and 20 °C) on the inactivation and removal of four indicator organisms (two traditional ones and two potential ones). Both algae (Mougeotia sp. and Nostochopsis sp.) that were collected from the Alkhademiyah sewage station in Baghdad WSP were noted to increase both pH and DO when cultivated under laboratory conditions. Their presence is believed to contribute to the high pH and DO levels in the WSP system. The inactivation of E. coli and total coliforms were found to increase with increasing in pH. The highest inactivation rates were observed at pH 10.6. Intermediate DO level appeared to support the survival of both E. coli and total coliforms, while both high and low DO levels could facilitate their removal and inactivation. Enterococci were significantly reduced at both intermediate and high DO concentrations. This would suggest that the presence of algae leading to high DO concentrations could potentially promote the removal of E. coli, total coliforms and Enterococci.
Two additional indicator organisms (Enterococci and C. perfringens) showed different inactivation trends compared to traditional indicator organisms. C. perfringens were tolerant to the pH, DO and temperature ranges investigated. This would suggest that E. coli and total coliforms are limited in their ability to represent the presence and concentration of Enterococci and C. perfringens. This finding supports the literature where they have been suggested to be potential indicator organisms of pathogens in addition to E. coli, fecal coliforms and total coliforms. Disinfection efficiencies should be expected to exhibit seasonal variations in WSP systems operated in temperate climates. Higher removal efficiencies of indicator organisms would be expected during the warmer seasons, which will continue to pose challenges to winter operation unless some engineered operational modifications can be adapted that will facilitate their retention and removal under colder conditions, which would normally be expected to offer more limited inactivation efficiencies.