The effect of biocides on the functioning of
microorganisms in constructed wetlands
A study to the influence of glutaraldehyde and DBNPA on microorganisms at
different temperatures
Patrick Luif
July 2018
BSc Thesis
Future Planet Studies
University of Amsterdam
Thesis supervisors:
John Parsons
&
Abstract
Freshwater is a fundamental resource for all living organisms. Nonetheless, traditional freshwater resources are decreasing. A large part of the global freshwater use is used by the industry in cooling towers. After cooling tower blow down, wastewater treatment via constructed wetlands can offer a sustainable form of treatment. By using constructed wetlands, the wastewater can be re-used in the cooling towers. However, biocides are added to cooling tower water in order to prevent biofilms to emerge. Hence, when treating the wastewater in constructed wetlands these biocides will enter the environment. This could have a negative impact on the functioning of the constructed wetland. Therefore, this study focusses on whether biocides found in cooling water towers, glutaraldehyde and 2,2-dibromo-3-nitrilopriopionamide, have an impact on the microorganisms present in the constructed wetlands. The study is conducted with 0.5, 1, 2.5 and 5 mg/L biocide concentrations and is performed under different temperature conditions in order to simulate seasonality. The main finding of this research is that a higher biocide concentration does influence the microbial activity in a negative way. Moreover, the difference in temperature shows a significant difference in microbial activity. Furthermore, the experimental set up does not adequately represent a constructed wetland. Hence, more study has to be done to the microbial interaction within a constructed wetland and the effect of biocides on these interactions.
Keywords: Biocide, glutaraldehyde, DBNPA, constructed wetland, microbial activity, cooling tower
Table of Contents
1. Introduction...4
1.1 Research and aim objectives...6
1.2 Research questions...6
2. Methods...7
2.1 Inoculum...7
2.2 Chemicals...7
2.3 Experimental set up...7
2.4 Benzoic acid analysis...8
2.5 Analysis in SPSS and Excel...8
3. Results...9
3.1 The 0, 0.5 and 1 mg/L biocide concentrations at 20 oC...9
3.2 The 2.5 and 5 mg/L biocide concentration at 20 oC...11
3.3 The 0, 0.5 and 1 mg/L biocide concentration at 5 oC...12
4. Discussion...14
4.1 Comparison of the results...14
4.2 The link between microbial activity, temperature and effectiveness of both biocides...14
4.3 The extent to which this experiment simulates a constructed wetland...16
5. Conclusion...17
Acknowledgements...18
1. Introduction
This study to constructed wetlands is induced by a five-year project called Water Nexus. The ambition of the Water Nexus program is to find solutions to the water scarcity problems in delta areas worldwide. A small part of this project is the research to purification methods of freshwater for reuse, i.e. constructed wetlands.
Freshwater is a fundamental resource, which is essential to all living organisms. Moreover, freshwater is integral for food and energy production, transportation, waste disposal and industrial purposes (Gleick, 1993). Despite the importance of freshwater, the resources are unevenly distributed which have led to regions with severe water scarcity (Gleick, 1993; Löwenberg et al., 2015). Yet, the traditional water resources are decreasing due to over exploiting and pollution and become saline due to salt water intrusion, especially in coastal areas (Löwenberg et al., 2015).
However, in these water scarce areas industrial processes often need a considerable amount of freshwater (Löwenberg et al., 2015). According to the AQUASTAT database of the Food and Agriculture Organization of the United Nations (FAO), the global industrial freshwater use is 19% of the total global annual freshwater use in 2010 (FAO, 2016). Although water depleted areas have a relatively high industrial freshwater use, most of the industrial freshwater use can be found in Northern America and South East Asia, respectively 37.6% and 29.2% of the global annual freshwater use in 2010 (FAO, 2016).
Among the large industrial freshwater consumers are Cooling Towers (CTs) (Löwenberg et al., 2015). These CTs are mainly used in power generation facilities. Wang et al. (2005) stated that 60%-70% of freshwater usage for industrial purposes is used in CTs. The cooling water system consists of the cooling tower, re-circulation system and a heat exchange system (Fig. 1) (Kim & Smith, 2001). Cold water used in the heat exchange system returns to the CTs where the boiling hot water is cooled. Flow rate losses in the system are caused by evaporation and blow down. This cooling tower blow down
(CTBD) is done to prevent accumulation of undesirable materials, such as salts which results in scaling and corrosion (Bott, 1998), in the re-circulating water as a result of evaporation. The flow rate loss is compensated by make-up water (Kim & Smith, 2001).
Moreover, the warm environment of CTs create the ideal circumstances for microorganisms to thrive in the piping systems and form biofilms (Bott, 1998). This reduces the overall efficiency of the CTs (Kim & Smith, 2001). Hence, a wide range of chemicals such as anti-scalants, corrosion inhibitors and biocides are added to the cooling water to mitigate this problem (Bott, 1998).
The chemicals added to the cooling water are seen as contaminants which are released during CTBD. You et al. (1999) stated that the relatively low amount of contaminants in very large volumes of water from CTBD establish the potential of re-using CTBD water when treated. This re-use potential has also been opted by Greenlee et al. (2009) and Fritzmann et al. (2007). Water from CTBD is treated as typical wastewater (Mohsen, 2004). However, the chemical composition differs from wastewater from other sources due to the chemical additives such as biocides, anti-scalants and corrosion inhibitors (Löwenberg et al., 2005). Resulting in relatively high treatment costs (Löwenberg et al., 2005).
However, constructed wetland systems can be seen as a more sustainable and cost efficient substitute for wastewater treatment (Richardson and Davis, 1987). Specifically, engineered wetlands
Figure 1. Cooling water system (Kim & Smith, 2001)
are the primary type of wetland treatment systems, the so called constructed wetlands (Richardson and Davis, 1987). Constructed wetlands are seen as the “kidneys of the landscape” (Brix, 1994) and are arranged in such a way that it simulates natural wetlands in a more controlled way (Matamoros et al., 2017). These constructed wetlands have been proven to be an efficient treatment process for the removal of a wide range of both conventional and non-conventional contaminants from, e.g. industrial wastewater (Dunbabin and Bowmer, 1992; Avila et al., 2004). The wastewater treatment in (constructed) wetlands can occur either physical (photodegradation, sedimentation), chemical (adsorption) or biological (biodegradation, plant uptake) (Vymazal et al., 2010).
The way in which the constructed wetland will be arranged is dependent on which type of treatment is necessary to filter the wastewater. Conventionally constructed wetlands can be classified in surface flow (SF) and sub-surface flow (SSF) (Avila et al., 2014) (Fig.2). The SSF can be further divided into vertical SSF and horizontal SSF (Avila et al., 2014). Within the vertical SSF, the system operates under unsaturated and more oxidized conditions, whereas the conditions under horizontal SSF are anaerobic and saturated (Avila et al., 2014). In general, vertical SSF is the most effective way of treating wastewater, as there are relatively higher contamination concentrations in the vertical flow (Garcia et al., 2010). All in all, the most dominant form of treatment is dependent on the design of the constructed wetland.
The active reaction zone for the biological degradation by microorganisms is the rhizosphere (root zone). The rhizosphere is the area where the biological processes, which are induced by interaction between microorganisms and pollutants take place (Stottmeister et al., 2003). Microorganisms play a
major role in the transformation process of organic pollutants (Li et al., 2014) and are thus very important for the biodegradation of contaminants.
However, cooling tower wastewater contains biocides (Bott, 1998). These biocides are used to prevent microorganisms to grow in the piping systems (Bott, 1998). Hence, when using a constructed wetland for the cooling tower wastewater treatment process, the biocides in the wastewater can have a negative effect on the microorganisms in the constructed wetland. Therefore, this study will focus on the effect of biocides on the functioning and existence of microorganisms during the industrial wastewater treatment in constructed wetlands.
Hence, for this study two types of biocides which are commonly used were chosen: glutaraldehyde and 2,2-dibromo-3-nitrilopropionamide (DNBPA). Glutaraldehyde is an electrophilic biocide (Kahrilas et al., 2014). Interaction with microorganisms will lead to cell wall damage and cytoplasmic coagulation, i.e. cell death (Kahrilas et al., 2014). The biocide DBNPA is also an electrophilic biocide (Kahrilas et al., 2014). This biocide releases small amounts of bromine which eventually result in disrupting the function of cell components (Kahrilas et al., 2014). Moreover, both biocides are generally used simultaneously as this will increase the overall biocidal efficiency (Kahrilas et al., 2014). In this study the effect of simultaneously addition of both glutaraldehyde and DBNPA will be studied using benzoic acid as a reference compound. Furthermore, the research will be conducted at both 5°C and 20°C in order to study the effect of winter and summer temperature of
constructed wetlands on the functioning of microorganisms under different conditions.
1.1 Research and aim objectives
This study focusses on the sustainability of using constructed wetlands for industrial wastewater treatment. Previous studies have concluded that wastewater treatment by constructed wetlands can be seen as a more sustainable and cost efficient solution. However, the presence of the biocides glutaraldehyde and DBNPA in industrial wastewater can possibly have a negative effect on microorganisms in constructed wetlands and thus make the use of constructed wetlands for industrial wastewater treatment impractible.
1.2 Research questions
This study consists of the following two main research questions:
Main research question – What is the effect of both glutaraldehyde and DBNPA on the functioning of microorganisms in constructed wetlands?
Main research question – In what way does temperature influence the effect of biocides on the functioning of microorganisms in constructed wetlands?
2. Methods
In order to address the research question, biodegradation experiments were performed. This chapter describes the used chemicals, the experimental setup, benzoic acid analysis and successive analysis in SPSS and Excel.
2.1 Inoculum
In this study a constructed wetland and its microbial activity were simulated by using inoculum from a constructed wetland near Hapert (Noord-Brabant, the Netherlands). The material was taken from the sludge in the first compartment. Before use the sludge was sieved with a 2 mm filter in order to remove remaining plant fragments. Finally, the microbial inoculum was stored at 4 oC to minimize
microbial activity.
2.2 Chemicals
The biocides glutaraldehyde and DBNPA were acquired form Sigma Aldrich and were stored at a chemical storage room. DBPNPA was stored at room temperature (20 oC) and glutaraldehyde was
stored in the freezer. Stock solutions of the compounds of 100 mg/L concentration were prepared in ultra pure water acquired from Purelab. Furthermore, the stock solution of benzoic acid was set at 500 mg/L concentration. This stock was also prepared with ultra pure water acquired from Purelab. Both stock solutions were used once. The sodium azide (NaN3) was already prepared at 7,8 mg/L.
Finally, the 99,7% pure acetic acid (CH3COOH) was acquired from Sigma Aldrich.
2.3 Experimental set up
Aerobic biodegradation experiments with benzoic acid were performed in triplicate in 250 ml serum bottles. These bottles were covered with tin foil in order to prevent photo degradation whilst being shaken at approximately 120 rpm. The bottles were stored at both 5 oC and 20 oC to simulate the
summer and winter temperature circumstances in a constructed wetland.
In the bottles a 1:100 ratio of inoculum/medium a total volume of 60 ml is used. The mineral medium which was used is composed of macro nutrients (NH4Cl, 170 g/L; CaCl2.2H2O, 8 g/L; MgSO4.7H2O, 9 g/L), trace elements (FeCl2.4H2O, 2 g/L; CoCl2.6H2O, 2 g/L; MnCl2.4H2O, 0.5 g/L; CuCl2.2H2O, 30 mg/L; ZnCl2, 50 mg/L; HBO3, 50 mg/L; (NH4)6Mo7O24.4H2O, 90 mg/L; Na2SeO3.5H2O, 100 mg/L; NiCl2.6H2O, 50 mg/L; EDTA (tripex 2), 1 g/L, HCL 36%, 1 ml/L, Na-Resaruzin, 0.5 g/L) and 50 mM phosphate buffer in MilliQ water.
The 250 ml serum bottles were filled with 0.6 ml inoculum and a variable amount of medium. Glutaraldehyde and DBNPA concentrations of 0.5, 1, 2.5 and 5 mg/L were added simultaneously. These biocide concentrations were chosen as these concentrations are representative for the biocide concentration in cooling tower wastewater (T. Wagner, personal communication, June 22, 2018). The control experiment of 0 mg/L of the biocides were performed twice to control the ability of the microbial inoculum to degrade benzoic acid. Benzoic acid of 50 mg/L concentration was added to the 250 ml serum bottles. The experiments which determine the effect of the different concentrations of glutaraldehyde and DBNPA were performed similar to the control experiments.
Furthermore, abiotic controls were performed to quantify other potential removal mechanisms as sorption and hydrolysis, moreover these abiotic controls could determine whether microbial activity is responsible for benzoic acid degradation Abiotic controls were performed in serum bottles with butyl stoppers closed with aluminium crimp caps. In order to exclude microbial activity, the serum bottles were put in an autoclave at 120 oC and approximately 1 ml of NaN3 was
added. Biotic controls were performed to ensure microbial activity of the inoculum.
0.22 m filters and acidified with acetic acid. The samples were stored in 1.5 ml LC vials at 4 oC until
analysis by the HPLC-DAD.
2.4 Benzoic acid analysis
Benzoic acid is analysed by a High Performance Liquid Chromatographer (HPLC-DAD). Chromatographic separation was achieved with a Phenomenex Kinetex XB-C18 column (100 x 3.00 mm with a mean particle size of 2.6μm) installed in a Prominence HPLC-DAD (Shimadzu, Japan) consisting of two LC-20AD XR pumps, a SIL-20AC XR auto-sampler and a CTO-20AC column oven. The mobile phase consisted of A: H2O with 0,1% acetic acid and B: methanol and an isocratic method of 30% B was used with a column temperature of 45°C. Quantification will performed at a wavelength of 246 nm excited by a deuterium lamp using an external calibration curve in LCSolutions software (Shimadzu, Japan).
2.5 Analysis in SPSS and Excel
The results from the HPLC-DAD were further analysed in SPSS and Excel. The absolute benzoic acid concentration, which was analysed in triplicate has been converted into relative numbers with sample time zero (T0) set at 100%. By creating a nonlinear regression line through the measured benzoic acid
concentration over time, a clear indication of the effect of biocides on the functioning of microorganisms can be given. The nonlinear regression line was made according to a ecotoxicological study by Haanstra et al. (1985). The following formula was used:
B∗(log 10 (time)−log 10 ( A ))
(¿¿)
e
¿1+
¿
¿
y=
C
¿
In this formula three parameters are used. Parameter A represents the exact time in which the benzoic acid is 50% degraded. The second parameter B is an indication for the steepness of the modelled line. The last parameter C is by definition 100 as this indicates the starting concentration of benzoic acid.
The formula of Haanstra et al. (1985) was used in SPSS to obtain the estimated values for both parameters A and B. Finally, the obtained parameters were used in the formula in excel to make a figure.
3. Results
In this section the results of the experiments for both 5 and 20 oC are given. All figures consist of the
actual measured benzoic acid concentration over time, the model fit, i.e. nonlinear regression line and the abiotic control. First, the results of both 5 and 20 oC are separately shown. Followed by a
comparison between the results.
3.1 The 0, 0.5 and 1 mg/L biocide concentrations at 20
oC
The first experiment gives an estimation on the time which is needed for the microorganisms to degrade benzoic acid without the effect of biocides (Fig. 1). Hence, it represents a control experiment, i.e. this nonlinear regression line can be used as comparison to the experiments with addition of a certain concentration of biocides. Moreover, this experiment has been performed twice, both resulting in the same outcome. Furthermore, the relative benzoic acid concentration has been rounded to two decimals (Table. 1).
However, when adding 0.5 mg/L (Fig. 4) and 1 mg/L (Fig. 5) concentrations of both glutaraldehyde and DBNPA, there is a significant effect visible. As can be seen for the 0.5 mg/L concentration, the average time for the benzoic acid to completely degrade lays between 20-22 hours, whereas in the control group this point lays between 17-20 hours. It can be said that the degradation of benzoic acid is effected by the biocides. Moreover, the abiotic control in these experiments show the expected results, as no benzoic acid has been degraded over the measured time and the biotic experiments did show degradation of benzoic acid within the measured time. Hence, the degradation of benzoic acid is accounted for by microorganisms.
0 h
17 h
20 h
22 h
24 h
42 h
0 mg/L
100
4,43
0
0
0
0
0,5 mg/L
100
78,26
23,17
0
0
0
1 mg/L
100
91,38
64,19
20,36
1,25
0
Table 1. Relative benzoic acid concentration over measured time in hours at 20 oC.
Figure 3 relative benzoic acid degradation over time at 0 mg/L biocide concentration at 20 oC.
5 11 2 8 14 17 20 23 26 29 32 35 38 41 44 47 50 53 56 59 62 0 20 40 60 80 100 Model fit Abiotic con-trol Data Time (hour) B e n zo ic a ci d ( % )
Figure 4. In this figure the relative benzoic acid degradation over time at 0.5 mg/L concentration at 20 oC.
Figure 5. In this figure the relative benzoic acid degradation over time at 1 mg/L concentration at 20 oC.
10
5 11 2 8 14 17 20 23 26 29 32 35 38 41 44 47 50 53 56 59 62 0 20 40 60 80 100 Model Fit Abiotic con-trol Data Time (hour) B e n zo ic a ci d ( % ) 5 11 2 8 14 17 20 23 26 29 32 35 38 41 44 47 50 53 56 59 62 0 20 40 60 80 100 Model Fit Data Abiotic Con-trol Time (hour) B e n zo ic a ci d ( % )3.2 The 2.5 and 5 mg/L biocide concentration at 20
oC
Next to the 0, 0.5 and 1 mg/L concentration experiments, the 2.5 and 5 mg/L experiments at 20 oC
has been conducted (Fig. 6) and (Fig. 7). The obtained data shows no sign of benzoic acid degradation within the measured time. According to previous experiments conducted by Wagner (2018), the results of this experiment are not in line with the expected results. An explanation for the unexpected results could be the fact that a higher glutaraldehyde concentration was used. When the stock solution was made, an error has resulted in a 2.5x higher glutaraldehyde concentration. Hence, instead of 2.5 mg/L and 5 mg/L concentrations, a concentration of respectively 6.25 mg/L and 12.5 mg/L glutaraldehyde concentrations were used. The higher glutaraldehyde concentrations could have affected this experiment in two ways. The first hypothesis is that the higher glutaraldehyde concentrations have led to a longer timespan in which benzoic acid degrades. This means that the degradation of benzoic acid did not start within the measured time. The other theory is that the higher biocide concentration killed all the microorganisms, hence no biodegradation could have taken place. However, due to lack of time, these experiments were not repeated.
Figure 6. In this figure the relative benzoic acid degradation over time at 2.5 mg/L concentration at 20 oC.
Figure 7. In this figure the relative benzoic acid degradation over time at 2.5 mg/L concentration at 20 oC.
0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 99.95 100.14 100.09 100.18100.03 100.15 Data Abiotic control Time (hour) B e n zo ic a ci d ( % ) 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 96.58 94.33 96.69 97.77 96.7 Data Abiotic control Time (hour) B e n zo ic a ci d ( % )
3.3 The 0, 0.5 and 1 mg/L biocide concentration at 5
oC
In order to compare the effect of glutaraldehyde and DBNPA on the functioning of microorganisms in constructed wetlands in summer and winter conditions, the same experiment at 5 oC has been
conducted. However, due to lack of time, the 2.5 and 5 mg/L experiments were not executed. Nonetheless, the experiments at 0, 0.5 and 1 mg/L have resulted in data which can be used for comparison. The experiments at 5 degrees have been executed twice, as the first time the results were compromised by the timespan in which the samples were taken. The first time the measured time was too short, resulting in unusable data.
When looking at the results of the 0 mg/L concentration experiment, a clear difference with the 20 oC experiment with 0 mg/L concentration can be seen (Fig. 8). The time before benzoic acid
starts to degrade at 5 oC is approximately 7x later than at the same experiment at 20 oC. Furthermore,
within the measured time of 158 hours, the point of no benzoic acid left has not been reached. Therefore, the model fit is not as precise as the model fit for the 20 oC experiments. The reason is the
incapability of SPSS to determine the parameters for the model when there is no point of zero. Hence, the model fit line is determined by estimating the correct parameters, making the model fit a rough estimation.
The experiments at 0.5 mg/L and 1 mg/L concentrations have led to similar results (Fig. 9) and (Fig. 10). In both datasets the point before benzoic acid starts to degrade is respectively 8.5x and 7.6x later than the 20 oC experiments. Moreover, at the 0.5 mg/L experiment the benzoic acid degradation
started after approximately 120 hours, whereas the benzoic acid degradation at 1 mg/L experiment does not even start within the measured time. Furthermore, the model fit of the 0.5 mg/L and the 1 mg/L do both have the same complications as with the 0 mg/L model fit.
Figure 8. In this figure the relative benzoic acid degradation over time at 0 mg/L concentration at 5 oC.
12
8 17 26 35 44 53 62 71 80 89 98107 116125134143152161170179188197206215224233242 0 20 40 60 80 100 Model Fit Data Abiotic Con-trol Time (hour) B e n zo ic a ci d ( % )Figure 9. In this figure the relative benzoic acid degradation over time at 0.5 mg/L concentration at 5 oC.
Figure 10. In this figure the relative benzoic acid degradation over time at 1 mg/L concentration at 5 oC.
8 17 26 35 44 53 62 71 80 89 98107 116125134143152161170179188197206215224233242 0 20 40 60 80 100 Model Fit Data Abiotic Con-trol Time (hour) B e n zo ic a ci d ( % ) 8 17 26 35 44 53 62 71 80 89 98107 116125134143152161170179188197206215224233242 0 20 40 60 80 100 Model Fit Data Abiotic Con-trol Time (hour) B e n zo ic a ci d ( % )
4. Discussion
In this section the set up of the experiments and the results will be discussed. First of all, a comparison between the 20 oC and the 5 oC experiment will be made. In specific, the model fit of all
concentrations are compared and discussed. Secondly, the link between temperature, microbial activity and effectiveness of glutaraldehyde and DBPNA will be given. Finally, the experimental set up is discussed. Especially, the resemblance/dissimilarity between the processes in the serum bottles and a constructed wetland will be debated. Hence, the extent to which this experiment simulates a constructed wetland will be discussed.
4.1 Comparison of the results
In order to make a proper comparison between the results for the 5 oC and the 20 oC experiments,
complete datasets are necessary. In this study the datasets are not complete. First of all, useful data for both 2.5 mg/L and 5 mg/L concentrations at 5 and 20 oC are missing. Hence, no comparison can
be made.
Moreover, only the results of the 0 mg/L, 0.5 mg/L and 1 mg/L concentrations at 20 oC are
complete, as for the same concentrations at 5 oC the point of zero benzoic acid was not reached
within the measured timespan. Due to this incomplete results for the 5 oC experiments, the model fit
data is based on a rough estimation of the parameters.
When using the model of Voshaar et al. (1985) in SPSS, the time in which the benzoic acid concentration is 50% has to be known. Only when this time can be estimated correctly, SPSS can calculate the exact parameters for the model fit. However, when considering the used nonlinear regression equation, it can be debated whether this type of regression analysis is correct for this study. According to Leatherbarrow (1990) the main advantage of nonlinear regression is that a curve can be drawn through each point, resulting in the best fit to the data. However, a wide range of nonlinear regression analysis can be used in different situations (Leatherbarrow, 1990). For this specific study it is assumed that the nonlinear equation of Voshaar et al. (1985) can be used. However, more study to a possible more suitable nonlinear regression analysis can be considered needed. Nonetheless, a rough comparison can be made.
The main empirical finding of this experiment is the difference time before benzoic acid starts to degrade. Another main finding is the steepness of the model fit line. When looking at the 20
oC experiments and the 5 oC experiments, it can be seen that once the benzoic acid starts to degrade,
the concentration decreases exponentially. However, the extent of exponential decline is higher at the 20 oC experiments than at the 5 oC experiments. However, due to incomplete data the model fit is
considered a rough estimation. Therefore, the latter finding cannot be considered a fact and more study has to be done to prove this finding.
4.2 The link between microbial activity, temperature and
effectiveness of both biocides
When looking at the main findings, it can be concluded that there is a link between temperature and microbial activity. In microbiology it has been recognized that temperature is a fundamental factor controlling the rate of development of microbial populations (Ratkowski et al., 1982). According to the Arrhenius law the relation between microbial growth and temperature can be described best via a curve instead of linear regression (Arrhenius, 1989). An Arrhenius curve was made by Johnson et al. (1974) for six sets of data, 5 bacteria’s and 1 fungus (Fig. 11). In this plot it is clearly shown that there is an exponential relationship between temperature and microbial growth. However, due to the complexity of microbial processes, the Arrhenius law does not adequately describe the effect of temperature on the growth of microbes.
Besides the Arrhenius law the specific growth rate (SGR) is often used as an indication for the microbial activity (Price and Sowers, 2004). The relationship between SGR and temperature can be described best via linear regression (White et al., 1991) (Fig. 12). According to White et al. (1991) an increase of 10 oC results in a higher SGR per day with a factor 2.1 for freshwater microorganisms. For
salt water microorganisms this factor is 3.9 (White et al., 1991). Furthermore, these results did not include the size of the microorganisms (White et al., 1991).
Figure 11. The Arrhenius curve for six sets of data. (Johnson et al. 1974 retrieved from Ratkowsky et al. 1982)
Figure 12. The relationship between specific growth rate (day-1) and temperature (oC) in fresh and saltwater
Hence, there is a strong link between microbial activity and temperature. However, the relation between microbial activity, temperature and effectiveness of both biocides is not a strong link. According to the empirical results of this study, the effect of adding biocides with different concentrations does slower the degradation of benzoic acid, hence, an effect of biocides on the microbial activity is found. However, the experiments at both temperatures show the same results considering the effect of higher biocide concentrations. Hence, a relation between the effectiveness of biocides and temperature is not clear. In order to find out whether the impact of biocides on a constructed wetland is higher under lower concentrations, more study to the effect of biocides under different temperature conditions has to be done.
Furthermore, the effectiveness of using both glutaraldehyde and DBNPA simultaneously should increase the overall biocidal effect (Kahrilas et al., 2014). However, a similar study conducted by Shenoy (2017) shows that the biocidal effect of using both biocides simultaneously is lower. In the study of Shenoy (2017) the biodegradation of 1H-Benzotriazole and the influence of biocides was studied. In the first five days, the experiment with solely DBNPA showed a 1H-Benzotriazole degradation of 30%, whereas the 1H-Benzotriazole degradation with both glutaraldehyde and DBNPA addition showed a degradation of 23% (Shenoy, 2017).
4.3 The extent to which this experiment simulates a
constructed wetland
Finally, it should be debated whether this experiment is representative for the processes that occur in constructed wetlands. When analysing the experimental set up it could be said that this experiment does simulate a constructed wetland, as the microorganisms in the inoculum are a representation of the population in a specific constructed wetland. Moreover, the 5 oC experiments correspond to the
winter conditions in a constructed wetland, whereas the summertime conditions are represented with the 20 oC experiments.
However, the processes that influence the microorganisms in a constructed wetland are not taken into account. For example, a major influence is the nutrient availability and composition (Truu et al., 2009). In the conducted experiments there was an abundance of nutrients available due to the addition of microbial medium and benzoic acid. In constructed wetlands, however, the quality of the nutrients is not always as high as the used medium (Truu et al., 2009). Hence, this could result in a different outcome, as the microorganisms need time to grow before the benzoic acid degradation starts. Moreover, the microbial processes are also dependent on plant types, hydraulic conditions and multiple environmental factors (Truu et al., 2009).
Hence, more study to the impact of the processes within a constructed wetland on the effect of biocides on the functioning of microorganisms has to be done.
5. Conclusion
The study to the effect of biocides on the functioning of microorganisms in a constructed wetland has given important insight in the possibility for CTs wastewater to be treated in a constructed wetland. When looking at the outcome of this study, it can be said that biocides do have an effect on the functioning of microorganisms. Moreover, the experiments at different temperatures show a significant difference in benzoic acid degradation over time. With a far greater timespan before benzoic starts to degrade for the lower temperature. However, the model fit of the 5 oC experiments
cannot be made properly due to missing data. Therefore, a comparison of the regression lines of the experiment of both temperatures cannot be made. Furthermore, datasets for both temperatures at 2.5 mg/L and 5 mg/L concentrations are missing or incomplete due to lack of time.
Finally, to refer back to the two main questions:
Main research question – What is the effect of both glutaraldehyde and DBNPA on the functioning of microorganisms in constructed wetlands?
Main research question – In what way does temperature influence the effect of biocides on the functioning of microorganisms in constructed wetlands?
It can be concluded that glutaraldehyde and DBNPA do have an effect on the functioning of microorganisms. However, it is not clear whether these results are representative for a constructed wetland. This is mainly due to the processes which occur in constructed wetlands, which has not been accounted for in the experiments. Considering the second main question, temperature does influence the functioning of microorganisms significantly. This is mainly due to the relation between microbial activity and temperature, as lower temperature means lower microbial activity. However, the results did not show a significantly different effect of temperature on the effect of biocides on microorganisms.
All in all, it can be concluded that the biocides do have an effect on the functioning of microorganisms in a serum bottle. Hence, more study has to be done in order to find out whether biocides do have an effect on the functioning of microorganisms in a constructed wetland.
Acknowledgements
I would first like to thank my thesis supervisor PhD-candidate Thomas Wagner of the University of Amsterdam. Whenever I ran into a trouble spot or had a question about my research or writing, Thomas always provided valuable feedback. He consistently allowed this paper to be my own work, but steered me in the right the direction whenever he thought I needed it.
I would also like to acknowledge dr. John Parsons and dr. Boris Jansen of the Faculty of Science Institute for Biodiversity and Ecosystem Dynamics at the University of Amsterdam as the first and second assessor of this thesis, and I am gratefully indebted to them for their very valuable comments on this thesis.
Furthermore, I would also like mention my lab partner Timo van den Berg and the lab technician of the University of Amsterdam who have helped me getting around the lab and ensured the presence of enough materials throughout the whole lab work.
Finally, I must express my very profound gratitude to my parents Maarten and Ciska, my brother Dion and my girlfriend Anne for providing me with unfailing support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been possible without them. Thank you.
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