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Interaction of chlorine concentration and shear stress on chlorine consumption, biofilm growth rate and particle number
Bioresource Technology 97 (2006) 1912–1919
Interaction of chlorine concentration and shear stress on chlorine consumption, biofilm growth rate and particle number Yung-Pin Tsai
*
Department of Civil Engineering, National Chi Nan University, 1, University Road, PuLi, NanTou 545, Taiwan, ROC Received 12 February 2005; received in revised form 9 August 2005; accepted 16 August 2005 Available online 30 September 2005
Abstract Eleven test runs (including two replicates) were carried out to explore the interaction of shear stress and chlorine concentration on the growth of heterotrophic microorganisms. Experimental results revealed that influent chlorine concentration and shear stress had no interaction on biofilm formation. Biofilm bacterial numbers decreased with the increase of influent chlorine concentration. Increasing the shear stress up to a specific level could significantly reduce the potential of biofilm formation. A strong interaction on bacterial quality or chlorine consumption rate of bulk water existed. With non-chlorinated and lower chlorinated conditions, the specific growth rate of biofilm increased with the increase of shear stress. However, an inverse relation occurred at higher chlorine conditions. No significant interaction of chlorine concentration and shear stress existed for particle numbers with 2–5, 5–15, 50–100 and >100 lm diameters. However, a significant interaction existed on particle numbers of 15–25 and 25–50 lm diameters. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Biofilm; Chlorine; Detachment; Flow velocity; Particle number; Shear stress
1. Introduction During the distribution of drinking water, bacterial regrowth may lead to a deterioration of bacterial water quality, increased corrosion, generation of bad tastes and odors, and proliferation of macroinvertebrates (Volk and LeChevallier, 1999). Piriou et al. (1998) indicated that the presence of a substantial and active attached biomass can protect pathogenic microorganisms, create anaerobic zones, lead to the formation of high biocorrosion zones and consume residual chlorine. It has been shown that chlorine has limited penetration into biofilms (De Beer et al., 1994), even though extremely high chlorine concentration (Lomander et al., 2004) and that the maintenance of an appropriate chlorine residual has little effect on biofilm growth (Chandy and Angles, 2001). On the other
*
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0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.08.003
hand, the extracellular polymeric substances (EPS) of biofilm and assimilable organic carbon (AOC) in water can react with chlorine (Angles et al., 1999) and increase the chlorine demand in distribution systems. However, the interaction between other factors and chlorine concentration on biofilm formation has seldom been studied. Shear stresses (SS) have been shown to be instrumental in affecting microbial growth on pipe surfaces for potable water. It is generally considered that high shear stresses cause (1) a greater flux of nutrients to a pipe surface, (2) a greater transport of disinfectants, and (3) a greater shearing of biofilms (Percival et al., 2000). Experimental results have shown that the subsequent growth of the biolayer is mainly due to the activity of the microorganisms in the attached film, and not to the transport of new bacteria from the liquid to the biofilm surface (Bott and Miller, 1983). Thus, increasing shear stress helps to transport the nutrients of bulk water into the biofilm, causing the bacteria within the biofilms to multiply more quickly. Conversely, increasing shear stress is also helpful to the sloughing of biofilms and the penetration
Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
of disinfectants into biofilms, thus suppressing biofilms formation. These complex phenomena explain the contradictions and inconsistencies of previous studies. Most past studies focused on the impacts of AOC concentration on the growth of biofilm bacteria. However, the impacts of shear stress condition have seldom been explored, especially the effect of shear stress on the function of disinfectant to biofilm and bulk bacteria. In a drinking water distribution system, the flow velocities of pipes may be variable, which is assumed to affect the behavior of the disinfectant. However, the interaction between shear stress and disinfectant on biofilm formation and/or detachment is not yet well known. In this study, the experimental design focused on the comparison of the amount of biofilm bacteria, effluent bulk bacteria and particle numbers when shear stress and chlorine concentration were varied. Biofilm structures, which may be destroyed by chlorine, were examined by the particle numbers which were assumed as the detached biofilm pieces. The influences of the shear stress and chlorine concentration on the specific growth (detachment) rate of biofilm and chlorine consumption rate were also examined. 2. Methods 2.1. Experimental system An annular reactor system (AR system; BioSurfaces Technologies, Bozeman, MT) was used to monitor the interaction of shear stress and chlorine concentration on biofilm formation and chlorine demand. Fig. 1 shows the scheme of the AR system used in the study. The detailed characteristics of the AR system and its operational conditions can be found in Tsai et al. (2004). The rotating speeds
1.6 mL/min
rotational speed controller
rotational number counter
nutrient
slide sampling port slide
AR system Volume 1.15 L carbon water temperature controller
1.6 mL/min drainage
constant temperature water feed in
carbon and nutrient feed in
solid drum
slide surface
vertical view
Fig. 1. Scheme of the AR system used in the study.
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in this study were 27, 55 and 82 rpm (measured). Based on the experimental data from the original manufacturer of this AR system, the equivalent shear stresses acting on the slide surface by liquid were about 0.07, 0.17 and 0.29 N m2, respectively. At those shear stress conditions, the AR system was used to simulate the flow condition of a 600 mm diameter pipe whose flow velocities were about 18, 30 and 40 cm/s, respectively and whose Reynolds numbers were about 105,910, 176,225 and 235,518, respectively. 2.2. Synthetic influent water The carbon substrate consisted of equimolar concentrations on the basis of carbon of sodium acetate, sodium benzoate, propionaldehyde, parahydroxybenzoic acid and ethanol, as shown in Table 1. These chemicals were chosen because (1) they represent major classes of compounds, found in drinking water; (2) they were relatively non-reactive with chlorine (Camper, 1995), and (3) many treatment processes utilizing chemical coagulation are not effective in the removal of low molecular weight compounds, and only partially effective in removal of higher molecular weight humic substances. Thus, these compounds can make their way into water distribution systems and serve as biodegradable substrates for biofilm growth (Bell-Ajy et al., 2000). These compounds were assumed as the major AOC composition in the study. The synthetic influent water was prepared by sterilized ultra-pure water, and its equivalent AOC concentration was about 0.5 mg l1-C. The nitrate/phosphate compositions and concentrations were the same as used by LeChevallier et al. (1990), as shown in Table 2. Under these conditions, the nitrate/phosphate would not be a limiting factor of the growth of biofilm bacteria. All chemicals were supplied by Merck Chemical Co. and were filter sterilized prior to addition to the synthetic water. Sterilized ultrapure water was used for dilution (maxima, Elga). The sodium hypochlorite solution was prepared using commercial bleach (6%) diluted with sterilized ultra-pure water (maxima, Elga) to about 50 mg l1 and was automatically added to the synthetic influent water to give the studied free chlorine concentration in influent water. It was controlled by an on-line monitor instrument of free chlorine (RC300, Water Zone, Korea; ATC500S Detector, Japan). The residual chlorine concentration of the reactor effluent was manually measured daily. 2.3. Reactor operation At the beginning of each experimental run, the AR system (taken apart), slides, influent water and tanks, tubes and related accessories were cleaned thoroughly and autoclave sterilized. Then, a slide and influent water samples were randomly sampled to analyze the bacteria count to confirm that the experimental system was not polluted by bacteria. Finally, the AR system and its accessories were assembled carefully on the sterile table and sterile
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Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
Table 1 Compositions and concentrations of carbon source in influent water Chemicals
Molecular formula Molecular weight
Sodium acetate
Sodium benzoate
Propionaldehyde
Parahydroxybenzoic acid
Ethanol
CH3COONa 82
C7H5NaO2 144
CH3CH2CHO 58
C7H6O3 138
CH3CH2OH 64
328.6
383.3
Experimental conditions—individual concentration (lg l1) in synthetic influent water 1000 lg l1-C 683.3 342.9 322.2
Table 2 Compositions and concentrations of nitrate/phosphate in influent water Chemicals (conc. mg l1)
1
P (mg l -P) N (mg l1-N) S (mg l1-S) Na (mg l1-Na) Ca (mg l1-Ca) Fe (mg l1-Fe) Mg (mg l1-Mg) K (mg l1-K)
K2HPO4
KH2PO4
(NH4)2SO4
MgSO4 Æ 7H2O
NaCl
CaCl2
FeSO4
1.4
0.6
0.02
0.02
0.02
2
0.2
0.2494
0.1368 0.0042 0.0048
0.0026
0.0042 0.0079 0.7207 0.0737
0.0020 0.6276
0.1721
ultra-pure water was used as influent water to confirm that there was no leakage. The leakage test proceeded for at least one day. Before starting up an experimental run, a certain quantity of bacteria-containing water (about 103 HPC CFU/ml for each experiment) was added into the AR system for 1 h as the source of the bacterial inoculum of biofilm (slides). In order to exclude the impact of the transportation of new bacteria from the liquid to the slide surfaces and in order to minimize the bacterial number of influent water, influent water and storage tanks were renewed and autoclave sterilized every 2 days and the influent water tanks were covered tightly at all times, except during sampling periods. 2.4. Analyzing methods Samples of the influent and effluent water of the AR system were routinely collected every 2 days. Chlorine was neutralized by sodium thiosulfate (0.13%). Standard spread plate procedure was used to enumerate HPC bacteria, based on APHA (1998) 9215C, and R2A agar (DifcoTM) was used for incubation after appropriate dilution by sterile buffer. Plates were incubated for 48 ± 3 h at 35 ± 0.5 °C. TOC was measured by a TOC analyzer (model Phoenix 8000, Tekmar Dohrmann, USA) with the detection limit of 0.005 mg l1. Turbidity was measured by a Turbidity Meter (2100N, Hack, USA). Other physicochemical parameters were also analyzed based on APHA (1998). Attached microorganisms were released by sonication (Levi, 1997) in a water bath for two minutes (Branson Ultrasonics Co., USA). The number of particles was estimated by a Particle Counter (KL-11A, Rion, Japan).
Total 0.3862 0.0042 0.0496 0.0079 0.7207 0.0737 0.0020 0.7997
2.5. Quality control and data analysis All water samples for the examination of bacteria were diluted by sterile buffer to at least three different dilution ratios, and every diluted sample was analyzed in duplicate. Due to the considerable variance usually experienced in microbiological water quality data and particle numbers, a modified log transformation was used based on the formula log10(x + 1), where x was the measured value of biofilm, bulk HPC bacterial number or particle number. Data series obtained from the routinely sampling every two days were used as the data sources of the following statistical tests. To compare variation in treatments, analysis of variance (ANOVA) F-test was applied, with the bacterial counts, chlorine consumptions and particle numbers as the dependent variables, to test whether the shear stress, chlorine concentration and their interaction significantly influence the behaviors of biofilm formation, chlorine demand and particle variation. If significant factors (e.g. treatments) were found, then the least significant difference test (LSD) was applied to establish which particular levels of a factor were different. The paired sample t-test was also used to determine if means of interested variables were not statistically equal. All tests were performed at a significance level (p-value) of 0.05 using the standard statistical software (SPSS release 10.1.0, SPSS Inc.). Using HPC bacterial numbers of influent and effluent water in reactor and assuming the difference was the result of balanced growth and detachment of biofilm biomass, a specific growth rate (lb) was calculated for biofilm based on biofilm biomass within the reactor, as follows (Butterfield et al., 2002):
Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
F X X0 ¼ lb A Xb
ð1Þ
where F and A were volumetric flow rate (ml h1) and surface area available for biofilm development (cm2) of the reactor, respectively; X0 and X were influent and effluent bulk bacterial numbers (cfu ml1) of the reactor, respectively; and Xb was the biofilm bacterial number (cfu cm2) within reactor. 3. Results The experimental conditions and basic water quality data of all test runs are listed in Table 3. Influent water quality was good due to the use of ultra-pure water. Influent chlorine concentrations of RUNs 1–3 and RUNs 4–6 were automatically maintained at the levels of 0.26 ± 0.08 mg l1 and 0.57 ± 0.08 mg l1, respectively. 3.1. Examination of repeatability of experiments Bacterial number of influent water at each test run was between 0 and 0.766 log cfu ml1, which is a very low level
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of HPC bacteria. The means and standard deviations of biofilm and effluent bulk bacteria of all test runs at pseudo steady state are shown in Fig. 2. Results of the t-test showed that there was no significant difference between RUNs 0–2 and RUNs 0–2r for both biofilm and effluent bulk bacterial numbers (p values were 0.294 and 0.192, respectively). In addition, there was also no significant difference between RUNs 0–3 and RUNs 0–3r for both bacterial numbers (p values were 0.991 and 0.852, respectively). This meant that the test runs conducted in the study had a good repeatability. 3.2. Biofilm formation Raw data in Fig. 2(a) were used to examine the effects of shear stress, influent chlorine concentration and their interaction on biofilm bacterial counts by two-factor ANOVA statistical test. The error bars in Fig. 2 are large in chlorinated system due to the interference of chlorine. Results showed that influent chlorine concentration and shear stress had no interaction on biofilm formation (p = 0.108), although the former significantly influenced
Table 3 Water qualities data sets of experimental runs Run no.
Run conditions CL conc.a (mg l1) b
SS (N m2) c
TOC (mg l1)
Turbidity (NTU)
Conductivity (lS cm1)
pH
Influent
Effluent
Influent
Effluent
Influent
Effluent
Influent
Effluent
RUNs 0–1
ND
0.07
Ave. Stdev.d
0.270 0.075
0.251 0.033
3.150 0.212
3.527 0.233
6.245 0.163
5.963 0.286
0.640 0.051
0.737 0.084
RUNs 0–2
ND
0.17
Ave. Stdev.
0.270 0.075
0.281 0.062
3.150 0.212
3.418 0.244
6.245 0.163
6.159 0.385
0.640 0.051
0.757 0.018
RUNs 0–2re
ND
0.17
Ave. Stdev.
0.118 0.036
0.297 0.109
2.880 1.640
3.250 1.310
6.140 0.110
6.150 0.130
0.468 0.093
0.459 0.091
RUNs 0–3
ND
0.29
Ave. Stdev.
0.372 0.128
0.474 0.166
10.840 12.808
6.493 3.691
6.449 1.074
6.271 0.397
0.616 0.137
0.682 0.210
RUNs 0–3r
ND
0.29
Ave. Stdev.
0.185 0.093
0.381 0.029
3.750 3.590
4.790 4.050
6.620 0.220
6.380 0.610
0.467 0.047
NAf NA
RUN 1
0.26 ± 0.08
0.07
Ave. Stdev.
0.222 0.062
0.284 0.104
14.440 8.582
18.506 10.923
7.109 0.675
6.998 0.646
0.540 0.140
0.752 0.252
RUN 2
0.26 ± 0.08
0.17
Ave. Stdev.
0.222 0.062
0.474 0.565
14.440 8.582
21.201 13.930
7.109 0.675
5.900 2.149
0.540 0.140
0.787 0.394
RUN 3
0.26 ± 0.08
0.29
Ave. Stdev.
0.222 0.062
0.350 0.162
14.440 8.582
31.526 44.610
7.109 0.675
6.603 0.951
0.544 0.140
0.795 0.381
RUN 4
0.57 ± 0.08
0.07
Ave. Stdev.
0.218 0.087
0.365 0.150
35.611 52.722
37.665 50.302
6.392 0.490
6.254 0.470
0.484 0.096
0.614 0.209
RUN 5
0.57 ± 0.08
0.17
Ave. Stdev.
0.218 0.087
0.578 0.341
35.611 52.722
35.688 46.604
6.392 0.490
6.168 0.395
0.484 0.096
0.639 0.179
RUN 6
0.57 ± 0.08
0.29
Ave. Stdev.
0.218 0.087
1.352 1.995
35.611 52.722
37.418 47.870
6.392 0.490
5.984 0.373
0.484 0.096
0.632 0.208
a b c d e f
Free chlorine conc. of influent water. Not detected. Average. Standard deviation. Replicate run. Not available.
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Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
7
SS 0.07 SS 0.17 SS 0.29 SS 0.17 (replicate) SS 0.29 (replicate)
(a)
6 5 4
3.4. Chlorine consumption
2 1 0.00 0.26 0.57 Chlorine conc. of influent water (mg l-1) 8 7
SS 0.07 SS 0.17 SS 0.29 SS 0.17 (replicate) SS 0.29 (replicate)
(b)
6 5 4 3 2 1 0 0.00
0.26
0.57
Chlorine conc. of influent water (mg l -1) Fig. 2. Biofilm (a) and effluent bulk (b) bacteria numbers.
the potential of biofilm formation (p < 0.001). Biofilm bacterial numbers decreased with the increase of influent chlorine concentration. Statistical testing also showed that shear stress was an important factor affecting the formation of biofilm (p < 0.001) and that increasing the shear stress up to a specific level could significantly reduce the potential of biofilm formation. Biofilm counts were nearly equal between SS0.07 and 0.17 N m2 (3.54 and 3.34 log cfu cm2) (p = 0.485). But, biofilm count of SS0.29 N m2 (2.13 log cfu cm2) was significantly less than those of SS0.07 and 0.17 N m2 (p < 0.001). 3.3. Effluent bulk bacteria Analyzing data in Fig. 2(b) by ANOVA showed the existence of a strong interaction of chlorine and shear stress on bacterial quality of effluent water (p = 0.004). Shear stress at CL0 unchlorinated and CL0.26 chlorinated conditions did not significantly affect the effluent bulk bacterial number (p = 0.805 and 0.887, respectively). However, it had a significant impact at the condition of CL0.57 (p = 0.001). Bulk bacterial number at unchlorinated condition (CL0) was significantly larger than those at chlorinated conditions (CL0.26 and CL0.57).
Chlorine in the AR system was consumed mainly by the oxidations of bulk bacteria, biofilm bacteria and biofilm extracellular polymeric substances. Shear stress and concentration gradient provided the driving forces for the penetration of chlorine into inner parts of biofilm and then further affected the consumption of chlorine in bulk water. Data in Fig. 3 was used to examine (by one-way ANOVA) the effect of shear stress on residual chlorine concentration (RCC). At lower influent chlorine concentration (CL0.26), the effluent RCC at SS0.07 condition was significantly higher than those at SS0.17 and SS0.29 conditions (p = 0.013). In addition, shear stress had no significant impact on effluent RCC at the higher level of CL0.57 (p = 0.328). Two-factor ANOVA test showed the existence of a significant interaction (p = 0.030) between shear stress and influent chlorine concentration on chlorine consumption rate. Significant impact of shear stress on chlorine consumption rate occurred only at lower influent chlorine level (p = 0.019), and lower shear stress level (SS0.07) had a smaller consumption rate than higher levels (SS0.17 and SS0.29). However, shear stress had no significant impact on chlorine consumption rate while the initial chlorine concentration was further increased up to 0.57 mg l1. The lowest consumption rate (57.6%) occurred at the conditions of lowest shear stress and lowest initial chlorine concentration (SS0.07 and CL0.26) in this study. This indicates that chlorine consumption rate was substantially reduced if the driving forces provided by both shear stress and chlo-
100
0.7
90
0.6
80 0.5
70 60
0.4
CL 0.26 (effluent conc.) CL 0.57 (effluent conc.) CL 0.26 (consumption rate) CL 0.57 (consumption rate)
0.3
50 40 30
0.2
20 0.1
Chlorine consumption rate (%)
3
0
Effluent bulk bacteria (log CFU/ml)
At the conditions of CL0.57 and SS0.29, the effluent bulk HPC bacteria were not detected over the whole experimental period (52 days). This indicated that in addition to high chlorine concentration, it is still necessary to provide sufficient shear stress on the pipe wall if a substantial reduction of bulk bacteria is expected.
Effluent chlorine conc. (mg l-1)
Biofilm bacteria (log CFU/ cm2)
8
10
0.0
0 0.07
0.17 Shear stress (Nm-1)
0.29
Fig. 3. Experimental results of effluent chlorine concentration and chlorine consumption rate.
Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
rine concentration gradient were insufficient at the same time. Otherwise, shear stress would not significantly affect the chlorine consumption rate, until one of them (shear stress or gradient) was large enough. 3.5. Specific growth rate of biofilm (lb) Pseudo-steady-state data (influent, effluent and biofilm HPC bacterial numbers) of every test run were used to estimate the specific growth rate of biofilm (lb), basing on Eq. (1) and its assumptions, as shown in Fig. 4. For the non-chlorinated condition, lb was between 0.005 and 0.070 h1. With lower chlorine addition, lb was between 0.003 and 0.53 h1. The lb value increased with the increase of the shear stress for both conditions. The difference of lb value between non-chlorinated (CL0) and lower chlorine (CL0.26) conditions also increased with the increase of shear stress. On the contrary, the lb value significantly decreased with the increase of shear stress while the initial chlorine concentration was further increased to higher level (0.57 mg l1). At SS0.07 and CL0.57 conditions, lb was relatively high, about 0.986 h1; although by increasing shear stress up to 0.29 N m2, lb was substantially decreased to a negative value. This implied that the formation of biofilm was nearly restrained if both shear stress and chlorine concentration were high enough. This might be the reason why no effluent bulk bacteria were found at this condition over the whole experimental period (RUN 6).
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conditions re-distribute these three behaviors in the AR system. The major sources of bacteria within bulk water include (1) the bacteria originally surviving in bulk water, (2) the planktonic bacteria released from biofilm and (3) the bacteria hidden within the detached biofilm pieces, which are resuspended into bulk water. The third source might be the major reason for larger variations of bacterial quality for drinking water. However, it is difficult to distinguish the detached biofilm bacteria from total bulk bacteria. Consequently, particle number monitoring was used in this study to replace the understanding of the effects of shear stress and chlorine concentration on the biofilm detachment behavior. Fig. 5 shows the particle numbers of influent and effluent water in diameter of 2–100 lm, which was divided into six ranges (2–5, 5–15, 15–25, 25–50, 50–100 and >100 lm). Statistical method t-test was used to compare the means of particle numbers between influent and effluent water. At lower chlorine level (Fig. 5(a)), only particle number of effluent water in diameter of 15–25 lm was significantly larger than that of influent water. This might imply that most of detached biofilm were at this diameter range. At higher chlorine level (Fig. 5(b)), particle numbers of effluent water at all diameter ranges were not significantly larger than those of influent water at lowest shear stress level (0.07 N m2),
3.6. Characteristics of particle numbers in diameter between 2 and 100 lm It was assumed in this study that (1) planktonic bacteria and detached biofilm pieces existing within bulk water were in the particle form; (2) the diameters of most planktonic bacteria were less than 2 lm; (3) the diameters of most detached biofilm pieces were larger than 2 lm. Biofilm problems in drinking water system are the combinations of biofilm growth, biofilm detachment and bulk bacteria survival. Different shear stress and chlorine concentration
Specific growth rate (1/hr)
2.5 CL0 CL0.26 CL0.57
2.0 1.5 1.0 0.5 0.0 -0.5 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Shear stress (Nm-2) Fig. 4. Estimations of biofilm specific growth rate at pseudo-steady state.
Fig. 5. Particle numbers with diameter of 2–100 lm. (a) CL 0.26; (b) CL 0.57.
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Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
but significances occurred at all diameter ranges while increasing shear stress over 0.17 N m2. This result implied that higher chlorine level produced a more fragile biofilm structure, so that the damaged biofilm could be detached more easily by the increase of shear stress. Statistical method analysis of covariance (ANCOVA) was further used to examine the effects of chlorine concentration, shear stress and their interaction on the variation of particle number. Particle number data of influent water were used as co-variate for ANCOVA analysis to exclude the influence of initial particles. No significant interaction between chlorine concentration and shear stress on particle numbers of 2–5 lm diameters (p = 0.103) was found. The main effect of chlorine concentration was significant (p < 0.001). The increased amount (the difference of influent and effluent water) of 2–5 lm particle numbers at 0.57 mg l1 chlorine was significantly larger than that at 0.26 mg l1. However, the shear stress did not significantly influence 2–5 lm particle numbers (p = 0.064). For 5– 15 lm particle numbers, there was also no interaction (p = 0.302) between these two factors (chlorine concentration and shear stress) but the main effects of both factors were individually significant (p < 0.001 and p = 0.002, respectively). The chlorine effect was same as on 2–5 lm particle. Lower shear stress (SS0.07) produced lower levels of 5–15 lm particles than did higher shear stress (SS0.17 and SS0.29). Interaction of these two factors occurred on 15–25 and 25–50 lm particles (p < 0.014 and p = 0.004, respectively). For 15–25 lm particles, chlorine effect was insignificant at lower shear stress condition (SS0.07), but significant while increasing shear stress to 0.17 or 0.29 N m2 (p = 0.004 and 0.001, respectively). Further more, shear stress did not show any significant influence on 15–25 lm particles at lower chlorine level, but significance occurred at lower chlorine level (p < 0.001). No interaction between these two factors existed on 50– 100 and >100 lm particles (p = 0.507 and 0.538, respectively). Shear stress did not show any significant main effect on particle numbers of these two ranges (p = 0.507 and 0.695, respectively). Only the main effect of chlorine concentration was significant for both ranges of particle (p < 0.001 and p = 0.013, respectively). The increased amount of 50–100 or >100 lm particle at higher chlorine condition was significantly larger than that at lower chlorine level. 4. Discussion This study showed that shear stress between 0.07 and 0.29 N m2 did not significantly influence the transmission of chlorine into biofilm, implying that the biofilm attached on pipe walls might be rigid. Either increasing chlorine concentration or shear stress level had a positive inhibition effect on biofilm formation. Thus, biofilm count was minimized under highest chlorine concentration and largest shear stress conditions. However, a significant interaction
on effluent bacterial water quality was found in this study, implying that the detached biofilm pieces might be more fragile than attached biofilm and the disinfection ability of chlorine for bulk water depends on the driving force provided by shear stress. To kill bacteria surviving within the detached biofilm pieces, a sufficient driving force might be necessary to transmit the chlorine more deeply into biofilm pieces. Butterfield et al. (2002) found that biofilm biomass appeared to be related to the chlorine demand, so high demand occurred with high biomass. Biofilms and the non-cell organic matter that accumulated on pipe walls have a high reducing potential, which leads to high chlorine consumption at the surface of the material (Thogersen and Dahi, 1996; Lu et al., 1999). Such oxidation by chlorine leads to the sloughing off of part of the biofilm (Characklis, 1990). However, no significant correlation existed between biofilm bacterial number and chlorine demand in the study. In this study, it was found that the presence of free chlorine resulted in less biofilm biomass, but in a higher specific growth rate of biofilm bacteria. This agreed with the results obtained by van der Wende et al. (1989) and Butterfield et al. (2002). The former determined the growth rate of biofilm to be greater in rotating annular reactors using 0.2 and 0.8 mg l1 residual chlorine compared to non-chlorinated systems. The latter showed that the specific growth rate was on the average five times greater for chlorinated biofilm compared to the control. However, observed yield values were less for chlorinated biofilm. The increase of the specific growth rate in this study did not indicate the increase of the net production for biofilm bacteria. On the contrary, the net production of biofilm bacteria reduced in the chlorinated system because more biofilm bacteria were killed by chlorine. It is believed that the chlorination products may be more available to microorganisms than non-chlorinated organic substances because lower molecular weight compounds require less cell energy for their uptake and utilization (Butterfield et al., 2002). That would be why lower chlorine concentration (0.26 mg l1) led to an increase in biofilm growth rates in this study, compared to non-chlorinated system. Furthermore, growth rate of biofilm bacteria also increased with the increase of shear stress, implying that the increase of shear stress could promote the penetration of nutrient into biofilm. However, increasing shear stress might also increase the penetration of chlorine into biofilm, killing more biofilm bacteria. Although the biofilm bacterial number in chlorinated system was significantly less than that in nonchlorinated system, the net specific growth rate of biofilm would be positive and increased if the increased amount of biofilm bacteria was larger than the amount of biofilm bacteria killed by chlorine, as the result of lower chlorine conditions in this study. An inverse relation between shear stress and growth rate of biofilm was found in this study, while the initial chlorine concentration was further increased from 0.26 to 0.57 mg l1. Specific biofilm growth rate was significantly
Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
reduced with the increase of shear stress. At this time, growth rate at lower shear stress (0.07 N m2) condition was relatively large. This implied that the increased amount of biofilm bacteria (due to the production of lower molecular weight organic substances) was far larger than the amount of being killed biofilm bacteria. Clearly, the driving force for penetration of chlorine was too low to kill much biofilm bacteria at lower shear stress condition. However, increasing shear stress up to 0.29 N m2, the specific growth rate of biofilm was substantially decreased to a negative value. This implied that higher chlorine concentration and higher driving force for penetration of chlorine resulted in the amount of being killed biofilm bacteria was inversely larger than the increased amount of biofilm bacteria by uptake of lower molecular weight organic substances. In this study, using the variations of particle number to examine the behaviors of biofilm detachment is a new technique that has not been previously used. The results have shown some important relations between the sloughing of biofilm and environmental conditions (shear stress and chlorine concentration). The data of particle numbers at different diameters are important, if they can give some important implications about biofilm behaviors, because the particle counter can be used as an on-line instrument and has a high potential for on-line monitoring of the biofouling problem of water supplies. Particle numbers are especially important to the fate of heterotrophic bacteria carried by particles that can penetrate the disinfection barrier, attach to or detach from pipe surface (Morin and Camper, 1997). This becomes critical if pathogens are involved. 5. Conclusions Influent chlorine concentration and shear stress had no interaction on biofilm formation. However, a strong interaction of chlorine and shear stress on bacterial quality of bulk water or on chlorine consumption rate existed. In addition to high chlorine concentration, it is still necessary to provide enough shear stress on the pipe wall if a substantial reduction of bulk bacteria is expected. Chlorine consumption rate was substantially reduced when the driving forces provided by both shear stress and chlorine concentration gradient were insufficient at the same time. For non-chlorinated or lower chlorine addition condition, lb was increased with the increase of shear stress, although the opposite result occurred at higher chlorine condition. No significant interaction of chlorine concentration and shear stress existed on particle numbers of 2–5, 5–15, 50–100 and >100 lm diameters. Higher chlorine concentrations produced significantly larger particle numbers than lower chlorine levels for all of the above ranges. A significant interaction of chlorine concentration and shear stress existed on particle numbers with 15–25 and 25–50 lm diameters.
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Acknowledgements This study was supported by a grant received from the National Science Council of the Republic of China (No. NSC 92-2211-E-260-002). References Angles, M.L., Chandy, J., Kastl, G., Jegatheesan, V., Cox, P., Fisher, I., 1999. Biofilms in drinking water: influence of organic carbon and disinfection. Water 26 (4), 30–33. APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association/American Water Works Association/Water Environment Federation, Washington, DC, USA. Bell-Ajy, K., Abbaszadegan, M., Ibrahim, E., Verges, D., LeChevallier, M.W., 2000. Conventional and optimized coagulation for NOM removal. J. Am. Water Works Assoc. 92 (10), 44–58. Bott, T.R., Miller, P.C., 1983. Mechanisms of biofilm formation on aluminium tubes. J. Chem. Tech. Biotech. 33B, 177–184. Butterfield, P.W., Camper, A.K., Ellis, B.D., Jones, W.L., 2002. Chlorination of model drinking water biofilm: implications for growth and organic carbon removal. Water Res. 36, 4391–4405. Camper, A.K., 1995. Factors influencing biofilm growth in drinking water distribution systems. Ph.D. Dissertation. Department of Civil Engineering. Montana State University, Bozeman, MT. Chandy, J.P., Angles, M.L., 2001. Determination of nutrients limiting biofilm formation and the subsequent impact on disinfectant decay. Water Res. 35 (11), 2677–2682. Characklis, W.G., 1990. Microbial biofouling control. In: Characklis, W.G., Marshall, K.C. (Eds.), Biofilms. Wiley, New York, pp. 523–633. De Beer, D., Srinivasan, R., Stewart, P.S., 1994. Direct measurement of chlorine penetration into biofilms during disinfection. Appl. Environ. Microbiol. 60, 4339–4344. LeChevallier, M.W., Lowry, C.D., Lee, R.G., 1990. Disinfecting biofilms in a model distribution system. J. Am. Water Works Assoc. 82 (7), 87–99. Levi, Y., 1997. Standard method to evaluate aquatic biofilms. In: International Conference on Biofilms in Aquatic systems. Warwick, UK. Lomander, A., Schreuders, P., Russek-Cohen, E., Ali, L., 2004. Evaluation of chlorines impact on biofilms on scratched stainless steel surfaces. Biores. Technol. 94, 275–283. Lu, W., Kie´ne´, L., Levi, Y., 1999. Chlorine demand of biofilms in water distribution systems. Water Res. 33 (3), 827–835. Morin, P., Camper, A.K., 1997. Attachment and fate of carbon fines in simulated drinking water distribution system biofilms. Water Res. 31, 399–410. Percival, S.L., Walker, J.T., Hunter, P.R., 2000. Microbiological aspects of biofilms and drinking water. CRC press, Washington, DC. Piriou, P., Dukan, S., Kiene, L., 1998. Modelling bacteriological water quality in drinking water distribution systems. Water Sci. Technol. 38 (8/9), 299–307. Thogersen, J., Dahi, E., 1996. Chlorine decay and bacterial inactivation kinetics in drinking water in the tropics. World J. Microbial. Biotechnol. 12 (5), 549–556. Tsai, Y.P., Pai, T.Y., Qiu, J.M., 2004. The impacts of the AOC concentration on biofilm formation under higher shear force condition. J. Biotechnol. 111 (2), 155–167. van der Wende, E., Characklis, W.G., Smith, D.B., 1989. Biofilms and bacterial drinking water quality. Water Res. 23, 1313–1322. Volk, C.J., LeChevallier, M.W., 1999. Impacts of the reduction of nutrient levels on bacterial water quality in distribution systems. Appl. Environ. Microbiol. 65 (11), 4957–4966.
Interaction of chlorine concentration and shear stress on chlorine consumption, biofilm growth rate and particle number Yung-Pin Tsai
*
Department of Civil Engineering, National Chi Nan University, 1, University Road, PuLi, NanTou 545, Taiwan, ROC Received 12 February 2005; received in revised form 9 August 2005; accepted 16 August 2005 Available online 30 September 2005
Abstract Eleven test runs (including two replicates) were carried out to explore the interaction of shear stress and chlorine concentration on the growth of heterotrophic microorganisms. Experimental results revealed that influent chlorine concentration and shear stress had no interaction on biofilm formation. Biofilm bacterial numbers decreased with the increase of influent chlorine concentration. Increasing the shear stress up to a specific level could significantly reduce the potential of biofilm formation. A strong interaction on bacterial quality or chlorine consumption rate of bulk water existed. With non-chlorinated and lower chlorinated conditions, the specific growth rate of biofilm increased with the increase of shear stress. However, an inverse relation occurred at higher chlorine conditions. No significant interaction of chlorine concentration and shear stress existed for particle numbers with 2–5, 5–15, 50–100 and >100 lm diameters. However, a significant interaction existed on particle numbers of 15–25 and 25–50 lm diameters. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Biofilm; Chlorine; Detachment; Flow velocity; Particle number; Shear stress
1. Introduction During the distribution of drinking water, bacterial regrowth may lead to a deterioration of bacterial water quality, increased corrosion, generation of bad tastes and odors, and proliferation of macroinvertebrates (Volk and LeChevallier, 1999). Piriou et al. (1998) indicated that the presence of a substantial and active attached biomass can protect pathogenic microorganisms, create anaerobic zones, lead to the formation of high biocorrosion zones and consume residual chlorine. It has been shown that chlorine has limited penetration into biofilms (De Beer et al., 1994), even though extremely high chlorine concentration (Lomander et al., 2004) and that the maintenance of an appropriate chlorine residual has little effect on biofilm growth (Chandy and Angles, 2001). On the other
*
Tel.: +886 939337630; fax: +886 492918386. E-mail address: [email protected]
0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.08.003
hand, the extracellular polymeric substances (EPS) of biofilm and assimilable organic carbon (AOC) in water can react with chlorine (Angles et al., 1999) and increase the chlorine demand in distribution systems. However, the interaction between other factors and chlorine concentration on biofilm formation has seldom been studied. Shear stresses (SS) have been shown to be instrumental in affecting microbial growth on pipe surfaces for potable water. It is generally considered that high shear stresses cause (1) a greater flux of nutrients to a pipe surface, (2) a greater transport of disinfectants, and (3) a greater shearing of biofilms (Percival et al., 2000). Experimental results have shown that the subsequent growth of the biolayer is mainly due to the activity of the microorganisms in the attached film, and not to the transport of new bacteria from the liquid to the biofilm surface (Bott and Miller, 1983). Thus, increasing shear stress helps to transport the nutrients of bulk water into the biofilm, causing the bacteria within the biofilms to multiply more quickly. Conversely, increasing shear stress is also helpful to the sloughing of biofilms and the penetration
Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
of disinfectants into biofilms, thus suppressing biofilms formation. These complex phenomena explain the contradictions and inconsistencies of previous studies. Most past studies focused on the impacts of AOC concentration on the growth of biofilm bacteria. However, the impacts of shear stress condition have seldom been explored, especially the effect of shear stress on the function of disinfectant to biofilm and bulk bacteria. In a drinking water distribution system, the flow velocities of pipes may be variable, which is assumed to affect the behavior of the disinfectant. However, the interaction between shear stress and disinfectant on biofilm formation and/or detachment is not yet well known. In this study, the experimental design focused on the comparison of the amount of biofilm bacteria, effluent bulk bacteria and particle numbers when shear stress and chlorine concentration were varied. Biofilm structures, which may be destroyed by chlorine, were examined by the particle numbers which were assumed as the detached biofilm pieces. The influences of the shear stress and chlorine concentration on the specific growth (detachment) rate of biofilm and chlorine consumption rate were also examined. 2. Methods 2.1. Experimental system An annular reactor system (AR system; BioSurfaces Technologies, Bozeman, MT) was used to monitor the interaction of shear stress and chlorine concentration on biofilm formation and chlorine demand. Fig. 1 shows the scheme of the AR system used in the study. The detailed characteristics of the AR system and its operational conditions can be found in Tsai et al. (2004). The rotating speeds
1.6 mL/min
rotational speed controller
rotational number counter
nutrient
slide sampling port slide
AR system Volume 1.15 L carbon water temperature controller
1.6 mL/min drainage
constant temperature water feed in
carbon and nutrient feed in
solid drum
slide surface
vertical view
Fig. 1. Scheme of the AR system used in the study.
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in this study were 27, 55 and 82 rpm (measured). Based on the experimental data from the original manufacturer of this AR system, the equivalent shear stresses acting on the slide surface by liquid were about 0.07, 0.17 and 0.29 N m2, respectively. At those shear stress conditions, the AR system was used to simulate the flow condition of a 600 mm diameter pipe whose flow velocities were about 18, 30 and 40 cm/s, respectively and whose Reynolds numbers were about 105,910, 176,225 and 235,518, respectively. 2.2. Synthetic influent water The carbon substrate consisted of equimolar concentrations on the basis of carbon of sodium acetate, sodium benzoate, propionaldehyde, parahydroxybenzoic acid and ethanol, as shown in Table 1. These chemicals were chosen because (1) they represent major classes of compounds, found in drinking water; (2) they were relatively non-reactive with chlorine (Camper, 1995), and (3) many treatment processes utilizing chemical coagulation are not effective in the removal of low molecular weight compounds, and only partially effective in removal of higher molecular weight humic substances. Thus, these compounds can make their way into water distribution systems and serve as biodegradable substrates for biofilm growth (Bell-Ajy et al., 2000). These compounds were assumed as the major AOC composition in the study. The synthetic influent water was prepared by sterilized ultra-pure water, and its equivalent AOC concentration was about 0.5 mg l1-C. The nitrate/phosphate compositions and concentrations were the same as used by LeChevallier et al. (1990), as shown in Table 2. Under these conditions, the nitrate/phosphate would not be a limiting factor of the growth of biofilm bacteria. All chemicals were supplied by Merck Chemical Co. and were filter sterilized prior to addition to the synthetic water. Sterilized ultrapure water was used for dilution (maxima, Elga). The sodium hypochlorite solution was prepared using commercial bleach (6%) diluted with sterilized ultra-pure water (maxima, Elga) to about 50 mg l1 and was automatically added to the synthetic influent water to give the studied free chlorine concentration in influent water. It was controlled by an on-line monitor instrument of free chlorine (RC300, Water Zone, Korea; ATC500S Detector, Japan). The residual chlorine concentration of the reactor effluent was manually measured daily. 2.3. Reactor operation At the beginning of each experimental run, the AR system (taken apart), slides, influent water and tanks, tubes and related accessories were cleaned thoroughly and autoclave sterilized. Then, a slide and influent water samples were randomly sampled to analyze the bacteria count to confirm that the experimental system was not polluted by bacteria. Finally, the AR system and its accessories were assembled carefully on the sterile table and sterile
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Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
Table 1 Compositions and concentrations of carbon source in influent water Chemicals
Molecular formula Molecular weight
Sodium acetate
Sodium benzoate
Propionaldehyde
Parahydroxybenzoic acid
Ethanol
CH3COONa 82
C7H5NaO2 144
CH3CH2CHO 58
C7H6O3 138
CH3CH2OH 64
328.6
383.3
Experimental conditions—individual concentration (lg l1) in synthetic influent water 1000 lg l1-C 683.3 342.9 322.2
Table 2 Compositions and concentrations of nitrate/phosphate in influent water Chemicals (conc. mg l1)
1
P (mg l -P) N (mg l1-N) S (mg l1-S) Na (mg l1-Na) Ca (mg l1-Ca) Fe (mg l1-Fe) Mg (mg l1-Mg) K (mg l1-K)
K2HPO4
KH2PO4
(NH4)2SO4
MgSO4 Æ 7H2O
NaCl
CaCl2
FeSO4
1.4
0.6
0.02
0.02
0.02
2
0.2
0.2494
0.1368 0.0042 0.0048
0.0026
0.0042 0.0079 0.7207 0.0737
0.0020 0.6276
0.1721
ultra-pure water was used as influent water to confirm that there was no leakage. The leakage test proceeded for at least one day. Before starting up an experimental run, a certain quantity of bacteria-containing water (about 103 HPC CFU/ml for each experiment) was added into the AR system for 1 h as the source of the bacterial inoculum of biofilm (slides). In order to exclude the impact of the transportation of new bacteria from the liquid to the slide surfaces and in order to minimize the bacterial number of influent water, influent water and storage tanks were renewed and autoclave sterilized every 2 days and the influent water tanks were covered tightly at all times, except during sampling periods. 2.4. Analyzing methods Samples of the influent and effluent water of the AR system were routinely collected every 2 days. Chlorine was neutralized by sodium thiosulfate (0.13%). Standard spread plate procedure was used to enumerate HPC bacteria, based on APHA (1998) 9215C, and R2A agar (DifcoTM) was used for incubation after appropriate dilution by sterile buffer. Plates were incubated for 48 ± 3 h at 35 ± 0.5 °C. TOC was measured by a TOC analyzer (model Phoenix 8000, Tekmar Dohrmann, USA) with the detection limit of 0.005 mg l1. Turbidity was measured by a Turbidity Meter (2100N, Hack, USA). Other physicochemical parameters were also analyzed based on APHA (1998). Attached microorganisms were released by sonication (Levi, 1997) in a water bath for two minutes (Branson Ultrasonics Co., USA). The number of particles was estimated by a Particle Counter (KL-11A, Rion, Japan).
Total 0.3862 0.0042 0.0496 0.0079 0.7207 0.0737 0.0020 0.7997
2.5. Quality control and data analysis All water samples for the examination of bacteria were diluted by sterile buffer to at least three different dilution ratios, and every diluted sample was analyzed in duplicate. Due to the considerable variance usually experienced in microbiological water quality data and particle numbers, a modified log transformation was used based on the formula log10(x + 1), where x was the measured value of biofilm, bulk HPC bacterial number or particle number. Data series obtained from the routinely sampling every two days were used as the data sources of the following statistical tests. To compare variation in treatments, analysis of variance (ANOVA) F-test was applied, with the bacterial counts, chlorine consumptions and particle numbers as the dependent variables, to test whether the shear stress, chlorine concentration and their interaction significantly influence the behaviors of biofilm formation, chlorine demand and particle variation. If significant factors (e.g. treatments) were found, then the least significant difference test (LSD) was applied to establish which particular levels of a factor were different. The paired sample t-test was also used to determine if means of interested variables were not statistically equal. All tests were performed at a significance level (p-value) of 0.05 using the standard statistical software (SPSS release 10.1.0, SPSS Inc.). Using HPC bacterial numbers of influent and effluent water in reactor and assuming the difference was the result of balanced growth and detachment of biofilm biomass, a specific growth rate (lb) was calculated for biofilm based on biofilm biomass within the reactor, as follows (Butterfield et al., 2002):
Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
F X X0 ¼ lb A Xb
ð1Þ
where F and A were volumetric flow rate (ml h1) and surface area available for biofilm development (cm2) of the reactor, respectively; X0 and X were influent and effluent bulk bacterial numbers (cfu ml1) of the reactor, respectively; and Xb was the biofilm bacterial number (cfu cm2) within reactor. 3. Results The experimental conditions and basic water quality data of all test runs are listed in Table 3. Influent water quality was good due to the use of ultra-pure water. Influent chlorine concentrations of RUNs 1–3 and RUNs 4–6 were automatically maintained at the levels of 0.26 ± 0.08 mg l1 and 0.57 ± 0.08 mg l1, respectively. 3.1. Examination of repeatability of experiments Bacterial number of influent water at each test run was between 0 and 0.766 log cfu ml1, which is a very low level
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of HPC bacteria. The means and standard deviations of biofilm and effluent bulk bacteria of all test runs at pseudo steady state are shown in Fig. 2. Results of the t-test showed that there was no significant difference between RUNs 0–2 and RUNs 0–2r for both biofilm and effluent bulk bacterial numbers (p values were 0.294 and 0.192, respectively). In addition, there was also no significant difference between RUNs 0–3 and RUNs 0–3r for both bacterial numbers (p values were 0.991 and 0.852, respectively). This meant that the test runs conducted in the study had a good repeatability. 3.2. Biofilm formation Raw data in Fig. 2(a) were used to examine the effects of shear stress, influent chlorine concentration and their interaction on biofilm bacterial counts by two-factor ANOVA statistical test. The error bars in Fig. 2 are large in chlorinated system due to the interference of chlorine. Results showed that influent chlorine concentration and shear stress had no interaction on biofilm formation (p = 0.108), although the former significantly influenced
Table 3 Water qualities data sets of experimental runs Run no.
Run conditions CL conc.a (mg l1) b
SS (N m2) c
TOC (mg l1)
Turbidity (NTU)
Conductivity (lS cm1)
pH
Influent
Effluent
Influent
Effluent
Influent
Effluent
Influent
Effluent
RUNs 0–1
ND
0.07
Ave. Stdev.d
0.270 0.075
0.251 0.033
3.150 0.212
3.527 0.233
6.245 0.163
5.963 0.286
0.640 0.051
0.737 0.084
RUNs 0–2
ND
0.17
Ave. Stdev.
0.270 0.075
0.281 0.062
3.150 0.212
3.418 0.244
6.245 0.163
6.159 0.385
0.640 0.051
0.757 0.018
RUNs 0–2re
ND
0.17
Ave. Stdev.
0.118 0.036
0.297 0.109
2.880 1.640
3.250 1.310
6.140 0.110
6.150 0.130
0.468 0.093
0.459 0.091
RUNs 0–3
ND
0.29
Ave. Stdev.
0.372 0.128
0.474 0.166
10.840 12.808
6.493 3.691
6.449 1.074
6.271 0.397
0.616 0.137
0.682 0.210
RUNs 0–3r
ND
0.29
Ave. Stdev.
0.185 0.093
0.381 0.029
3.750 3.590
4.790 4.050
6.620 0.220
6.380 0.610
0.467 0.047
NAf NA
RUN 1
0.26 ± 0.08
0.07
Ave. Stdev.
0.222 0.062
0.284 0.104
14.440 8.582
18.506 10.923
7.109 0.675
6.998 0.646
0.540 0.140
0.752 0.252
RUN 2
0.26 ± 0.08
0.17
Ave. Stdev.
0.222 0.062
0.474 0.565
14.440 8.582
21.201 13.930
7.109 0.675
5.900 2.149
0.540 0.140
0.787 0.394
RUN 3
0.26 ± 0.08
0.29
Ave. Stdev.
0.222 0.062
0.350 0.162
14.440 8.582
31.526 44.610
7.109 0.675
6.603 0.951
0.544 0.140
0.795 0.381
RUN 4
0.57 ± 0.08
0.07
Ave. Stdev.
0.218 0.087
0.365 0.150
35.611 52.722
37.665 50.302
6.392 0.490
6.254 0.470
0.484 0.096
0.614 0.209
RUN 5
0.57 ± 0.08
0.17
Ave. Stdev.
0.218 0.087
0.578 0.341
35.611 52.722
35.688 46.604
6.392 0.490
6.168 0.395
0.484 0.096
0.639 0.179
RUN 6
0.57 ± 0.08
0.29
Ave. Stdev.
0.218 0.087
1.352 1.995
35.611 52.722
37.418 47.870
6.392 0.490
5.984 0.373
0.484 0.096
0.632 0.208
a b c d e f
Free chlorine conc. of influent water. Not detected. Average. Standard deviation. Replicate run. Not available.
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Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
7
SS 0.07 SS 0.17 SS 0.29 SS 0.17 (replicate) SS 0.29 (replicate)
(a)
6 5 4
3.4. Chlorine consumption
2 1 0.00 0.26 0.57 Chlorine conc. of influent water (mg l-1) 8 7
SS 0.07 SS 0.17 SS 0.29 SS 0.17 (replicate) SS 0.29 (replicate)
(b)
6 5 4 3 2 1 0 0.00
0.26
0.57
Chlorine conc. of influent water (mg l -1) Fig. 2. Biofilm (a) and effluent bulk (b) bacteria numbers.
the potential of biofilm formation (p < 0.001). Biofilm bacterial numbers decreased with the increase of influent chlorine concentration. Statistical testing also showed that shear stress was an important factor affecting the formation of biofilm (p < 0.001) and that increasing the shear stress up to a specific level could significantly reduce the potential of biofilm formation. Biofilm counts were nearly equal between SS0.07 and 0.17 N m2 (3.54 and 3.34 log cfu cm2) (p = 0.485). But, biofilm count of SS0.29 N m2 (2.13 log cfu cm2) was significantly less than those of SS0.07 and 0.17 N m2 (p < 0.001). 3.3. Effluent bulk bacteria Analyzing data in Fig. 2(b) by ANOVA showed the existence of a strong interaction of chlorine and shear stress on bacterial quality of effluent water (p = 0.004). Shear stress at CL0 unchlorinated and CL0.26 chlorinated conditions did not significantly affect the effluent bulk bacterial number (p = 0.805 and 0.887, respectively). However, it had a significant impact at the condition of CL0.57 (p = 0.001). Bulk bacterial number at unchlorinated condition (CL0) was significantly larger than those at chlorinated conditions (CL0.26 and CL0.57).
Chlorine in the AR system was consumed mainly by the oxidations of bulk bacteria, biofilm bacteria and biofilm extracellular polymeric substances. Shear stress and concentration gradient provided the driving forces for the penetration of chlorine into inner parts of biofilm and then further affected the consumption of chlorine in bulk water. Data in Fig. 3 was used to examine (by one-way ANOVA) the effect of shear stress on residual chlorine concentration (RCC). At lower influent chlorine concentration (CL0.26), the effluent RCC at SS0.07 condition was significantly higher than those at SS0.17 and SS0.29 conditions (p = 0.013). In addition, shear stress had no significant impact on effluent RCC at the higher level of CL0.57 (p = 0.328). Two-factor ANOVA test showed the existence of a significant interaction (p = 0.030) between shear stress and influent chlorine concentration on chlorine consumption rate. Significant impact of shear stress on chlorine consumption rate occurred only at lower influent chlorine level (p = 0.019), and lower shear stress level (SS0.07) had a smaller consumption rate than higher levels (SS0.17 and SS0.29). However, shear stress had no significant impact on chlorine consumption rate while the initial chlorine concentration was further increased up to 0.57 mg l1. The lowest consumption rate (57.6%) occurred at the conditions of lowest shear stress and lowest initial chlorine concentration (SS0.07 and CL0.26) in this study. This indicates that chlorine consumption rate was substantially reduced if the driving forces provided by both shear stress and chlo-
100
0.7
90
0.6
80 0.5
70 60
0.4
CL 0.26 (effluent conc.) CL 0.57 (effluent conc.) CL 0.26 (consumption rate) CL 0.57 (consumption rate)
0.3
50 40 30
0.2
20 0.1
Chlorine consumption rate (%)
3
0
Effluent bulk bacteria (log CFU/ml)
At the conditions of CL0.57 and SS0.29, the effluent bulk HPC bacteria were not detected over the whole experimental period (52 days). This indicated that in addition to high chlorine concentration, it is still necessary to provide sufficient shear stress on the pipe wall if a substantial reduction of bulk bacteria is expected.
Effluent chlorine conc. (mg l-1)
Biofilm bacteria (log CFU/ cm2)
8
10
0.0
0 0.07
0.17 Shear stress (Nm-1)
0.29
Fig. 3. Experimental results of effluent chlorine concentration and chlorine consumption rate.
Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
rine concentration gradient were insufficient at the same time. Otherwise, shear stress would not significantly affect the chlorine consumption rate, until one of them (shear stress or gradient) was large enough. 3.5. Specific growth rate of biofilm (lb) Pseudo-steady-state data (influent, effluent and biofilm HPC bacterial numbers) of every test run were used to estimate the specific growth rate of biofilm (lb), basing on Eq. (1) and its assumptions, as shown in Fig. 4. For the non-chlorinated condition, lb was between 0.005 and 0.070 h1. With lower chlorine addition, lb was between 0.003 and 0.53 h1. The lb value increased with the increase of the shear stress for both conditions. The difference of lb value between non-chlorinated (CL0) and lower chlorine (CL0.26) conditions also increased with the increase of shear stress. On the contrary, the lb value significantly decreased with the increase of shear stress while the initial chlorine concentration was further increased to higher level (0.57 mg l1). At SS0.07 and CL0.57 conditions, lb was relatively high, about 0.986 h1; although by increasing shear stress up to 0.29 N m2, lb was substantially decreased to a negative value. This implied that the formation of biofilm was nearly restrained if both shear stress and chlorine concentration were high enough. This might be the reason why no effluent bulk bacteria were found at this condition over the whole experimental period (RUN 6).
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conditions re-distribute these three behaviors in the AR system. The major sources of bacteria within bulk water include (1) the bacteria originally surviving in bulk water, (2) the planktonic bacteria released from biofilm and (3) the bacteria hidden within the detached biofilm pieces, which are resuspended into bulk water. The third source might be the major reason for larger variations of bacterial quality for drinking water. However, it is difficult to distinguish the detached biofilm bacteria from total bulk bacteria. Consequently, particle number monitoring was used in this study to replace the understanding of the effects of shear stress and chlorine concentration on the biofilm detachment behavior. Fig. 5 shows the particle numbers of influent and effluent water in diameter of 2–100 lm, which was divided into six ranges (2–5, 5–15, 15–25, 25–50, 50–100 and >100 lm). Statistical method t-test was used to compare the means of particle numbers between influent and effluent water. At lower chlorine level (Fig. 5(a)), only particle number of effluent water in diameter of 15–25 lm was significantly larger than that of influent water. This might imply that most of detached biofilm were at this diameter range. At higher chlorine level (Fig. 5(b)), particle numbers of effluent water at all diameter ranges were not significantly larger than those of influent water at lowest shear stress level (0.07 N m2),
3.6. Characteristics of particle numbers in diameter between 2 and 100 lm It was assumed in this study that (1) planktonic bacteria and detached biofilm pieces existing within bulk water were in the particle form; (2) the diameters of most planktonic bacteria were less than 2 lm; (3) the diameters of most detached biofilm pieces were larger than 2 lm. Biofilm problems in drinking water system are the combinations of biofilm growth, biofilm detachment and bulk bacteria survival. Different shear stress and chlorine concentration
Specific growth rate (1/hr)
2.5 CL0 CL0.26 CL0.57
2.0 1.5 1.0 0.5 0.0 -0.5 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Shear stress (Nm-2) Fig. 4. Estimations of biofilm specific growth rate at pseudo-steady state.
Fig. 5. Particle numbers with diameter of 2–100 lm. (a) CL 0.26; (b) CL 0.57.
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but significances occurred at all diameter ranges while increasing shear stress over 0.17 N m2. This result implied that higher chlorine level produced a more fragile biofilm structure, so that the damaged biofilm could be detached more easily by the increase of shear stress. Statistical method analysis of covariance (ANCOVA) was further used to examine the effects of chlorine concentration, shear stress and their interaction on the variation of particle number. Particle number data of influent water were used as co-variate for ANCOVA analysis to exclude the influence of initial particles. No significant interaction between chlorine concentration and shear stress on particle numbers of 2–5 lm diameters (p = 0.103) was found. The main effect of chlorine concentration was significant (p < 0.001). The increased amount (the difference of influent and effluent water) of 2–5 lm particle numbers at 0.57 mg l1 chlorine was significantly larger than that at 0.26 mg l1. However, the shear stress did not significantly influence 2–5 lm particle numbers (p = 0.064). For 5– 15 lm particle numbers, there was also no interaction (p = 0.302) between these two factors (chlorine concentration and shear stress) but the main effects of both factors were individually significant (p < 0.001 and p = 0.002, respectively). The chlorine effect was same as on 2–5 lm particle. Lower shear stress (SS0.07) produced lower levels of 5–15 lm particles than did higher shear stress (SS0.17 and SS0.29). Interaction of these two factors occurred on 15–25 and 25–50 lm particles (p < 0.014 and p = 0.004, respectively). For 15–25 lm particles, chlorine effect was insignificant at lower shear stress condition (SS0.07), but significant while increasing shear stress to 0.17 or 0.29 N m2 (p = 0.004 and 0.001, respectively). Further more, shear stress did not show any significant influence on 15–25 lm particles at lower chlorine level, but significance occurred at lower chlorine level (p < 0.001). No interaction between these two factors existed on 50– 100 and >100 lm particles (p = 0.507 and 0.538, respectively). Shear stress did not show any significant main effect on particle numbers of these two ranges (p = 0.507 and 0.695, respectively). Only the main effect of chlorine concentration was significant for both ranges of particle (p < 0.001 and p = 0.013, respectively). The increased amount of 50–100 or >100 lm particle at higher chlorine condition was significantly larger than that at lower chlorine level. 4. Discussion This study showed that shear stress between 0.07 and 0.29 N m2 did not significantly influence the transmission of chlorine into biofilm, implying that the biofilm attached on pipe walls might be rigid. Either increasing chlorine concentration or shear stress level had a positive inhibition effect on biofilm formation. Thus, biofilm count was minimized under highest chlorine concentration and largest shear stress conditions. However, a significant interaction
on effluent bacterial water quality was found in this study, implying that the detached biofilm pieces might be more fragile than attached biofilm and the disinfection ability of chlorine for bulk water depends on the driving force provided by shear stress. To kill bacteria surviving within the detached biofilm pieces, a sufficient driving force might be necessary to transmit the chlorine more deeply into biofilm pieces. Butterfield et al. (2002) found that biofilm biomass appeared to be related to the chlorine demand, so high demand occurred with high biomass. Biofilms and the non-cell organic matter that accumulated on pipe walls have a high reducing potential, which leads to high chlorine consumption at the surface of the material (Thogersen and Dahi, 1996; Lu et al., 1999). Such oxidation by chlorine leads to the sloughing off of part of the biofilm (Characklis, 1990). However, no significant correlation existed between biofilm bacterial number and chlorine demand in the study. In this study, it was found that the presence of free chlorine resulted in less biofilm biomass, but in a higher specific growth rate of biofilm bacteria. This agreed with the results obtained by van der Wende et al. (1989) and Butterfield et al. (2002). The former determined the growth rate of biofilm to be greater in rotating annular reactors using 0.2 and 0.8 mg l1 residual chlorine compared to non-chlorinated systems. The latter showed that the specific growth rate was on the average five times greater for chlorinated biofilm compared to the control. However, observed yield values were less for chlorinated biofilm. The increase of the specific growth rate in this study did not indicate the increase of the net production for biofilm bacteria. On the contrary, the net production of biofilm bacteria reduced in the chlorinated system because more biofilm bacteria were killed by chlorine. It is believed that the chlorination products may be more available to microorganisms than non-chlorinated organic substances because lower molecular weight compounds require less cell energy for their uptake and utilization (Butterfield et al., 2002). That would be why lower chlorine concentration (0.26 mg l1) led to an increase in biofilm growth rates in this study, compared to non-chlorinated system. Furthermore, growth rate of biofilm bacteria also increased with the increase of shear stress, implying that the increase of shear stress could promote the penetration of nutrient into biofilm. However, increasing shear stress might also increase the penetration of chlorine into biofilm, killing more biofilm bacteria. Although the biofilm bacterial number in chlorinated system was significantly less than that in nonchlorinated system, the net specific growth rate of biofilm would be positive and increased if the increased amount of biofilm bacteria was larger than the amount of biofilm bacteria killed by chlorine, as the result of lower chlorine conditions in this study. An inverse relation between shear stress and growth rate of biofilm was found in this study, while the initial chlorine concentration was further increased from 0.26 to 0.57 mg l1. Specific biofilm growth rate was significantly
Y.-P. Tsai / Bioresource Technology 97 (2006) 1912–1919
reduced with the increase of shear stress. At this time, growth rate at lower shear stress (0.07 N m2) condition was relatively large. This implied that the increased amount of biofilm bacteria (due to the production of lower molecular weight organic substances) was far larger than the amount of being killed biofilm bacteria. Clearly, the driving force for penetration of chlorine was too low to kill much biofilm bacteria at lower shear stress condition. However, increasing shear stress up to 0.29 N m2, the specific growth rate of biofilm was substantially decreased to a negative value. This implied that higher chlorine concentration and higher driving force for penetration of chlorine resulted in the amount of being killed biofilm bacteria was inversely larger than the increased amount of biofilm bacteria by uptake of lower molecular weight organic substances. In this study, using the variations of particle number to examine the behaviors of biofilm detachment is a new technique that has not been previously used. The results have shown some important relations between the sloughing of biofilm and environmental conditions (shear stress and chlorine concentration). The data of particle numbers at different diameters are important, if they can give some important implications about biofilm behaviors, because the particle counter can be used as an on-line instrument and has a high potential for on-line monitoring of the biofouling problem of water supplies. Particle numbers are especially important to the fate of heterotrophic bacteria carried by particles that can penetrate the disinfection barrier, attach to or detach from pipe surface (Morin and Camper, 1997). This becomes critical if pathogens are involved. 5. Conclusions Influent chlorine concentration and shear stress had no interaction on biofilm formation. However, a strong interaction of chlorine and shear stress on bacterial quality of bulk water or on chlorine consumption rate existed. In addition to high chlorine concentration, it is still necessary to provide enough shear stress on the pipe wall if a substantial reduction of bulk bacteria is expected. Chlorine consumption rate was substantially reduced when the driving forces provided by both shear stress and chlorine concentration gradient were insufficient at the same time. For non-chlorinated or lower chlorine addition condition, lb was increased with the increase of shear stress, although the opposite result occurred at higher chlorine condition. No significant interaction of chlorine concentration and shear stress existed on particle numbers of 2–5, 5–15, 50–100 and >100 lm diameters. Higher chlorine concentrations produced significantly larger particle numbers than lower chlorine levels for all of the above ranges. A significant interaction of chlorine concentration and shear stress existed on particle numbers with 15–25 and 25–50 lm diameters.
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