Controlled experiments were conducted to examine the validity of the linear partition model for faecal coliform (FC) attachment to suspended sediments and assess the impact of sediment concentration on the attachment. Sediments were collected from a river in Beijing, China and the suspended particles <62 μm were separated out and mixed with FC suspensions. The experimental mass specific concentration of attached FC linearly increased with the free-floating FC concentration, with the partition coefficient for four different sediment concentrations ranging from 0.2286 to 0.2695 L/g. Actually, the results can be well described using a single partition coefficient of 0.2565 L/g. The rate of sediment particle surface covered by attached FC was in the range of 0.32 × 10−5–0.58 × 10−5, and the relatively low rate gave a possible explanation of the linear relationship. The experimental fraction of attached FC significantly increased with increasing sediment concentration, ranging from 7.5 to 54.2%, and this was well explained by the linear partition model. These results proved that the linear partition model was valid in describing FC attachment to suspended sediments in surface waters and the attached fraction was sediment concentration dependent, while the impact of suspended sediment concentration on the partition coefficient was insignificant.
Microbial pathogenic pollution of surface waters is a major concern for public health worldwide. Drinking, recreational and irrigation water contaminated by pathogens will result in waterborne disease epidemics. Difficulties and expenses involved in the testing for specific pathogens have led to the use of indicator bacteria to monitor pathogenic pollution of water and other kinds of environment. Escherichia coli is one of the most commonly used indicator bacteria to evaluate water quality and manage water resources (Carson et al. 2001). According to WHO guidelines (2011), faecal coliforms (FC) can be an acceptable substitute for E. coli and therefore are still used by many countries, e.g. China.
Bacterial indicators existing in surface waters are either in free-floating form or attached to suspended sediments. Settling of sediment-attached faecal bacteria would improve microbial water quality due to the higher settling rate (Matson et al. 1978). On the other hand, bottom sediment harbours much higher faecal bacteria concentration due to its hospitable environment for bacterial survival and poses a major risk for quality of the overlying water (Koirala et al. 2007). Therefore, sediment-bacteria associations must be incorporated into microbial water quality models to accurately obtain fate and transport of attached bacteria for better management of water resource. The attached fraction, defined as the ratio of attached FC concentration to total FC concentration in the water column, was usually employed to quantitatively represent the attachment in modelling, but findings based on field sampling in surface waters demonstrated that this fraction was highly variable. A large number of researchers found that only 15–38% of faecal bacteria existed in the attached form (Fries et al. 2006; Cizek et al. 2008). However, some other researchers have reported that as high as 90–100% of faecal bacteria were attached to suspended sediments (Auer & Niehaus 1993; Mahler et al. 2000).
Sediment concentration may be an important factor responsible for the highly variable attached fraction, but it has not yet been fully understood and characterized (Pachepsky & Shelton 2011). While Sayler et al. (1975) did not find a significant correlation between the concentration of suspended sediment and the percentage of attached FC, George et al. (2004) reported that the percentage of FC associated with suspended particles >5 μm increased linearly with the suspended solid concentration. Since the attached fraction was highly variable and its values may be dependent on sediment concentration, which was also highly variable in surface waters (Chien & Wan 2003), it was inappropriate to assume that the attached fraction was constant in many modelling efforts (Pachepsky & Shelton 2011).
To address these problems, controlled laboratory experiments were conducted to: (1) examine the validity of the linear model as in Equation (1) and evaluate sediment concentration impacts on the fitted partition coefficients; and (2) further identify the sediment concentration impacts on the attached fraction and attempt to explain these impacts using the linear partition model and fitted partition coefficients.
MATERIALS AND METHODS
The sediment samples were taken from the Yongding River and the sampling site was in the suburbs of Beijing, China. Then, they were soaked in distilled water for several days followed by drying up in an oven at 45 °C. Afterwards, the size distribution was analysed by a laser particle analyser. The sediment fraction that passed a 0.0625 mm sieve was regarded as the suspended sediments (<62 μm) and used in the experiments. The organic matter, cation exchange capacity, zeta potential, specific gravity, Brunauer–Emmett–Teller (BET) specific surface area and average pore size of the suspended sediments were, respectively, 2.5%, 7.61 × 10−3 cmol/g, −44.27 mV, 2.650, 4,700 dm2/g and 9.615 nm. The mineral composition of the suspended sediments determined by X-ray diffraction was quartz, feldspar, chlorite and illite.
Bacteria and bacteria-sediment-water mixture
The FC bacteria in the experiments were isolated from local waste water. A loop of FC was transferred to two 1,000 mL Erlenmeyer flasks, each containing 200 mL of beef extract peptone. The flasks were incubated at 44.5 °C and shaken at 150 rpm for 24 h. Then, the bacteria were harvested by centrifugation at 12,000 r/min for 15 minutes and washed twice with sterile distilled water. The harvested bacteria were mixed with 500 mL sterile distilled water to obtain FC suspension.
Four sediment concentrations S1, S2, S3 and S4 were achieved by adding, respectively, 0.05, 0.1, 0.15 and 0.2 g of dried sediments to 100 mL FC suspension in a 300 mL Erlenmeyer flask, and 12 initial FC concentrations ranging from 101 to 106 cfu/L were used in the experiments. The pH value of mixture in the flask was 7.0 ± 0.5. Then, the flask was stirred at 280 rpm on a rotary shaker at 25 °C for 1 h. An initial experiment suggested that 1 h was long enough for FC to attach to sediments and another indicated that the impacts of die-off/regrowth at 25 °C were negligible. Each experiment was conducted in triplicate.
Separation of unattached/attached bacteria
The filtration technique used by many researchers (Sayler et al. 1975; Matson et al. 1978; Soupir et al. 2010) was employed to separate the free-floating and attached bacteria. Ideally, the selected filter membrane should allow free-floating FC to pass and retain almost all the sediment particles. The mass percentage of particles <5 μm was only about 0.75% and negligible in the experiment. So, a pore size of 5 μm was selected, and the FC passing the 5 μm pore were regarded as the free-floating group. In each experiment 100 mL of the mixture was poured through a filter in a vacuum filtration apparatus. The filtrate was collected to analyse the free-floating FC bacteria number Nf using the membrane filtering (MF) technique and the filter was removed from the filtration apparatus to further analyse the number of attached bacteria.
An initial experiment was conducted to evaluate the impact of clogging of filters on classification of attached and unattached FC. A series of 100 mL bacteria-sediment-water mixtures were analysed, each sample being divided into two parts of 5 mL and 95 mL, respectively, and then filtered. Since both the number of bacteria cells and sediment particles in the 5 mL parts were much lower than in the 95 mL parts, there was little possibility of clogging for the 5 mL parts. The free-floating FC concentration obtained from the 5 mL parts was found essentially the same as that from the 95 mL parts (data not shown). This indicated that the incorrect classification of attached and unattached FC caused by clogging of filters was insignificant in the experiments.
Enumeration of attached bacteria
A physical procedure was designed in this study to enumerate the attached bacteria. After separation of unattached and attached bacteria, the filter retaining the sediments with FC attached was placed in a beaker containing 100 mL of sterile water and the beaker was agitated on a motor stirrer for 5 min to wash the attached FC off into the water. Then, a new filter was placed in the filtration apparatus and the mixture in the beaker was filtered again, and the filtrate was collected to analyse the attached FC number Ns1 using the MF technique. The above steps were repeated for another two times to analyse the attached FC numbers Ns2 and Ns3. The FC numbers obtained from the three repetitions were added up to get the total attached FC number Ns. An initial experiment indicated that about 76.55%, 22.05% and 1.40% of the attached bacteria were, respectively, recovered from the first, second and third repetition. Therefore, three repetitions were sufficient to obtain the attached FC number.
RESULTS AND DISCUSSION
Linear partition model validation and sediment concentration effects
The measured mass specific concentration of the attached bacteria P was plotted against the free-floating bacteria concentration Cw in Figure 1. An obvious trend that P linearly increased with Cw can be observed; the dots according to each sediment concentration were fitted using a straight line and the values of the coefficient of determination R2 were 0.9939, 0.9862, 0.9916 and 0.9666, respectively. The high values of R2 suggested that the attachment of faecal bacteria to suspended sediments can be described using the linear partition model as in Equation (1). The fitted values of partition coefficient k were 0.2695 L/g, 0.2471 L/g, 0.2425 L/g and 0.2286 L/g for the sediment concentrations S1, S2, S3 and S4, respectively. Furthermore, the dots corresponding to all the four sediment concentrations can also be fitted by a single straight line, and the value of R2 was as high as 0.9863. The fitted value of k was 0.2565 L/g, and it was only 7.4% smaller than the fitted value for S1 and, respectively, 3.4%, 5.7%, and 11.5% larger than the values for S2, S3 and S4. The relatively small difference between the overall fitted value of k and the values for S1, S2, S3 or S4 indicated that the impact of sediment concentration on the partition coefficient k was insignificant. The partition coefficients from the investigations for bacterial attachment to soil particles ranged from 0.001 to 0.1 L/g (Bai & Lung 2005). The partition coefficient in the water column was believed to be much higher than that in groundwater according to the study of Mahler et al. (2000). The measured partition coefficients in the present experiments were close to the upper limiting value in groundwater.
Theoretical analysis on validity of linear partition model
The linear partition model was believed to be only applicable when the surface coverage rate (SCR) of the particles was relatively low, under which the saturation adsorption was far from reached and P would linearly increase with the increase of Cw (Stumm 1992). SCR, defined as the ratio of the area covered by bacterial cells Ac to the total surface area Aa available for attachment of the particles, has been employed by researchers to study the adsorption state of particles in colloid and interface chemistry.
The pores on the sediment surface will increase the specific surface area by orders of magnitude, and the BET specific surface area includes the surface area of the pores. The averaged pore size of the suspended sediments in the experiments was 9.615 nm, which was much smaller than the cell size of faecal bacteria. This meant that faecal bacteria had little chance to enter the pores on the surface of the suspended sediments. Thus, instead of using the BET specific surface area to calculate the total surface area of the particles available for attachment Aa and then calculating SCR, we determined the total surface area by classifying the particles into 11 fractions as in Table 1 and assuming all the particles were spherical and of the same density of 2.65 g/cm3. The assumptions and detailed calculating procedures can be found in Oliver et al. (2007). In the experiments, the calculated values of Aa were 4.0383 dm2, 8.0766 dm2, 12.1149 dm2 and 16.1532 dm2 for S1, S2, S3 and S4, respectively, and the values were linearly proportional to the sediment concentration S. We can speculate that the actual values of Aa should be higher than these estimated values, for the irregularity of surface would increase the specific surface area of suspended sediments (Hunter & Liss 1982).
|Size fraction (equivalent spherical diameter D)/μm .||Median diameter used in calculations Dm/μm .||Mass percentage fi/% .|
|Size fraction (equivalent spherical diameter D)/μm .||Median diameter used in calculations Dm/μm .||Mass percentage fi/% .|
Based on the values of Acmax and Aa, the calculated values of SCR were 0.58 × 10−5, 0.50 × 10−5, 0.44 × 10−5 and 0.32 × 10−5 for S1, S2, S3 and S4, respectively, and they significantly decreased with increasing S. It can be regarded as reaching saturation adsorption if the value of SCR is equal to unity. The calculated values of SCR in the experiments were five orders of magnitude lower than unity, and this indicated that saturation adsorption of the suspended sediments was far from reached. Furthermore, the value of SCR might approach unity if the free bacteria concentration in the water column was five orders of magnitude higher. In that case, the bacteria concentration should be at least as high as 1010–1011 cfu/L, and this extremely high concentration was very rarely seen in surface waters (Bai & Lung 2005; Fries et al. 2006; Cizek et al. 2008). Therefore, the linear partition model was justified to describe the attachment of FC to suspended sediments in surface waters.
Impact of sediment and bacteria concentration on attached fraction
The averaged values of fp were 15.00%, 23.46%, 27.65% and 33.09% for S1, S2, S3 and S4, respectively. The averaged values of fp significantly increased with the increase of sediment concentration in the experiments (R2 = 0.6291, n = 144, p < 0.001). Although the values of initial bacteria concentration CT were in geometric progression growth, fp showed no monotone increasing or decreasing trend. The averaged values of fp were 28.15%, 28.65%, 24.53%, 28.8%, 28.57%, 23.00%, 23.73%, 20.86%, 20.02%, 18.57%, 21.06% and 23.66% for C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12, respectively. This indicated that the impact of initial bacteria concentration CT on the attachment fraction was insignificant (R2 = −0.0607, n = 144, p > 0.05).
The positive correlation between attached fraction and sediment concentration has been reported in the literature. George et al. (2004) presented that the attached FC fraction would linearly increase from 0 to 80% with the suspended sediment content increasing from zero to 80 mg/L. Characklis et al. (2005) reported that there was seemingly a positive relationship between the fraction of attached FC and particle concentration. Particle properties, including organic matter, surface charge and specific surface area, were acknowledged to affect the partition coefficient of bacterial attachment to particles (Guber et al. 2005). These properties would not be affected by the sediment concentration, since the increase of sediment concentration only increased the number of particles in the suspension. Therefore, it was reasonable that the partition coefficients in the experiments remained fairly constant with the increase of sediment concentration. However, the increase of sediment concentration significantly increased the surface area available for the attachment, and this explained why the total number of attached FC and the correspondent attached fraction were increased.
Accurate physical representations of sediment-bacteria association are necessary for microbial water quality modelling. The study provided experimental evidence for the validity of the linear partition model to describe the attachment of FC to suspended sediments in surface waters. The theoretical analysis based on the surface coverage rate gave a possible explanation for the validity. The impacts of sediment concentration on the partition coefficient were insignificant, and this indicated that sediment concentration had little impact on mass specific attached concentration of FC. The fraction of attached FC significantly increased with the increase of the sediment concentration. The variation of the attached fraction with sediment concentration was well explained by the linear partition model and fitted partition coefficient. This implicated that the linear partition model, resulting in a sediment concentration dependent attached fraction, was better than a sediment concentration independent attached fraction to represent the sediment-bacteria attachment in modelling microbial water quality.
The financial support from Tsinghua University Initiative Scientific Research Program (No. 20121088082) and the National Natural Science Foundation of China (No. 51039002) is gratefully acknowledged.