IMPACT OF PARTICLE AGGREGATED MICROBES AND PARTICLE SCATTERING ON UV DISINFECTION Hadas Mamane-Gravetz and Karl G. Linden Duke University Department of Civil and Environmental Engineering Box 90287 Durham, NC 27708-0287 ABSTRACT There is a lack of fundamental information on the association of microorganisms with particulate matter within the aqueous system and the effects of such associations on UV disinfection efficacy. It has been recognized that non-aggregated dispersed microorganisms in wastewater are easier to disinfect than aggregated ones (Parker and Darby, 1995). Inactivation of the microbes aggregated within particles can therefore be hindered. Studies on the effect of turbidity on inactivation of microbes in the past have related mainly to the effect of disperse particles on UV irradiance, and have not directly investigated the impact of particle-microbe association on UV performance. This was the driving force for studying the discrepancy between the effects of clay particle turbidity as compared to actual clay-spore aggregates, on spore inactivation. Spores within spore-clay aggregates were more protected from UV irradiation compared to non-aggregated spores co-suspended with particles. Increasing the UV dose or fluence that penetrated through the aggregate resulted in decreasing viable spore count within the aggregate. In addition, advanced microscopy techniques and particle analysis allowed viewing the aggregate shape, size, size distribution and elemental make-up. Ultraviolet (UV) absorbance measurements are subject to significant error using a standard spectrophotometer when particles or aggregates that scatter light are present. Absorbance of highly turbid waters was measured using integrating sphere (IS) spectrophotometry to account for scattering of particles. Proper considering of scattering resulted in determining the correct dose for UV disinfection systems. The survival of microbes from UV with an aggregate depends on the particle characteristics. Spore clay aggregates protected spores embedded within them to a certain extent, however aggregates of spores with natural particles protected spores to a much greater extent. Extent of protection of microbes within an aggregate correlated to particle distribution of aggregates, where natural aggregates contained a larger mean particle size. KEYWORDS Bacillus; ultraviolet; particle-size; turbidity; scattering, integrating sphere, absorbance.
INTRODUCTION The impact of particles on ultraviolet (UV) disinfection has typically been studied by spiking particles that affect water turbidity and microorganisms into water and contacting them by stirring, essentially measuring the effect of particles on UV performance. Of ultimate concern, however, is the impact of particle-microbe association on disinfection performance. According to the USEPA UV disinfection guidance manual (US EPA, 2003) studies on the effect of turbidity on inactivation of microbes relates mainly to the scattering effect of disperse particles on UV irradiance, however these types of studies do not directly investigate the impact of particle-microbe association on UV. Therefore, studies in which microorganisms are spiked into a solution with particles or flocs not associated with each other, cannot substitute for studies that relate to microorganisms trapped within the floc network due to natural or induced flocculation. The use of UV irradiation for water disinfection purposes relies on accurate measurements of light absorption in water. Natural waters contain various substances that can affect its optical properties such as mineral particles, particulate organic matter, and microorganisms. UV absorbance measurements are subject to significant error using a standard spectrophotometer when particles or aggregates that scatter light are present. True UV absorbance for turbid waters should be measured using integrating sphere (IS) spectrophotometry that allows the collection of reflected and transmitted radiation simultaneously. This is especially important when the effects of scattering impact UV disinfection such as with the presence of aggregates. This study aimed to develop better understanding of the impact of particle-microbe aggregation on UV disinfection by (a) comparing the effect of dispersed spores, dispersed spores mixed with montmorillonite clay particles (non aggregated) and spore-clay aggregates in a jar test apparatus on UV disinfection performance; and (b) by relating the impact of the scattering properties of suspended particles, and spore-clay aggregates, on UV disinfection performance. Investigations were also performed with natural particles, and the effect of scattering on fluence determination was demonstrated experimentally. MATERIALS AND METHODS Clay and spore preparation Montmorillonite suspensions in the size range of 1 µm were used to represent inorganic natural particles in water sources. The clay type used was SWy-2 which is Na-rich Montmorillonite (Crook County, Wyoming, USA) obtained from the Clay Minerals Society (Source Clays Repository). Bacillus subtilis spores originated from ATCC 6633. Experimental design The makeup of simulated drinking water (SDW) was designed to model natural surface water, yet control the basic water quality parameters. The combined properties of the spore-clay aggregate in simulated waters were compared to the initial state of a mix of
dispersed spores and particles. Aggregation due to addition of chemical coagulants was measured in bench-scale studies using a Jar Test apparatus over a matrix of alum added at different concentrations. Types of waters used in this experiment were SDW that were spiked with spores and clay particles and natural waters (NW) that contained their original particles and therefore were spiked only with spores. The following procedure describes formation of spore-particle aggregates: Jar A no alum added to SDW or NW, rapid and slow mixing without settling. Suspension represents a suspended non-aggregated system and termed Sus. Jar B - alum added at optimum concentration to SDW or NW, rapid and slow mixing without settling to result in a suspension of aggregates. Suspension of aggregates was termed Agg. Each system was exposed to UV fluence of 0-60 mj/cm 2. The spores within the different aggregates were enumerated after physical separation techniques that allowed obtaining counts of all surviving spores within aggregates. Integrating sphere spectroscopy Absorbance measurements of water samples were performed with a UV-Vis dual beam spectrophotometer (Varian, Model Cary 100BIO, Victoria, Australia) equipped with 150 mm diameter integrating-sphere (IS) attachment (Labsphere Diffuse Reflectance accessory (DRA)-CA-30) and a center mount sample holder used to position the sample inside the IS. The integrating sphere is a highly efficient collector of scattered radiation due to its geometry and coating. The conventional absorbance mode, termed direct was performed with the same spectrophotometer without the integrating sphere attachment in place. True absorbance measurements measured with the same spectrophotometer equipped with the integrating sphere attachment, will be termed IS. Particle size analysis A Multisizer 3 (Beckman Coulter, Miami, FL) was used to size particles and yield a particle size distribution. Distributions of counts, count per ml (µm/ml) or volume per ml (µm 3 /ml) of suspensions and aggregates were obtained. RESULTS AND DISCUSSION Impact of spores suspended and aggregated with clay particles on UV Two different treatments were examined with an identical concentration of spores and clay particles. The impact of suspended spores with clay particles (without alum, constant mixing) and spore-clay aggregate (constant mixing) on UV fluence-response curves of
spores, is illustrated in Figure 1. A fluence of 30 mj/cm 2 resulted in log 10 inactivation of 1.24 and 1.5 for aggregated and suspended spores respectively. The inactivation rate constants (slopes) for suspended and aggregated spores (with clay) were 617 and 579 cm 2 /mj respectively. The difference between the inactivation rate constants for the suspended and aggregated spores when clay particles are added to the system is highly significant. UV inactivation of aggregated spores or aggregates of spores associated with clay particles indicate that spores in an aggregate are protected from UV. The difference in the counts between alum-aggregated spores and dispersed spores indicates the significance of particle-associated spores in the water samples presumably being shielded from UV light. Based on mathematical analysis, approximately 50% of spores in the aggregates are protected from UV light across UV fluences of 20 to 60 mj/cm 2. Inactivation of spores in an aggregate will depend on location of spores in the aggregate and the existence of zones that allow a pathway for penetration of UV light. Figure 1 - UV inactivation of suspended vs. aggregated system, with clay log(n0/nd) 0.5 1.5 2.0 2.5 3.0 3.5 5 NTU, Sus. 5 NTU, Agg. 4.0 10 20 30 40 50 60 Fluence, mj/cm 2 log(n 0/Nd) 0 1 2 3 0 10 20 30 40 50 60 Fluence, mj/cm 2
Homogeneous clay particles were suspended in deionized water to investigate the effect of 254 nm absorbance measurements conducted by mounting an integrated spheres setup as compared to using a regular spectrophotometer as illustrated in Figure 2. Low pressure UV lamps emit 254 nm as typically used in water disinfection of microorganisms. Above values of approximately 3 NTU a difference is observed, with higher values of absorbance with the use of the regular spectrophotometer utilized in typical laboratories. Research by Christensen and Linden (2003), emphasized the difference in the average irradiance as measured from IS absorbance compared to irradiance from direct absorbance and concluded that direct absorbance measurements underestimated the average irradiance and subsequent UV fluence by 3.5% at 5 NTU for 1 cm depth of raw water. In the current research the direct measurement underestimated the average irradiance by 5% for all synthetic waters with 5 NTU clay and spore particles. Figure 2 - Impact of turbidity on direct and IS absorbance 100 Absorbance, 254 nm 0.8 0.6 0.4 0.2 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 11 % Transmittance, 254 nm Turbidity of Clay, NTU Direct abs IS abs Direct UVT IS UVT IS integrating sphere. UVT 254 nm transmittance measurements. Impact of spores suspended with 5 NTU particles on direct and IS absorbance throughout a range of wavelengths is illustrated in Figure 3. Absorbance measurements of water suspended with spores (without clay particles) demonstrate that the clay particles are the cause for scattering in simulated waters. It is also evident that the difference in spectral absorbance between direct and IS absorbance scan is due to light scattering not accounted by direct measurements.
Figure 3 - Impact of wavelength on direct and IS absorbance Absorbance 0.8 0.6 0.4 Direct absorbance, spore (without clay) IS absorbance, 5 NTU clay + spores Direct absorbance, 5 NTU clay + spores 0.2 200 225 250 275 300 325 350 Wavelength, nm Figure 4 illustrates the difference in the UV response of suspended spores without particles and spores suspended with 5 NTU clay particles (without alum addition). The average irradiance or fluence rate was calculated considering absorbance measured by direct method or by IS method. The fluence-response curve is a function of fluence rate and differed substantially between different scenarios. Initially a lag in inactivation exists at a low UV fluence up to 10 mj/cm 2, which was similar for all different scenarios. The difference in inactivation of spores suspended with clay particles increased with UV fluence from 10 to 60 mj/cm 2, starting at 0.1 log difference at 20 mj/cm 2 up to 0.5 log difference at 60 mj/cm 2. Fluence determined with the direct absorbance measurement is conservative as it provides additional inactivation compared to the IS technique at the same apparent fluence. Thus, because of the falsely high absorbance value in the presence of particles by the direct measurement technique, the delivered fluence is too high for inactivation when clay particles at 5 NTU are suspended with spores. Interestingly, the fluence response of suspended spores (without clay) as measured by direct technique, compared to the (true) fluence response of spores suspended with clay particles as measured by IS technique, 5 NTU(IS) are not statistically significant, indicating that there is no difference between the inactivation rate constants for the fluence calculated using direct measurement of spores in the absence of clay particles and IS measurements of spores with clay particles. This findings provides further evidence
that scattering of particles is correctly taken into consideration when using IS technique for fluence determination of scattering particles. Figure 4 - Effect of absorbance measurement on spore UV inactivation Log(N0/Nd)) 0.5 1.5 2.0 2.5 3.0 3.5 Direct: y=58-0.472 Clay (Direct): y=67-0.518 Clay (IS): y=59-0.487 0 10 20 30 40 50 60 Fluence mj/cm 2 Direct 5 NTU Clay, Direct 5 NTU Clay, IS The impact of natural particles on absorbance and UV inactivation of spores The impact of 6.3 NTU natural particles suspended or aggregated with spores at optimum alum concentration on absorbance, and UV inactivation at 40 mj/cm 2, determined using direct and IS absorbance is illustrated in Figure 5. Results show that absorbance measurements are higher using direct compared to IS spectroscopy both for aggregates and suspended spores. In natural waters with natural particles, inactivation of aggregates of spores as measured by direct and IS absorbance at 40 mj/cm 2 is 0.63 log and 0.69 log respectively. With natural waters no difference in spore inactivation was apparent with aggregated systems using direct or IS absorbance for fluence calculations. With suspended systems, no difference in spore inactivation was evident with natural particles using direct or IS absorbance for fluence calculations. In natural systems microbes in coagulated aggregates are protected from UV light as indicated by lower inactivation compared to suspended systems. In SDW the difference in log inactivation between the suspended and aggregated system is 0.2 to 0.4 log
inactivation whereas this difference was about 1.4 log in natural waters. Therefore, aggregates formed with natural particles were much more effective at protecting the spores from UV irradiation as compared to aggregates formed with added clay particles. Figure 5 - UV inactivation in natural water (NW) samples, 6.3 NTU log(n0/nd) at 40 mj/cm 2 2.5 2.0 1.5 0.5 2.14 2.12 dp=0.91 log(no/nd) abs. 254 nm 0.63 0.69 dp = 4.89 0.35 0.30 0.25 0.20 0.15 0.10 5 Absorbance, 254 nm sus D sus IS agg D agg IS 0 Nature of particles as organic vs. inorganic of natural and simulated waters respectively and the particle distribution may affect the extent of protection of spores in an aggregate as illustrated in figure 6. Relative particle count per ml of aggregates and suspensions was divided into different size bands from 0.5-2.5, 2.5-4.5, 4.5-6.5, 6.5-8.5 and 8.5-10.5 µm. For all suspended particles the fraction between 0.5-2.5 µm is the most dominant fraction. For the aggregated system, dominant particle fraction is between 2.5-4.5 µm and 0.5-2.5 µm for natural and simulated waters respectively. The aggregated system shows a wider distribution compared to the suspended system. In addition aggregates of natural water contain particles in a larger size band (8.5-10.5 µm) compared to the spore-clay (SC) system in simulated DW, with largest size band between 4.5-6.5 µm. This difference likely has an effect on protection of spores in aggregates as the largest aggregates (in natural water) could protect spores more efficiently compared to smaller ones.
Figure 6 Particle size change following aggregation Normalized count per ml 1.2 0.8 0.6 0.4 0.2 0.5 2.5 µ 2.5 4.5 µ 4.5 6.5 µ 6.5 8.5 µ 8.5 10.5 µ Natural sus Natural agg Simulated sus Simulated agg Natural environments consist of many particles that each scatter light differently as measured by the integrating sphere. Therefore not only the chemical nature of the particle affects the scattered field but also the particle concentration, which eventually impacts the UV fluence and the extent to which spores are protected from UV inactivation. The extent of scattering depends on geometrical factors as scattering direction, size and shape of particles, in addition to particle chemical composition and concentration Practical considerations for water treatment plants The outcomes from this research can be applied to actual practices of UV disinfection of unfiltered waters; however it is unlikely that water utilities would routinely employ sophisticated UV absorbance measurement and particle distribution analysis to try and carefully characterize the physical state of microorganisms and scattering ability of their waters. Observations in this study can contribute to actual UV disinfection practices if unfiltered water samples or wastewater effluents at the inlet of the UV reactor were periodical analyzed at specialized labs that have an integrating sphere to determine if scattering is an issue leading to UV overdosing. To evaluate aggregation, various parameters should be measured, such as mean particle size and volume, particle size distribution (PSD) and filtered PSD. Dividing particle counts to fraction of counts below and above certain value (probably 2-3 µm), will differentiate between dispersed and aggregated particles. Further distinction into different size bands can help define the extent which microbes may be protected in an aggregate. Light scattering of dispersed particles and aggregates will lead to overdosing the bulk water. Perhaps it is preferable to overdose water with UV if components in water actually do scatter light. Delivering an increased UV fluence due to light scattering of particles is an advantage for public health and would provide a safety net in terms of
disinfection specifically because light scattering could indicate microbe protection due to the presence of particles. Unfiltered systems are those that meet the filtration avoidance criteria of the US EPA Surface Water Treatment Rule (SWTR). The SWTR allows turbidities of up to 5 NTU for unfiltered supplies prior to disinfection. Since unfiltered supplies do not have the treatment barrier of filtration, the bias of the UV dose measurement will provide some safety margin, offsetting the potential risk of particles interfering with disinfection in these unfiltered supplies or for wastewater effluents. REFERENCES Christensen, J.; Linden, K.G. (2003). How particles affect UV light in the UV disinfection of unfiltered drinking water. J. Am. Water Works Assoc., 95(4), 179. Parker, J.A.; Darby, J.L. (1995). Particle-associated coliform in secondary effluents: shielding from ultraviolet light disinfection. Water Environ. Res., 67(7), 1065. U.S. Environmental Protection Agency (U.S. EPA). (2003). Ultraviolet disinfection guidance manual. EPA 815-D-03-007, Office of Water, Washington, D.C.