Date

2015

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Environmental Engineering

First Adviser

Jellison, Kristen L.

Other advisers/committee members

SenGupta, Arup K.; Brown, Derick; Fox, John; Jedlicka, Sabrina; Hargreaves, Bruce R.

Abstract

Biofilms are communities of microorganisms that form on a solid surface. It has been shown previously that Cryptosporidium parvum is able to accumulate and survive in association with bacterial biofilms in aquatic environments. The long-term survival and sudden release of pathogenic microorganisms due to biofilm sloughing may lead to waterborne disease transmission. The fact that biofilms can immobilize C. parvum oocysts from the water column poses fundamental questions regarding interactions of oocysts with biofilm surfaces. Consequently, the main objectives of this study were to: 1) investigate the mechanisms that govern C. parvum oocyst attachment to environmental biofilms; 2) examine the role of light exposure, water chemistry, and wall shear stress on oocyst attachment to biofilms; and 3) identify C. parvum oocysts by scanning electron microscopy (SEM) using an immunogold labeling technique.Mechanisms of C. parvum oocyst attachment to environmental biofilms grown on polycarbonate coupon surfaces were investigated. Biofilms were formed in rotating annular bioreactors using water and biomaterials collected from different streams in northeastern Pennsylvania with different water chemistries under conditions of dark or light exposure. Biofilm physical structure was characterized by optical microscopy for mean biofilm thickness and roughness, and by SEM for characterization of biofilm architecture. A calcium-mediated psudo-second-order kinetic model for C. parvum oocyst deposition was developed to derive kinetic parameters, such as total number of oocysts retained on biofilm surfaces at equilibrium and the initial deposition rate constant, based on oocyst deposition efficiency. The pseudo-second-order kinetic model for oocyst deposition enabled correlation of water chemistry, hydrodynamic shear stress, and biofilm properties with the derived kinetic parameters. On mature environmental biofilm surfaces (150 days old), the adhesion of C. parvum oocysts was found to be independent of biofilm characteristics (e.g., dry mass, organic mass, protein content, mean biofilm thickness and roughness), solution chemistry (e.g., conductivity, alkalinity and hardness), and growth conditions (e.g., light or dark). However, in this study, mature biofilms developed in bioreactors with the same hydrodynamic shear stress but with different environmental waters had similar surface roughness, suggesting that the range of the roughness coefficient in our bioreactors may have been too narrow to observe any impacts on oocyst attachment. Calcium was observed to play a significant role in oocyst attachment to biofilms.In addition to water chemistry and growth conditions, the impact of wall shear stress on oocyst deposition was also investigated. Dense, compact and smooth biofilms formed under high wall shear stress, whereas fluffy, loose and rough biofilms developed under low wall shear stress. The initial attachment rate constant of C. parvum oocysts on the biofilm surface was found to increase as a function of wall shear stress, presumably due to enhanced mass transport between oocyst and surface; however, the value dropped when the wall shear exceeded a certain limit. The total number of oocysts retained on the biofilms decreased with increasing wall shear stress, possibly due to the smooth biofilm surface and increased drag force acting on the biofilm surface discouraging oocysts from adhering. Biomaterials collected from different seasons (i.e., summer and winter) resulted in different biofilms with respect to roughness, mean thickness and biomass, however these differences were not observed to impact the effect of wall shear stress on oocyst deposition kinetics, suggesting that shear stress plays a critical role in oocyst deposition.Progress was made on the development of an immunogold labeling technique to identify C. parvum oocysts in environmental biofilms by SEM. The performance of different fixatives (i.e., formalin, a mixture of paraformaldehyde and glutaraldehyde, and glutaraldehyde), two drying approaches (hexamethyldisilazane (HMDS) and critical point drying (CPD)), four coating materials (iridium, gold, gold palladium, and carbon), and four scanning electron microscopes (Environmental SEM, Hitachi 4300 4300 SEM, Zeiss 1550 1550 SEM, and FIB-SEM) were assessed to find the optimum settings for C. parvum oocyst and gold marker visualization. In our study, fixation by glutaraldehyde, dehydration by HMDS, and then coating with iridium was found to effectively preserve the specimen architecture. The best combination of coating, resolution and signal-to-noise ratio for a C. parvum oocyst positive specimen in our study was 5 kV beam voltage plus -1.5 kV beam receleration at a working distance of less than 3 nm with 1.7 nm iridium coating imaged with the FIB-SEM.The overall goal of this study is an increased understanding of oocyst and biofilm interaction mechanisms. Understanding the mechanisms of oocyst attachment to biofilm surfaces, and how environmental conditions and biofilm surface topography influence oocyst-surface interactions, is critical to the development of anti-adhesive surfaces or materials and could enable enhanced removal of oocysts from surface water. Kinetic profiles obtained from this research will also enable more accurate predictions of C. parvum oocyst fate and transport in the environment. Additionally, the information obtained from this work may be used to guide the engineering of biomimetic surfaces for oocyst detection in the environment.Further research will focus on manufacturing a standardized biomimetic surface which can be used to detect oocysts quantitatively. The impact of factors like temperature and pH, as well as biofilm roughness and composition, will also be further investigated to confirm the oocyst adhesion kinetics obtained from this study. Viability of attached oocysts over time will also be considered in future work.

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