Continuous-flow high temperature and pressure water sterilizer

The human body contains as much as 75% water, and without it, can only survive for about 3 days, making clean water the most important factor for global public health. In 2010, almost 1 billion people had to rely on unprotected wells, lakes, or rivers as water sources, which are susceptible to contamination by raw sewage, undertreated wastewater discharges, and urban/agricultural runoff (UNICEF, W. U. 2013). These people are at high risk from waterborne, disease-causing microorganisms, including bacteria, viruses, and protozoa. Indeed, waterborne pathogens cause billions of diarrhea cases annually, of which more than 2 million result in death (UNICEF, W. U. 2005). Most of the casualties are children under the age of 5, who are dying at a rate of more than 2,000 per day (UNICEF, D. O. P. A. S. 2012).

Waterborne pathogens are not just a problem in developing nations. For example, in the United States, there have been 4 million (Mintz et al. 2001) to 33 million (Gundry et al. 2003) annual cases of acute gastrointestinal illness. Moreover, destruction of water supply and/or wastewater treatment infrastructure after major catastrophes, such as earthquakes, tsunamis, floods, and war, pose immediate threats of waterborne disease epidemics. These epidemics are started by infected people and animals that shed pathogens via feces into the environment that then contaminate water supplies through untreated, undertreated, or accidentally released sewage (Connelly & Baeumner 2012). The pathogens of greatest concern are those capable of causing severe disease, persisting in the environment, and withstanding common disinfection methods. In particular, pathogens that form resilient structures, such as spores, cysts, oocysts, and ova (Gundry et al. 2004), require more stringent sterilization regimens.

Since the 19th century, when Louis Pasteur and William Henry found that contaminated garments and contagion infected objects could be made safe for handling by treatment with pressurized steam (Fraise 2012a), simple techniques for treating water by filtration and chlorination have virtually eliminated most waterborne enteric diseases such as typhoid and cholera in North America and Europe (UNICEF, W. U. 2005). And in developing nations, it has been shown that diarrheal episodes can be reduced by 25% by improving water supply, and by 39% via household water treatment and safe storage (Fewtrell et al. 2005). However, the use of filtration or reverse osmosis is expensive, and poor maintenance or failure to replace cartridges can expose users to pathogens, while the use of chlorine to disinfect water may produce carcinogenic trihalomethanes and haloacetic acids (World Health Organization 1998; Pereira et al. 2001). Consequently, an urgent need remains for new technologies that can sterilize large volumes of water cheaply, without consumables or regular maintenance requirements, and without degradation of chemical water quality.

Toward this end, the high temperature and pressure sterilization system (HAPSS) sterilizer was developed and patented to provide high-volume, continuous-flow sterile water at a low operating cost (Bowen 2011, 2012). The HAPSS sterilizer heats water under pressure to achieve high temperatures for sufficient times to sterilize water, and the system used in this investigation is capable of producing 2,000 L of sterile water daily, without removing beneficial minerals (see supplementary material). Moreover, the HAPSS sterilizer contains no moving parts or filters, which essentially eliminates maintenance costs, and operational costs are limited to water and electricity consumption. The system uses less than 20 W h/L supplied by a 20 A outlet.

The effectiveness of the HAPSS sterilization process was measured and is reported here in terms of sterility assurance levels (SALs), which are defined as the expected probability that a surface remains contaminated with viable microorganisms after exposure to a validated sterilization process (Instrumentation, A. F. T. A. O. M. 2011). Confidence in achieving a required SAL is obtained by culturing biological indicators, typically bacterial spores of an appropriate species. Geobacillus stearothermophilus spores are employed for saturated steam sterilization due to their extreme high temperature resistance, and present a considerably greater population and resistance challenge than the expected pathogen that needs to be eliminated (Fraise 2012b). The results from G. stearothermophilus spore inactivation experiments with the HAPSS sterilizer are reported here as a function of temperature and flow rate (i.e., duration at given temperature) to validate its sterilization performance.

The HAPSS water sterilizer model (HAPSS, London, UK) tested here (Figure 1) can operate at continuous-flow rates of up to 1,400 mL/min while maintaining 140 °C. This is achieved by heating water in a copper pipe with an internal volume of 10 L immersed in paraffin maintained at the operating temperature, while maintaining a backpressure sufficient to prevent steam formation. Specifically, the system is a single path consisting of 1/2 inch (12.5 mm) copper tubing coiled into the heat exchanger and an additional coil that is submerged in a paraffin heat transfer medium (Figure 2). Water enters the system at room temperature through a pump that determines the flow rate and produces a pressure of up to 130 psi (896 kPa). The ambient temperature input fluid first flows through the heat exchanger, where the input water provides cooling for the output water flowing from the heat transfer coil, which is held at a steady temperature of 140 ± 2 °C. While cooling the output fluid, the input fluid is preheated prior to entering the heat transfer coil. The entire tubing circuit is maintained at a pressure in excess of 70 psi (483 kPa) by virtue of a backpressure valve that allows no fluid flow at a pressure below 70 psi and free fluid flow above 70 psi. Holding the system pressure above 70 psi, while heating the fluid to 140 °C, creates super-heated fluid with no phase change from liquid to gas. Eliminating water phase change and using an efficient heat exchanger results in a system capable of producing sterile fluid at a very low energy usage (<20 W h/L). On startup, a control mechanism is employed that does not allow fluid flow until both the pressure (>70 psi) and temperature (140 °C) are achieved, preventing any discharge of non-sterile fluid.

Geobacillus stearothermophilus spore suspensions were obtained from Mesalabs (Lakewood, CO) at 3 × 10⁸ CFU/mL, and 10 mL of spore stocks were diluted into 114 L of water with stirring (footnote: including using a 3″ magnetic stir bar, adding spores then water, and mixing with a glass rod) to ensure that the spore suspension was evenly distributed within the water tank. This yielded theoretical initial spore concentrations of 2.6 × 10⁴ CFU/mL, which governed the maximum SAL that was directly measurable, and was limited to this concentration due to the cost of spore stocks (∼$2,000/stock suspension). The external surfaces of the QMI (Oakdale, MN) aseptic sampling port (Figure 1) were cleaned with alcohol wipes prior to each sampling to eliminate contamination, and the internal surfaces of the sterilizer and sampling port were sterilized with steam from the HAPSS sterilizer just prior the experiment by eliminating the back pressure. The experimental design included two samples from the 114-L tank, collected before and after the sterilization runs and analyzed via tryptic soy agar (TSA, Becton, Dickinson and Company, Franklin Lakes, NJ) culturing population analysis to confirm uniformity of the tank spore concentration. Collected samples were stored at 2–4 °C for less than 8 h, and subsequently filtered using 0.2-μm membrane filters (Millipore, Billerica, MA). Spores on filters were re-suspended in 15 mL of sterile/deionized water by vortexing for 3 minutes, heat shocked at 100 °C for 15 minutes, plated in 0.5 mL aliquots onto TSA growth plates and incubated at 55–60 °C for 48 h, and then enumerated for viability assessment. This resulted in a theoretical plated population of 1.3 × 10⁴ spores per plate for 150 mL samples, and 1.4–1.7 × 10⁵ spores per plate for 1,640–1,900 mL samples. In addition, control positive samples containing a high spore population, and control negative samples containing sterile water were also plated onto TSA, while witness TSA plates were retained to assure that the growth media were not contaminated.

The HAPSS high-volume continuous-flow HAPSS water sterilizer (Figure 1) has been validated in a series of inactivation experiments and subsequent viability assessments with known concentrations of G. stearothermophilus spores as a function of sterilizer temperature and flow rate.

The triplicate witness TSA plates contained no growth, showing that the growth media were sterile. The triplicate control negative plates also produced no growth, showing that sample handling for the population assay protocol was aseptic, while the triplicate control positive plates contained the expected population, showing that the growth medium and conditions were appropriate for this organism. The tank spore concentration sampled before and after the inactivation experiments were 1.3 × 10⁴ and 1.0 × 10⁴ CFU/mL, respectively, which compares favorably to the theoretical value of 2.6 × 10⁴ spores/mL, based on Mesalab stock diluted into 114 L of water. Some loss of spore concentration due to surface adhesion is expected and unavoidable. In the SAL calculations, a value of 1.0 × 10⁴ CFU/mL was used as the more conservative lower concentration limit of the spore bio-indicator concentration.

Geobacillus stearothermophilus spore inactivation experiments at 140 °C (samples 1–4) were performed as a function of flow rate (i.e., thermal dosages), up to 1.4 L/min, beyond which this model of the sterilizer could not maintain the operating temperature of 140 °C (Table 1). Since the internal volume of the sample flow pipe immersed in the paraffin heater was 10 L, the duration of inactivation at 140 °C was approximately 7 minutes for the 1.4 L/min flow rate. The resultant efflux was analyzed for surviving G. stearothermophilus spores in terms of CFU on TSA growth plates. Samples 1–5 were completely sterilized, which validated the HAPSS sterilizer by direct measurements of G. stearothermophilus spore inactivation to SALs approaching 10⁻⁶ (i.e., complete inactivation of nearly 10⁶ spores plated onto the growth media) for operating temperatures at 140 and 130 °C. Not surprisingly, operating temperatures of 120 and 110 °C did not inactivate the spore suspensions to sterility.

Given that thermally induced cell death follows first order (i.e., logarithmic) dynamics at a given temperature, then a flow rate at half that rate (i.e., 700 mL/min), which would hold the sample at operating temperatures for twice as long, will yield a SAL of ∼10⁻¹¹. The temperature dependence of the decimal reduction value of G. stearothermophilus spores killed by steam when extrapolated to 140 °C indicates sterilization in less than 1 second, and given that the internal volume of the sterilizer at 140 °C is ∼10 L, then a theoretical maximum flow rate of 100 L/min should still sterilize a sample, assuming that the heat exchange is sufficient to maintain the temperature of the system.²

The HAPSS high-volume, continuous-flow, high temperature/pressure sterilizer by HAPSS inactivated G. stearothermophilus spores, the most thermally resilient organism known, when operating at 140 and 130 °C, at continuous-flow rates of up to 1.4 L/min, which directly demonstrated a SAL of almost 10⁻⁶. Based on logarithmic inactivation dynamics, as a function of temperature and dosage (i.e., flow rate), SALs of >> 10⁻⁶ (i.e., the gold standard for achieving sterility) can be achieved readily at 1 L/min at 140 °C. The HAPSS water sterilizer requires only high temperature, pressure, and electricity and eliminates the need for chlorine. Moreover, it is not laden with the maintenance and cost of consumable filters and consequently represents a potentially game changing technology for safe drinking water at the household and community level, and at the regional level with larger systems in development.

A.P. thanks John Bowen for assistance in operating the HAPSS sterilizer and for helpful discussions, Michael Papadopoulos for providing laboratory supplies and helpful discussion, and DeAnn Dallas and Nicholas Fingland for help in sample analysis in preliminary experiments.