Experimental Approaches and Optical Calculations in sensing Biological Pathogens

MANOJ HARIDAS
Research & Technology Group
Honeywell Technology Solutions Lab
151/1, Doraisanipalya,  Bannerghatta Road,  Bangalore  560076.
INDIA.
http://www.honeywell.com

Abstract: - Biological pathogens often consolidate in colonies and manifest as biofilm that affect many engineering applications.  Biofilm monitoring and removal are frequently difficult processes in large engineering scenarios and complicate sensing and process control.  The paper describes the development of a continuous biofilm monitoring and cleaning system based on optical detection and communication protocols.

Key-Words: - biofilm	pathogen	fluorescence	ultraviolet	microprocessor	optical path

1	Introduction
Sensors and monitoring systems are widely used in many industrial applications such as petroleum refining, pharmaceuticals, cooling towers, food processing and healthcare to validate process integrity.  The sensors often operate for extended periods in hostile environments of heat, humidity, corrosion, wear and microorganism contact.  During extended times of continuous operation, sensor surfaces that are in contact with fluids often become coated with deposits of different natures.  Depending on the environment of the sensor, the deposits can be inorganic scale such as manganese scale, calcia scale, iron deposits, chrome deposits, biofilms, organic contaminants, and many others.  Monitoring of fluid properties and validating the process integrity becomes less accurate due to deposits on the sensors.
Compared to inorganic scale and deposits, biofilm is of particular interest due to its high prevalence.  Biofilms are found in virtually every aquatic environment.  A biofilm is a complex structure of microorganisms that function as a community.  Bacteria usually comprise the majority of microbial types found in biofilms, but fungi, yeast, algae, and protozoa can also be the dominant microbial types present in some biofilms. The majority of bacteria in any fluid system is likely to be in the form of biofilms, often attached to internal surfaces such as paddles, mixers, mechanical components and sensors. Additionally, biofilms are the source of much of the free-floating bacteria, some of which can cause infection and disease.  One common biofilm bacteria, Pseudomonas aeruginosa, is a secondary pathogen that can lead to infections in humans or other animals.  Besides being a reservoir of bacteria that can affect health, biofilms can also cause corrosion within the fluid transport systems, such as stainless steel piping systems.
In general, a biofilm forms when bacteria adhere to surfaces in aqueous environments and begin to excrete a slimy, glue-like substance that can anchor them to all kinds of materials – such as metals, plastics, soil particles, medical implant materials, or tissue. A biofilm can be formed by a single bacterial species, but more often biofilms consist of many species of bacteria, as well as fungi, algae, protozoa, debris and corrosion products. Essentially, biofilm may form on any surface exposed to microbes and some amount of water. Once anchored to a surface, biofilm microorganisms can carry out a variety of detrimental reactions, depending on the surrounding environmental conditions.
Microbial biofilms on fluid surfaces cause billions of dollars in annual equipment damage, product contamination, energy losses and medical infections.  Biofilms frequently result in corroded pipes, reduced heat transfer or hydraulic pressure in industrial cooling systems, plugged water injection jets, and clogged water filters.  Conventional methods of killing bacteria (such as antibiotics, and disinfection chemicals) are often ineffective with biofilm bacteria. The huge doses of anti-microbials required to rid systems of biofilm bacteria are undesirable environmentally and impractical in application.  Electrical techniques that provide a small electrical charge along with anti-microbial dosage are not effective for large fluid volumes.
With regard to drinking water, biofilm is an especially critical issue, since once anchored to a surface (such as a sensor or a pipe), biofilm microorganisms can carry out a variety of detrimental reactions and actually interfere with the water treatment process.  Recent studies have shown, for example, that by forming itself into a biofilm, the organism responsible for outbreaks of cholera can survive chlorine concentrates 10 to 20 times higher than are normally used to treat drinking water.  Examples of common disease causing waterborne bacteria found in biofilm include Eschericha coli, Legionella pneumophila, Pseudomonas, Flavobacterium, Arthrobacter, Acinetobacter, Sarcina, Micrococcus, Porteus, Bacillus, Klebsiella and Enterobacter.
In addition to contamination concerns, biofilm can also affect water pressure.  Once attached to a pipeline wall, biofilm begins building upon itself, adding layer upon layer, forming a plaque-like coating.  The bacterial community traps nutrients, microbes, worms and viruses to form an almost impenetrable gunky material.  Such growth can clog water lines to the point of insufficient water pressure, creating a hazard for homes, businesses and even firefighters.
Microbial cells and their metabolic activities frequently cause bio-corrosion on metal surfaces. Bio-corrosion results in pitting, crevice corrosion, selective dealloying, stress corrosion cracking, and under-deposit corrosion and occurs by oxygen depletion (or differential aeration) that causes a rupture in stainless steel’s protective film and provides a condition for sulphate reducing bacteria (SRB) or acid producing bacteria to accelerate metal degradation.
Conventionally, biofilm monitoring on sensor and other surfaces has been done using a variety of visual examination techniques or probe assisted inspection.  Currently employed techniques for biofilm monitoring (or removal) include the addition of antibacterial compounds [1-6] or by introducing microbiological consortia to prevent consolidation of biofilm bacteria [7-8].  Other techniques for biofilm monitoring have focused on creating fluid turbulence such that biofilm formation is disrupted.  These techniques include creating a mechanical turbulence in the fluid [9], bubbling carbon dioxide through the fluid [10], powered jetting of inert liquid or gas through the fluid [11-14] and offline fluid flushing [15].  Certain chemical procedures also have been followed to monitor biofilm activity.  These include periodic chemical treatment of the fluid [16-18] or by ozonolysis of the fluid to create a highly oxidizing atmosphere that is unfavorable to biofilm activity [19].  Smaller fluid volumes can also be monitored for biofilm activity using electrical discharges [20-22], epifluorescence microscopy [23-24], DNA sequencing [25] or by optical transmittance measurements [26] followed by interfacially induced phase separation or filtration [13] or by heating [27].  In other controlled environments, biofilm response has been gauged  using differential weight increases of biofilm on test coupons before and after the addition of biocide [28], trace analysis [29], laser patterning of biofilm [30], biofouling measurements [31] and by using surface active coatings to interfacially disband biofilm formation [32].  On noticing a biofilm build-up, cleaning has been generally done using electrochemical methods, mechanical cleaning, hydraulic cleaning, ultrasound, and combinations thereof.
2	Problem Formulation
It is believed that currently practiced methods do not provide continuous monitoring and biofilm removal.  Besides, most of the known techniques do not eliminate the biofilm; many techniques only temporarily disrupt the critical biofilm mass.  With passage of time, the disrupted biofilm is liable to re-consolidate.  Moreover, the areal scope of biofilm monitoring currently provided is limited by the sensitivity of biofilm response received beyond a critical distance (i.e. attenuation and damping).  Electromagnetic stimuli that may be provided to distant biofilm locations to elicit measurable (and mappable) response are not suited to conveying through large fluid bodies such as municipal reservoirs.  There is also a need for ‘round-the-clock’ monitoring of biological activity in fluid systems such that immediate remedial measures are taken before a crisis develops (such as alerting law enforcement and shutting off a city water supply in case of an anthrax alert).  Therefore, there is a technological need for a realtime biofilm detection system.  What is also needed is a system for continuous biofilm monitoring and removal that may function without human intervention.  What is hence needed is a smart biosensor that can correct biological anomaly in fluids or draw attention to the occurrence of a biological event.
2.1	Methodology
The optical protocol proposed in this research provides: (i) a biofilm detection system comprising a source of electromagnetic radiation for exciting a biofilm in order to generate a characteristic electromagnetic response from the biofilm and (ii) a detection system for correlating the characteristic electromagnetic response to a respective biofilm.  The protocol also provides a biofilm cleaning system comprising a biofilm detection system that sends a biofilm response to electromagnetic radiation to a microprocessor that triggers a source of ultraviolet radiation to emit characteristic ultraviolet rays to destroy the biofilm.  The paper thus provides a smart sensor wherein a biofilm detection system and a biofilm cleaning system are provided with the sensor.
3	Problem Solution
The experimental approach and optical layout proposed in this paper is described in subsequent sections of this manuscript.  An exemplary biofilm detection system 10 comprises a source of electromagnetic radiation 12 for exciting a biofilm 14 and generating a characteristic electromagnetic response 16 therefrom and a detection system 18 for correlating the characteristic electromagnetic response 16 to a respective biofilm 14, as shown schematically in Figure 1.  Inorganic scale 34 may also be detected either separately, or along with biofilm 14.  The biofilm detection system 10 receives the biofilm response 16 to the electromagnetic radiation 12 and sends a signal 20 to a microprocessor 22.  The microprocessor 22 compares the response 16 to a collection of known responses and correlates the response 16 to a specific biofilm 14.  Once the microprocessor 22 makes a determination what type of biofilm 14 is present, a number of events can follow.  The microprocessor 22 can trigger an alarm 100, trigger an event 200, like adding or increasing the amount of additives to the water supply 300, sending the information to a local or remote engineer 400 or service team 500 or other scenarios including, but not limited to, activating a shut-off valve, activating a self-cleaning mechanism 56, and combinations thereof.  Upon detection of biofilm, the biofilm detection system 10 can quantify the amount of biofilm, determine the nature of biofilm, initiate a cleaning process of the sensor for subsequent measurement, provide a signal for a controller to act upon the detected level of biofilm, and combinations thereof.  The response can be in the form of an actuation of a sequence to add a reagent of choice into the monitored environment, or any other control action.
For biofilm detection events having consequences beyond the scope of routine operational events, a high-level alarm system directing authority attention to the occurrence is activated.  In instances where biofilm occurrence is in accordance with the biofilm amount and type expected for the particular operation, a self-cleaning mode 24 of the sensor is triggered by activating a uv (ultraviolet) source 28 that emits ultraviolet radiation to destroy biofilm 14.  In one experimental approach, the system 10 can be used as a stand-alone biofilm detection system.
Biofilm 14 may be detected on any surface where biofilm may form.  For example, the surface can be a sensor, a window of a flow-though flow cell or a viewing port of optical fiber or any other probe that detects signals from the surface.  The surface includes at least one of plastics, metal, glass, ceramic, biological tissue, coatings or wood.  In one embodiment, biofilm 14 is deposited on a smart sensor 26.  The biofilm 14 being detected can be any biofilm capable of generating a fluorescence signature i. e. the biofilm can include at least one of Pseudomonas aeruginosa, Legionella pneumophila, Staphylococcus aureus, Mycobacterium, coliform bacteria or artificially grown cultures.
The source of electromagnetic radiation 12 provides single or multiple wavelengths that interact with deposited biofilm 14 or inorganic scale 34 to provide a response 16 that is typically a collection of multiple wavelengths of radiation 16.  Data analysis of biofilm 14 (or inorganic scale 34) response is performed using multivariate analysis tools 32 (shown in Figure 3) to provide a required selectivity and sensitivity in the detection system 10.  In the optical interrogation of the biofilm 14, a range of electromagnetic sources, including ultra-violet, infrared, visible, and radio-frequency sources may be used.  The excitation wavelengths of particular interest to biofilm detection are usually in the regime of about 190 nm to about 550 nm.  In one embodiment, the biofilm response 16 comprises a fluorescence signal 36 that is conveyed to spectro-fluorometer 38 for identification of biofilm type and biofilm amount.  In one embodiment, the spectro-fluorometer 38 sends a signal 20 to a microprocessor 22.
The intensity and identity of a fluorescence signal depend upon the source of fluorescence.  Generally, biofilms 14 fluoresce in a much greater degree than inorganic deposits 34 and generate fluorescence signals of higher intensity.  Fluorescence signals generally comprise an inelastic component 42 of electromagnetic response 16.  The inelastic components of an electromagnetic response at different wavelengths form a fluorescence spectrum.  Upon selection of a certain excitation wavelength of fluorescence source, a fluorescence spectrum, its features (shape) and its intensity may serve as a discrimination means between different fluorescent species such as bacteria.  In addition, a fluorescence lifetime may serve as an extra component in discriminating between inorganic scale and biofilm and biofilm type.  In addition, the kinetics of fluorescence decay upon photobleaching serves as an additional component in discriminating between inorganic scale 34 and biofilm 14 and biofilm type.  The elastic component 40 of electromagnetic response 16 generally results from non-biological film specimens such as inorganic scale deposits 34.  Spectro-fluorometer 38 and microprocessor 22 employ a multivariate data analysis protocol 32 (shown in Figure 3) to resolve and differentiate the elastic 40 and the inelastic 42 components of the electromagnetic response 16 along steps 48 and 50 respectively (shown in Figure 3) to correlate film response with film type.  The multivariate data analysis protocol 32 utilizes several types of collected information such as elastic components of electromagnetic response at different source wavelengths, features of fluorescence spectrum (shape and its intensity), fluorescence lifetime, and kinetics of fluorescence decay upon photobleaching.  The biofilm detection system 10 is hence also able to detect non biofilm deposits formed on the surface.  Inorganic scale 34 may occur either separately, or along with biofilm 14.
In Figure 2, a representation of a smart biosensor immersed in aqueous fluid is shown.  The sensor 26 is immersed in aqueous fluid 44.  With continued immersion and contact with aqueous fluid 44 a biofilm 14 deposits on the sensing surface 30 of sensor 26.  Sensor 26 typically comprises a light source 12 and a spectro-fluorometer 38.  Light source 12 sends an optical interrogation to sensing surface 30 via optical fiber 46.  Biofilm 14 responds to optical signal 12 by generating a characteristic fluorescence signal 36 of biofilm type and biofilm amount that is relayed to spectro-fluorometer 38 and conveyed to microprocessor 22.
An additional feature of the biofilm detection system 10 is a biofilm cleaning system 56.  An exemplary biofilm cleaning system 56 comprises a detection system 18 that receives a biofilm response 16 to electromagnetic radiation 12 and sends a response signal 20 to a microprocessor 22 that triggers a uv source 28 upon receiving response 20. Uv source 28 is in electronic communication with microprocessor 22 and upon receiving a response 20 emits uv radiation that disintegrate the biofilm.  Uv source 28 is maintained in radiation contact with biofilm 14.  In one embodiment, the biofilm detection system 10 and the biofilm cleaning system 56 are both provided on one sensor 26 to provide a self-cleaning sensor 58.  Inorganic scale 34 may be cleaned either separately, or along with biofilm 14.
One embodiment of the biofilm cleaning system 56, shows the use of at least one uv source 28 coupled to a sensor 26 for cleaning of a sensing surface 30 during sensor operation based on a multivariate data analysis protocol 50 that distinguishes between a biofilm deposit 14 and an inorganic scale 34.  Uv source 28 generates uv radiation that disintegrate any deposited film (14 or 34)  (shown in Figure 2).
Depending upon the nature and amount of the biofilm 14 detected, a range of ultraviolet frequencies particularly designed for cleaning specific biofilm types are emitted.  In one embodiment, a range of roving probe frequencies are targeted at the surface 30 and a range of frequencies are emitted to disintegrate single or multiple biofilm moieties.  In another embodiment, the dynamic probe and response frequencies are manually controllable using a Look-Up Table created on the basis of excitation-emission fluorescence maps obtained for individual biofilm species. In another embodiment, the uv power (or intensity) and frequency required for biofilm removal, is automatically selected and emitted by uv source 28 on identification of the type of biofilm detected by spectro-fluorometer 38 and microprocessor 22 as shown in Figure 2.  Microprocessor 22, on the basis of a prior calibration and standardization, determines the uv frequency, uv power and the time required to disintegrate biofilm 14 from surface 30.
As shown in Figure 3, a multivariate data analysis protocol 32 to determine the distribution of inorganic film 34 and biofilm 14 is performed and simultaneous evaluation yields a result in step 52.  In one embodiment, only biofilm 14 is found to occur.  In another embodiment a variety of biofilms 14 are found to occur together.  In a third embodiment, only inorganic scale 34 is found to occur.  In a fourth embodiment, a variety of inorganic scales 34 are found to occur together.  In a fifth embodiment, a variety of biofilms 14 and a variety of inorganic scales 34 are found to occur together.  A self-cleaning operation 24 is triggered on receiving input 54 that provides a decision to start the self-cleaning action based on a specified number of measurements, the time of operation and the uv frequency.  The self-cleaning operation continues until an iterative operation via feedback step 60 indicates that deposited film 14 has been sufficiently removed.  The self-cleaning operation stops and automatically resumes on the buildup of fresh deposit of inorganic scale or biofilm 14 upon sensing surface 30.
Deposited biofilm 14 is generally easier to remove in the early stages of its formation while it adheres to a surface on the strength of physical attractions and weak interactions.  Aged biofilms 14 tend to chemically bond and undergo syneresis with the surface and are more difficult to remove.  The substrate often suffers mechanical and chemical damage in efforts to remove chemically bonded film deposits.  One embodiment provides a system for continuous film monitoring wherein the film is detected and removed in real-time with little or no delay since film formation, and thus provides a system for efficient biofilm management.
To aid continuous biofilm monitoring and possible cleaning, the prototype design provides a continuous biofilm monitoring and cleaning system that works without human intervention.  One embodiment of the present design provides an efficient extension of the service life of a sensor by providing a self-cleaning sensor that does not have any moving parts.  The self cleaning ability of the sensor provides a means of using the same sensor surface as a reference (immediately when it is cleaned) and as a measurement surface upon formation of biofilm.  The prototype design finds applications in food processing, bottled water plants, waste water treatment, hydrocarbon processing, aviation fuel tanks, cooling towers, pasteurizers, aqueous paints, drinking water, pharmaceutics, healthcare, gas phase reservoirs, fluid cylinders and other engineering instances.
4	Conclusion
Biological pathogens often consolidate in colonies and manifest as biofilm that affect many engineering applications.  Biofilm monitoring and removal are frequently difficult processes in large engineering scenarios and complicate sensing and process control.  The paper describes the development of a continuous biofilm monitoring and cleaning system based on optical detection and communication protocols.

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