BIOL 402: APPLIED and INDUSTRIAL MICROBIOLOGY

BIOL 402: APPLIED and INDUSTRIAL MICROBIOLOGY

BIOCIDES

I. REVIEW OF WATER, WASTEWATER , AND PROCESS WATER MICROBIOLOGY

A. Waste Water Treatment

Types of waste water treatment: domestic, lagoons (agricultural and industrial)

Types of treatment systems and how they work (specifics of systems) and how biofilms are important in them

Why treat wastewater?

B. Water Treatment

What are indicator organisms and how do we test for them?

Steps in water treatment and purification/disinfection

C. Process Water

What we mean by process water, where it is used, and how it is used

What types of microorganisms are involved and importance of biofilms

Legionella pneumophila: its characteristics, where found, why a problem, how is it detected?

Bottled water: treatments, types, difference from tap water, microbiological standards and microbial composition

II. Microbiology of Papermaking

Process for making paper

Where microbes can be found

What problems microbes cause

Cures for these problems

II. THE CONTROL OF MICROBIAL GROWTH

REF: Chapter 23 in textbook

Block, S. S. 1991. Disinfection, Sterilization, and Preservation, 4 ed. Lea & Febiger

A. Definitions

1. disinfectant; an agent which frees from infection by physical or chemical means that kills disease or harmful bacteria but not spores; substances applied to inanimate objects

2. antiseptic: substance that prevents or arrests the growth of microorganism by either inhibiting them or killing them; usually refers to materials applied to living tissue;

3. germicide: an agent that destroys microorganisms;

4. bactericide/fungicide/virucide/sporicide: an agent that kills bacteria/fungi/viruses/spores;

5. biocide: agent that kills all living things - pathogenic and nonpathogenic

6. bacteriostat: an agent (usually chemical) that prevents the growth of organisms but does not necessarily kill them;

7. sterilization: physical or chemical act or process that destroys all life, especially microorganisms;

8. antibiotic: organic chemical that is used to destroy or inhibit pathogenic bacteria or fungi

9. preservative: prevents growth but may or may not kill existing microbial community

B. Special terms

1. Thermal Death Time : time required to kill all spores at a specified temperature

2. D Value: time required to inactivate or kill 90% of the microbial population -OVERHEAD

3. F Value:time in minutes required to kill all the spores in a suspension when heated to 120 C or 250 F

4. Z Value: a measure of the way the D Value changes with temperature for a particular organism; i.e. the slope of the logarithm of the thermal death time vs temperature – OVERHEAD

5. C @ t : disinfectant effectiveness where C = disinfectant concentration and t = time required to inactivate a given percentage of organisms (typically 90 - 99%) -OVERHEADS FOR DIFFERENT CHLORINE COMPDS

6. Q₁₀ Value: it is the ratio of the D values at two temperature which are 10 C apart.

C. Physical Methods

1. Ionizing Radiation

types: alpha, beta, gamma, x-rays, protons

uses: beta and gamma from Co⁶⁰ typically used

resistant organisms: bacillus spores, Deinococcus radiodurans

mechanisms for resistance: low water content of spore, highly efficient repair mechanisms within spore over vegetative cells

mode of action: breaks single or double stranded DNA

2. Ultraviolet Light (328 to 210 nm) with maximal bactericidal activity at about 260nm

mode of action: thymine dimers formed - OVERHEAD

resistant cells: Deinococcus radiodurans, and spores but not in the frozen state - cells more sensitive in the frozen state, even D. radiodurans. This coccus forms TDHT

repair mechanisms: photoreactivation, excision repair, postreplication recombination repair, error prone repair

3. Moist Heat

mode of action: unknown

do know that when cells exposed to steam, moist heat get leakage of RNA and DNA and other molecules from the cells, also proteins coagulate (egg whites), and spores loose dipicolenic acid and calcium

also have isolated bacilli capable of withstanding autoclaving

4. Dry Heat

mode of action: dependent on the water content of the cells, may affect DNA

5. Cold

mode of action: probably get ice crystals within the cell which affects cytoplasmic membrane

influencing factors: cold shock without freezing is affected by nutritional state, age (log phase cells more sensitive), medium composition

Lyophilization & Liquid N₂

6. Membrane Filtration

D. Chemical Methods

1. Hypochlorous acid/Chlorine

mode of action: thought to be hypochlorous acid that is most active portion

pH, temperature and cations also affect efficacy

chemical reaction: Overheads of types of compounds

Residuals

2. Iodine/Iodophores

mode of action: I₂ can penetrate cells where it can react with basic N-H in amino acids LYS, HIS, ARG; nucleotide bases; oxidize SH groups; derivatize TYR; react with double bonds in fatty acids

structure of iodophores:

3. Peroxygen compounds

mode of action: peroxide may oxidize Cl ions to form hypochlorite in the presence of myeloperoxidase

superoxide ion (from reduction of oxygen to water) reacts with peroxide to produce OH radical

4. Ozone

mode of action: attacks double bonds, forms dimers OVERHEADS OF GENERATION & EFFECTS

kill times: E. coli 0.23 mg/L 99.9% reduction in 1.67 min., in waste water 19 min at 0.85 mg/L; enteric viruses 92% reduction in 19 min. at 1.4 initial conc. but 0 residual

5. Alcohols

mode of action: believed to be denaturation of proteins, inhibits spore germination, can cause lysis of cells - OVERHEAD

6. Quaternary Ammonium Compounds

general structure:

mode of action: may act as surfactants and disrupt cell membrane; more effective in slightly alkaline or acid than neutral environments; more effective against gram positive

best that can be said at present for CATIONIC antiseptics is:

cmpd sorbed onto surface of cell, diffuses through, binds to cytoplasmic membrane and disrupts it causing release of K+ and other cytoplasmic constituents

7. Amphoteric Compounds

OVERHEAD of some types

mode of action: penetrate cells and ????

8. Ethylene Oxide

Used to sterilize surgical instruments, packs, etc. and tissue culture equipment

Mode of action: breaks double bonds and combines with proteins ; is sporicidal, good penetration if some moisture present; may absorb into material therefore need to off-gas or aerate for prolonged period of time.

OVERHEAD - Gelman technical Information

Use range: 450 - 500 mg/L

9. A Few Commercial Biocides

vi. Brominated compounds

a. Bronopol

Unstable at pH>8; used in cosmetic industry - activated with parabens

b. Bromonitrostyrene (Givgard 8 NS)

preservative; oil & emulsion-based paints

vii. Morpholines (Bioban series)

APPLICATION MATRIX	BIOBAN CS-1135	TRIS NITRO 50% AQUEOUS *	BIOBAN -P1487 **
Metalworking Fluids	-	2000-4000 ppm	500-1000 ppm
Water Treatment	-	1000-2000 ppm	-
Latex Paints	1000 - 2000 ppm	-	-
Water-based Emulsions & Solutions	-	1000-2000 ppm	-
Petroleum Production & Recovery	5-150 ppm	1000-2000 ppm	-
Fuel & Hydrocarbon Preservation	-	-	500-1000 ppm

*Formaldehyde releaser

** Approved for metal working fluids, pulp & paper industry, petroleum production, jet fuels

viii. Phenylphenylates (Dowicide A)

preservative; used in protein based paints, metal working fluids, polished, adhesives, gums, latexes, textiles

E. Testing Methods

1. Summary of Cellular Targets - TABLE 23.1

OVERHEADS

2. Steps in Selecting a Biocide

a. Look at the regulations: FDA, EPA, CFTA

b. If have contamination problems, use organisms from the contaminated environment

c. Run test in PRODUCT NOT TSA

d. Test biocide for reaction with product to avoid changes in product quality

e. Test should determine how long biocide is effective ( multiple challenges)

how much biocide is need

how effective & against which organisms

don’t forget sulfate reducers (anaerobic bacteria)

f. Frequently it is best to develop a suite of biocides

g. Bactometer (impedance) very useful in these types of studies; also Cide-Trak which is based on luminescent bacterium for real-time analyses or the Vitek system Dr. Chadick talked about

3. If testing whether biocide is effective, if a neutralizer is available, must neutralize test sample

OVERHEAD - Neutralization Agents

4. Standard Methods do exist: AOAC Methods and EPA/FDA Methods depending on material

F. Where Apply Biocides?

HACCP = hazard analysis critical control point

Basis - can’t sample everything all the time, so evaluate where contamination can occur and concentrate on those points

Are there supplier and production issues

Do we need protocols & who will write them

Do we need QA/QC and at what points

What are the hazards

What are acceptable ranges

Do we test product before it leave the plant

What do we do if problems occur and what criteria do we use

What kind of and how do we keep records

What type of verification do we need to be assured the problem won’t occur again

What type of sampling/testing do we use and what parameters do we set: when, where, how much, validation

For food the FDA is looking for a 5-log reduction in microbial bioburden