Information

Lab 18: Use of Physical Agents to Control of Microorganisms - Biology


A. INTRODUCTION TO THE CONTROL OF MICROORGANISMS

The next two labs deal with the inhibition, destruction, and removal of microorganisms. Control of microorganisms is essential in order to prevent the transmission of diseases and infection, stop decomposition and spoilage, and prevent unwanted microbial contamination.

Microorganisms are controlled by means of physical agents and chemical agents. Physical agents include such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration. Control by chemical agents refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial chemicals.

Basic terms used in discussing the control of microorganisms include:

1. Sterilization
Sterilization is the process of destroying all living organisms and viruses. A sterile object is one free of all life forms, including bacterial endospores, as well as viruses.

2. Disinfection
Disinfection is the elimination of microorganisms, but not necessarily endospores, from inanimate objects or surfaces.

3. Decontamination
Decontamination is the treatment of an object or inanimate surface to make it safe to handle.

4. Disinfectant
A disinfectant is an agents used to disinfect inanimate objects but generally to toxic to use on human tissues.

5. Antiseptic
An antiseptic is an agent that kills or inhibits growth of microbes but is safe to use on human tissue.

6. Sanitizer
A sanitizer is an agent that reduces, but may not eliminate, microbial numbers to a safe level.

7. Antibiotic
An antibiotic is a metabolic product produced by one microorganism that inhibits or kills other microorganisms.

8. Chemotherapeutic antimicrobial chemical
Chemotherapeutic antimicrobial chemicals are synthetic chemicals that can be used therapeutically.

9. Cidal
An agent that is cidal in action will kill microorganisms and viruses.

10. Static
An agent that is static in action will inhibit the growth of microorganisms.

These two labs will demonstrate the control of microorganisms with physical agents, disinfectants and antiseptics, and antimicrobial chemotherapeutic agents. Keep in mind that when evaluating or choosing a method of controlling microorganisms, you must consider the following factors which may influence antimicrobial activity:

1. the concentration and kind of a chemical agent used;

2. the intensity and nature of a physical agent used;

3. the length of exposure to the agent;

4. the temperature at which the agent is used;

5. the number of microorganisms present;

6. the organism itself; and

7. the nature of the material bearing the microorganism.

B. TEMPERATURE

Microorganisms have a minimum, an optimum, and a maximum temperature for growth. Temperatures below the minimum usually have a static action on microorganisms. They inhibit microbial growth by slowing down metabolism but do not necessarily kill the organism. Temperatures above the maximum usually have a cidal action, since they denature microbial enzymes and other proteins. Temperature is a very common and effective way of controlling microorganisms.

1. High Temperature

Vegetative microorganisms can generally be killed at temperatures from 50°C to 70°C with moist heat. Bacterial endospores, however, are very resistant to heat and extended exposure to much higher temperature is necessary for their destruction. High temperature may be applied as either moist heat or dry heat.

a. Moist heat

Moist heat is generally more effective than dry heat for killing microorganisms because of its ability to penetrate microbial cells. Moist heat kills microorganisms by denaturing their proteins (causes proteins and enzymes to lose their three-dimensional functional shape). It also may melt lipids in cytoplasmic membranes.

1. Autoclaving

Autoclaving employs steam under pressure. Water normally boils at 100°C; however, when put under pressure, water boils at a higher temperature. During autoclaving, the materials to be sterilized are placed under 15 pounds per square inch of pressure in a pressure-cooker type of apparatus. When placed under 15 pounds of pressure, the boiling point of water is raised to 121°C, a temperature sufficient to kill bacterial endospores.

The time the material is left in the autoclave varies with the nature and amount of material being sterilized. Given sufficient time (generally 15-45 minutes), autoclaving is cidal for both vegetative organisms and endospores, and is the most common method of sterilization for materials not damaged by heat.

2. Boiling water

Boiling water (100°C) will generally kill vegetative cells after about 10 minutes of exposure. However, certain viruses, such as the hepatitis viruses, may survive exposure to boiling water for up to 30 minutes, and endospores of certain Clostridium and Bacillus species may survive even hours of boiling.

b. Dry heat

Dry heat kills microorganisms through a process of protein oxidation rather than protein coagulation. Examples of dry heat include:

1. Hot air sterilization

Microbiological ovens employ very high dry temperatures: 171°C for 1 hour; 160°C for 2 hours or longer; or 121°C for 16 hours or longer depending on the volume. They are generally used only for sterilizing glassware, metal instruments, and other inert materials like oils and powders that are not damaged by excessive temperature.

2. Incineration

Incinerators are used to destroy disposable or expendable materials by burning. We also sterilize our inoculating loops by incineration.

c. Pasteurization

Pasteurization is the mild heating of milk and other materials to kill particular spoilage organisms or pathogens. It does not, however, kill all organisms. Milk is usually pasteurized by heating to 71°C for at least 15 seconds in the flash method or 63-66°C for 30 minutes in the holding method.

2. Low Temperature

Low temperature inhibits microbial growth by slowing down microbial metabolism. Examples include refrigeration and freezing. Refrigeration at 5°C slows the growth of microorganisms and keeps food fresh for a few days. Freezing at -10°C stops microbial growth, but generally does not kill microorganisms, and keeps food fresh for several months.

C. DESICCATION

Desiccation, or drying, generally has a static effect on microorganisms. Lack of water inhibits the action of microbial enzymes. Dehydrated and freeze-dried foods, for example, do not require refrigeration because the absence of water inhibits microbial growth.

D. OSMOTIC PRESSURE

Microorganisms, in their natural environments, are constantly faced with alterations in osmotic pressure. Water tends to flow through semipermeable membranes, such as the cytoplasmic membrane of microorganisms, towards the side with a higher concentration of dissolved materials (solute). In other words, water moves from greater water (lower solute) concentration to lesser water (greater solute) concentration.

When the concentration of dissolved materials or solute is higher inside the cell than it is outside, the cell is said to be in a hypotonic environment and water will flow into the cell (Fig. 1). The rigid cell walls of bacteria and fungi, however, prevent bursting or plasmoptysis. If the concentration of solute is the same both inside and outside the cell, the cell is said to be in an isotonic environment (Fig. 2). Water flows equally in and out of the cell. Hypotonic and isotonic environments are not usually harmful to microorganisms. However, if the concentration of dissolved materials or solute is higher outside of the cell than inside, then the cell is in a hypertonic environment (Fig. 3). Under this condition, water flows out of the cell, resulting in shrinkage of the cytoplasmic membrane or plasmolysis. Under such conditions, the cell becomes dehydrated and its growth is inhibited.

Flash animation showing osmosis in an isotonic environment.

http5 version of animation for iPad showing osmosis in an isotonic environment.

Flash animation showing osmosis in a hypotonic environment.

html5 version of animation for iPad showing osmosis in a hypotonic environment.

Flash animation showing osmosis in a hypertonic environment.

html5 version of animation for iPad showing osmosis in a hypertonic environment.

The canning of jams or preserves with a high sugar concentration inhibits bacterial growth through hypertonicity. The same effect is obtained by salt-curing meats or placing foods in a salt brine. This static action of osmotic pressure thus prevents bacterial decomposition of the food. Molds, on the other hand, are more tolerant of hypertonicity. Foods, such as those mentioned above, tend to become overgrown with molds unless they are first sealed to exclude oxygen. (Molds are aerobic.)

For more information on antigens, antibodies, and antibody production, see the following Learning Objects in your Lecture Guide:

  • The Cytoplasmic Membrane; Unit 1, Section IIB1

E. RADIATION

1. Ultraviolet Radiation

The ultraviolet portion of the light spectrum includes all radiations with wavelengths from 100 nm to 400 nm. It has low wave-length and low energy. The microbicidal activity of ultraviolet (UV) light depends on the length of exposure: the longer the exposure the greater the cidal activity. It also depends on the wavelength of UV used. The most cidal wavelengths of UV light lie in the 260 nm - 270 nm range where it is absorbed by nucleic acid.

In terms of its mode of action, UV light is absorbed by microbial DNA and causes adjacent thymine bases on the same DNA strand to covalently bond together, forming what are called thymine-thymine dimers (see Fig. 4). As the DNA replicates, nucleotides do not complementary base pair with the thymine dimers and this terminates the replication of that DNA strand. However, most of the damage from UV radiation actually comes from the cell trying to repair the damage to the DNA by a process called SOS repair. In very heavily damaged DNA containing large numbers of thymine dimers, a process called SOS repair is activated as kind of a last ditch effort to repair the DNA. In this process, a gene product of the SOS system binds to DNA polymerase allowing it to synthesize new DNA across the damaged DNA. However, this altered DNA polymerase loses its proofreading ability resulting in the synthesis of DNA that itself now contains many misincorporated bases. In other words, UV radiation causes mutation and can lead to faulty protein synthesis. With sufficient mutation, bacterial metabolism is blocked and the organism dies. Agents such as UV radiation that cause high rates of mutation are called mutagens.

The effect of this inproper base pairing may be reversed to some extent by exposing the bacteria to strong visible light immediately after exposure to the UV light. The visible light activates an enzyme that breaks the bond that joins the thymine bases, thus enabling correct complementary base pairing to again take place. This process is called photoreactivation.

UV lights are frequently used to reduce the microbial populations in hospital operating rooms and sinks, aseptic filling rooms of pharmaceutical companies, in microbiological hoods, and in the processing equipment used by the food and dairy industries.

An important consideration when using UV light is that it has very poor penetrating power. Only microorganisms on the surface of a material that are exposed directly to the radiation are susceptible to destruction. UV light can also damage the eyes, cause burns, and cause mutation in cells of the skin.

2. Ionizing Radiation

Ionizing radiation, such as X-rays and gamma rays, has much more energy and penetrating power than ultraviolet radiation. It ionizes water and other molecules to form radicals (molecular fragments with unpaired electrons) that can disrupt DNA molecules and proteins. It is often used to sterilize pharmaceuticals and disposable medical supplies such as syringes, surgical gloves, catheters, sutures, and petri plates. It can also be used to retard spoilage in seafoods, meats, poultry, and fruits.

For more information on antigens, antibodies, and antibody production, see the following Learning Objects in your Lecture Guide:

  • Mutation; Unit 7 Section IIG

F. FILTRATION

Microbiological membrane filters provide a useful way of sterilizing materials such as vaccines, antibiotic solutions, animal sera, enzyme solutions, vitamin solutions, and other solutions that may be damaged or denatured by high temperatures or chemical agents. The filters contain pores small enough to prevent the passage of microbes but large enough to allow the organism-free fluid to pass through. The liquid is then collected in a sterile flask (Fig. 5). Filters with a pore diameter from 25 nm to 0.45 µm are usually used in this procedure. Filters can also be used to remove microorganisms from water and air for microbiological testing (see Appendix E).

PROCEDURE

A. OSMOTIC PRESSURE

MEDIA

2 plates of Trypticase Soy agar, 2 plates of 5% glucose agar, 2 plates of 10% glucose agar, 2 plates of 25% glucose agar, 2 plates of 5% NaCl agar, 2 plates of 10% NaCl agar, and 2 plates of 15% NaCl agar.

ORGANISMS

Trypticase Soy broth cultures of Escherichia coli and Staphylococcus aureus; a spore suspension of the mold Aspergillus niger.

A. OSMOTIC PRESSURE PROCEDURE (to be done by tables)

1. Divide one plate of each of the following media in half. Using your inoculating loop, streak one half of each plate with E. coli and the other half with S. aureus (see Fig. 6). Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

a. Trypticase Soy agar (control)
b. Trypticase Soy agar with 5% glucose
c. Trypticase Soy agar with 10% glucose
d. Trypticase Soy agar with 25% glucose
e. Trypticase Soy agar with 5% NaCl
f. Trypticase Soy agar with 10% NaCl
g. Trypticase Soy agar with 15% NaCl

2. Using a sterile swab, streak one plate of each of the following media with a spore suspension of the mold A. niger (see Fig. 7). Incubate the plates upside down at room temperature for 1 week.

a. Trypticase Soy agar with 15% NaCl

B. ULTRAVIOLET RADIATION

MEDIA

5 plates of Trypticase Soy agar

ORGANISM

Trypticase Soy broth culture of Serratia marcescens

ULTRAVIOLET RADIATION PROCEDURE (to be done by tables)

1. Using sterile swabs, streak all 5 Trypticase Soy agar plates with S. marcescens as follows:

a. Dip the swab into the culture.

b. Remove all of the excess liquid by pressing the swab against the side of the tube.

c. Streak the plate so as to cover the entire agar surface with organisms.

2. Expose 3 of the plates to UV light as follows:

a. Remove the lid of each plate and place a piece of cardboard with the letter "V" cut out of it over the top of the agar.

b. Expose the first plate to UV light for 1 second, the second plate for 3 seconds, and the third plate for 5 seconds.

c. Replace the lids and incubate the plates upside down at room temperature until the next lab period.

3. Leaving the lid on, lay the cardboard with the letter "V" cut out over the fourth plate and expose to UV light for 30 seconds. Incubate the plates upside down at room temperature with the other plates.

4. Use the fifth plate as a non-irradiated control and incubate the plates upside down at room temperature with the other plates.

NOTE: Do not look directly at the UV light as it may harm the eyes.

C. FILTRATION

MEDIUM

2 plates of Trypticase Soy agar

ORGANISM

Trypticase Soy broth cultures of Micrococcus luteus

FILTRATION PROCEDURE (demonstration)

1. Using alcohol-flamed forceps, aseptically place a sterile membrane filter into a sterile filtration device.

2. Pour the culture of M. luteus into the top of the filter set-up.

3. Vacuum until all the liquid passes through the filter into the sterile flask.

4. With alcohol-flamed forceps, remove the filter and place it organism-side-up on the surface of a Trypticase Soy agar plate.

5. Using a sterile swab, streak the surface of another Trypticase Soy agar plate with the filtrate from the flask.

6. Incubate the plates at 37°C until the next lab period.

RESULTS

A. Osmotic Pressure

Observe the 2 sets of plates from the osmotic pressure experiment and record the results below.

PlateEscherichia coliStaphylococcus aureusAspergillus
niger
Control

+++

+++

+++

5% NaCl
10% NaCl
15% NaCl
5% glucose
10% glucose
25% glucose

+ = Scant growth
++ = Moderate growth
+++ = Abundant growth
- = No growth

Escherichia coli and
Staphylococcus aureus
Aspergillus
niger

Control

Control

5% NaCl

5% NaCl

10% NaCl

10% NaCl

15% NaCl

15% NaCl

5% glucose

5% glucose

10% glucose

10% glucose

25% glucose

25% glucose

Conclusions:

B. Ultraviolet Radiation

1. Make drawings of the 5 plates from the ultraviolet light experiment.

Non-irradiated control

1 second UV exposure;
lid off

3 second UV exposure;
lid off

5 second UV exposure;
lid off

30 second UV exposure; lid on

Conclusions:

2. Observe the plates exposed to UV light for any non-pigmented colonies. Aseptically pick-off one of these nonpigmented colonies and streak it in a plate of Trypticase Soy agar. Incubate at room temperature until the next lab period.

3. After incubation, observe the plate you streaked with the nonpigmented colony.

Does the organism still lack chromogenicity?

What would account for this?

C. FILTRATION

Observe the 2 filtration plates and describe the results below.

Plate containing the filter
(growth or no growth)

Plate streaked with filtrate
(growth or no growth)

Conclusions:


Introduction, Methods, Definition of Terms

In the United States, approximately 46.5 million surgical procedures and even more invasive medical procedures&mdashincluding approximately 5 million gastrointestinal endoscopies&mdashare performed each year. 2 Each procedure involves contact by a medical device or surgical instrument with a patient&rsquos sterile tissue or mucous membranes. A major risk of all such procedures is the introduction of pathogens that can lead to infection. Failure to properly disinfect or sterilize equipment carries not only risk associated with breach of host barriers but also risk for person-to-person transmission (e.g., hepatitis B virus) and transmission of environmental pathogens (e.g., Pseudomonas aeruginosa).

Disinfection and sterilization are essential for ensuring that medical and surgical instruments do not transmit infectious pathogens to patients. Because sterilization of all patient-care items is not necessary, health-care policies must identify, primarily on the basis of the items&rsquo intended use, whether cleaning, disinfection, or sterilization is indicated.

Multiple studies in many countries have documented lack of compliance with established guidelines for disinfection and sterilization. 3-6 Failure to comply with scientifically-based guidelines has led to numerous outbreaks. 6-12 This guideline presents a pragmatic approach to the judicious selection and proper use of disinfection and sterilization processes the approach is based on well-designed studies assessing the efficacy (through laboratory investigations) and effectiveness (through clinical studies) of disinfection and sterilization procedures.

This guideline resulted from a review of all MEDLINE articles in English listed under the MeSH headings of disinfection or sterilization (focusing on health-care equipment and supplies) from January 1980 through August 2006. References listed in these articles also were reviewed. Selected articles published before 1980 were reviewed and, if still relevant, included in the guideline. The three major peer-reviewed journals in infection control&mdashAmerican Journal of Infection Control, Infection Control and Hospital Epidemiology, and Journal of Hospital Infection&mdashwere searched for relevant articles published from January 1990 through August 2006. Abstracts presented at the annual meetings of the Society for Healthcare Epidemiology of America and Association for professionals in Infection Control and Epidemiology, Inc. during 1997&ndash2006 also were reviewed however, abstracts were not used to support the recommendations.

Sterilization describes a process that destroys or eliminates all forms of microbial life and is carried out in health-care facilities by physical or chemical methods. Steam under pressure, dry heat, EtO gas, hydrogen peroxide gas plasma, and liquid chemicals are the principal sterilizing agents used in health-care facilities. Sterilization is intended to convey an absolute meaning unfortunately, however, some health professionals and the technical and commercial literature refer to &ldquodisinfection&rdquo as &ldquosterilization&rdquo and items as &ldquopartially sterile.&rdquo When chemicals are used to destroy all forms of microbiologic life, they can be called chemical sterilants. These same germicides used for shorter exposure periods also can be part of the disinfection process (i.e., high-level disinfection).

Disinfection describes a process that eliminates many or all pathogenic microorganisms, except bacterial spores, on inanimate objects (Tables 1 and 2). In health-care settings, objects usually are disinfected by liquid chemicals or wet pasteurization. Each of the various factors that affect the efficacy of disinfection can nullify or limit the efficacy of the process.

Factors that affect the efficacy of both disinfection and sterilization include prior cleaning of the object organic and inorganic load present type and level of microbial contamination concentration of and exposure time to the germicide physical nature of the object (e.g., crevices, hinges, and lumens) presence of biofilms temperature and pH of the disinfection process and in some cases, relative humidity of the sterilization process (e.g., ethylene oxide).

Unlike sterilization, disinfection is not sporicidal. A few disinfectants will kill spores with prolonged exposure times (3&ndash12 hours) these are called chemical sterilants. At similar concentrations but with shorter exposure periods (e.g., 20 minutes for 2% glutaraldehyde), these same disinfectants will kill all microorganisms except large numbers of bacterial spores they are called high-level disinfectants. Low-level disinfectants can kill most vegetative bacteria, some fungi, and some viruses in a practical period of time (&le10 minutes). Intermediate-level disinfectants might be cidal for mycobacteria, vegetative bacteria, most viruses, and most fungi but do not necessarily kill bacterial spores. Germicides differ markedly, primarily in their antimicrobial spectrum and rapidity of action.

Cleaning is the removal of visible soil (e.g., organic and inorganic material) from objects and surfaces and normally is accomplished manually or mechanically using water with detergents or enzymatic products. Thorough cleaning is essential before high-level disinfection and sterilization because inorganic and organic materials that remain on the surfaces of instruments interfere with the effectiveness of these processes. Decontamination removes pathogenic microorganisms from objects so they are safe to handle, use, or discard.

Terms with the suffix cide or cidal for killing action also are commonly used. For example, a germicide is an agent that can kill microorganisms, particularly pathogenic organisms (&ldquogerms&rdquo). The term germicide includes both antiseptics and disinfectants. Antiseptics are germicides applied to living tissue and skin disinfectants are antimicrobials applied only to inanimate objects. In general, antiseptics are used only on the skin and not for surface disinfection, and disinfectants are not used for skin antisepsis because they can injure skin and other tissues. Virucide, fungicide, bactericide, sporicide, and tuberculocide can kill the type of microorganism identified by the prefix. For example, a bactericide is an agent that kills bacteria. 13-18


Abstract

Summary: The responses of microorganisms (viruses, bacterial cells, bacterial and fungal spores, and lichens) to selected factors of space (microgravity, galactic cosmic radiation, solar UV radiation, and space vacuum) were determined in space and laboratory simulation experiments. In general, microorganisms tend to thrive in the space flight environment in terms of enhanced growth parameters and a demonstrated ability to proliferate in the presence of normally inhibitory levels of antibiotics. The mechanisms responsible for the observed biological responses, however, are not yet fully understood. A hypothesized interaction of microgravity with radiation-induced DNA repair processes was experimentally refuted. The survival of microorganisms in outer space was investigated to tackle questions on the upper boundary of the biosphere and on the likelihood of interplanetary transport of microorganisms. It was found that extraterrestrial solar UV radiation was the most deleterious factor of space. Among all organisms tested, only lichens (Rhizocarpon geographicum and Xanthoria elegans) maintained full viability after 2 weeks in outer space, whereas all other test systems were inactivated by orders of magnitude. Using optical filters and spores of Bacillus subtilis as a biological UV dosimeter, it was found that the current ozone layer reduces the biological effectiveness of solar UV by 3 orders of magnitude. If shielded against solar UV, spores of B. subtilis were capable of surviving in space for up to 6 years, especially if embedded in clay or meteorite powder (artificial meteorites). The data support the likelihood of interplanetary transfer of microorganisms within meteorites, the so-called lithopanspermia hypothesis.


Lab 18: Use of Physical Agents to Control of Microorganisms - Biology

PHYSICAL FACTORS THAT AFFECT MICROBIAL GROWTH

  • a MINIMUM GROWTH TEMPERATURE,
  • an OPTIMUM GROWTH TEMPERATURE,
  • and a MAXIMUM GROWTH TEMPERATURE.
  • PSYCHROPHILES grow best between -5 o C and 20 o C,
  • MESOPHILES grow best between 20 o C and 45 o C and
  • THERMOPHILES grow best at temperatures above 45 o C.
  • THERMODURIC organisms can survive high temperatures but don't grow well at such temperatures. Organisms which form endospores would be considered thermoduric.

Microbes display a great diversity in their ability to use and to tolerate oxygen. In part this is because of the paradoxical nature of oxygen which can be both toxic and essential to life.

  • OBLIGATE AEROBES rely on aerobic respiration for ATP and they therefore use oxygen as the terminal electron acceptor in the electron transport chain. Pseudomonas is an example of this group of organisms.
  • MICROAEROPHILES require O 2 for growth but they are damaged by normal atmospheric levels of oxygen and they don't have efficient ways to neutralize the toxic forms of oxygen such as superoxide (O 2- ) and hydrogen peroxide (H 2 O 2 ). The Streptococci are examples of this group.
  • OBLIGATE ANAEROBES will die in the presence of oxygen because they lack enzymes like superoxide dismutase and catalase. Superoxide dismutase catalyzes the following reaction:
    2O 2 - + 2H + ----> H 2 O 2 + O 2

    and catalase catalyzes:

    2H 2 O 2 ---> 2H 2 O + O 2

  • 1. The CO 2 jar or CANDLE JAR lowers O 2 levels below 10% (from 20%) and raises CO 2 levels from 0.03% to about 10%. In addition to lowered oxygen levels, some organisms flourish better in an enriched CO 2 environment. Such organisms are called CAPNOPHILIC.
  • 2. The ANAEROBE JAR uses hydrogen gas and a palladium catalyst to convert oxygen into water. An indicator strip impregnated with methylene blue is used to indicate when reducing conditions (anaerobic conditions) have been achieved. When reduced, methylene blue is colorless, when oxidized this dye is blue.
  • 3. THIOGLYCOLLATE is a chemical reducing agent often put into broth media to eliminate free oxygen.

Representative Anaerobic Pathogens:

1. Clostridium tetani - agent of tetanus, puncture wounds, produces a toxin which enters the spinal column and blocks the inhibitory spinal motor neurons. This produces generalized muscle spasms or spastic paralysis. The muscle of the jaw are often the first affected, hence the name LOCKJAW.

2. Clostridium botulinum - this soil organism is the causative agent of botulism which typically occurs after eating home canned alkaline vegetables which were not heated enough during canning. The neurotoxin blocks transmission across neuromuscular junctions and this results in flaccid paralysis.

3. Clostridium perfringes and Clostridium sporogenes - these organisms are associated with invasive infections known as GAS GANGRENE.

4. Clostridium difficile - the causative agent of pseudomembranous colitis, a side effect of antibiotic treatment which eliminates the normal flora.

The Streptococci - Microaerophiles

These organisms are all catalase negative, therefore the catalase test is useful in identification. They also have distinctive colonial morphology on blood agar which is differential for them. It is important to note if the colonies are alpha , beta , or gamma hemolytic .

1. Group A Streptococcus - Streptococcus pyogenes

This beta hemolytic organism is also bacitracin sensitive. It is the cause of strep throat, rheumatic fever, glomerulonephritis and scarlet fever.

2. Group D Streptococcus - Enterococcus - Streptococcus faecalis

This organism is a normal inhabitant of the large intestine. It is also a frequent cause of bladdder infections

3. Streptococcus pneumoniae .

This organism is a normal inhabitant of the repiratory tract. It is a frequent cause of pneumonia in people who have been compromised by other illness.


Liquid disinfection

Liquid disinfectants can be generally classified as halogens, acids, alkalis, heavy metal salts, quaternary ammonium compounds, phenolic compounds, aldehydes, ketones, alcohols, and amines.

Liquid disinfectant effectiveness varies with the organism, concentration, contact time, and other conditions of use. Select only liquid disinfectants that are confirmed to be effective against the organism(s) present. No liquid disinfectant is equally useful or effective under all conditions and for all viable agents.

Typical uses: Liquid disinfectants are used for surface decontamination and, when used in sufficient concentration, as a decontaminate for liquid wastes prior to final disposal in the sanitary sewer.

Precautions: The more chemically reactive a compound is, the more likely it is to be toxic and corrosive.

  • Consult Summary of Disinfectants (Excel) (PDF) for recommended disinfectants, their uses, and requirements. NOTE: The chart below provides guidelines for surface decontamination For chemical disinfection of liquid biohazardous waste, the only university-wide approved disinfectant for UCSD is bleach (1 part bleach to 9 parts liquid waste, 30 min. contact time, followed by sewering).
  • If your laboratory wishes to inquire about the use of alternative disinfectants to inactivate liquid biohazardous waste, please send an email with the following information. You will receive a response within five (5) working days regarding approval.
      • Material to be disinfected
      • Chemical to be used
      • Concentration of chemical
      • Contact time
      • Disposal method (sewering, hazardous waste pickup)

      Related links:


      Physical, Chemical, and Biological Methods for the Removal of Arsenic Compounds

      Arsenic is a toxic metalloid which is widely distributed in nature. It is normally present as arsenate under oxic conditions while arsenite is predominant under reducing condition. The major discharges of arsenic in the environment are mainly due to natural sources such as aquifers and anthropogenic sources. It is known that arsenite salts are more toxic than arsenate as it binds with vicinal thiols in pyruvate dehydrogenase while arsenate inhibits the oxidative phosphorylation process. The common mechanisms for arsenic detoxification are uptaken by phosphate transporters, aquaglyceroporins, and active extrusion system and reduced by arsenate reductases via dissimilatory reduction mechanism. Some species of autotrophic and heterotrophic microorganisms use arsenic oxyanions for their regeneration of energy. Certain species of microorganisms are able to use arsenate as their nutrient in respiratory process. Detoxification operons are a common form of arsenic resistance in microorganisms. Hence, the use of bioremediation could be an effective and economic way to reduce this pollutant from the environment.

      1. Introduction

      Arsenic is one of the toxic metalloids that exists in more than 200 different mineral forms, where 60% of them are normally arsenates 20% are sulphosalts and sulphides and the remaining 20% are arsenite, oxides, arsenide, silicates, and elemental arsenic [1, 2]. The intrusion of orogenesis and granitic magma have resulted in the formation of arsenopyrite [1]. Arsenic was first discovered by Albertus Magnus in the year 1250 [3]. Under natural condition, arsenic normally cycled at the earth surface where the breakdown of rocks has converted arsenic sulfides into arsenic trioxide [2, 4]. Furthermore, arsenic is known to have multiple oxidation states where they are present in either organic or inorganic compounds in an aquatic environment [5, 6]. Both Zobrist et al. [7] and Root et al. [8] indicated that the mobility of arsenic inorganic compound in contaminated aquatic and sediment environment is controlled by redox processes, precipitation, sorption, and dissolution processes. It is known that ferric iron phase plays an important role for the sorption of dissolved arsenate in oxic groundwater [8]. Meanwhile, the reduction of arsenate into arsenite in the transition from aerobic to anoxic pore waters is often mediated by microbial activity, which includes detoxification and metabolic mechanisms [8]. In another study, Saalfield and Bostick [9] proposed that the presence of calcium and bicarbonate from the byproducts of biological processes in the aquifers will enhance the release of arsenic and the correlations between calcium and bicarbonate with arsenic were then observed.

      Arsenic usually exists in four oxidation states: As −3 (arsine), As° (arsenic), As +3 (arsenite), and As +5 (arsenate) [4, 10]. In soil environment, arsenic is generally present in two oxidation states which are As +3 (arsenite) and As +5 (arsenate) and normally present as a mixture of As +3 (arsenite) and As +5 (arsenate) in air [2]. Of the two oxidation states, arsenate is the main species associated with soil arsenic contaminations, and it is often written as

      which is very similar to phosphate [11, 12]. Arsenate could act as a potential oxidative phosphorylation inhibitor. This is a cause for concern since oxidation phosphorylation is the main key reaction of energy metabolism in humans and metazoans [4]. Arsenite has been reported as the most toxic and soluble form of arsenic when compared to arsenate, and it can bind with reactive sulfur atoms present in many enzymes, including enzymes which are involved in respiration [4, 13]. Furthermore, it is known that soluble inorganic arsenic is often more toxic than the organic form [2]. Unlike arsenate and arsenite, arsine is often available as highly toxic gases such as (CH3)3 and H3As and often present at low concentration in the environment [4].

      Meanwhile, the average concentration of arsenic in fresh water is around 0.4 μg/L and could reach 2.6 μg/L in seawater [13]. However, the thermal activity in some places has caused high level of arsenic in waters with the concentration of arsenic in geothermal water in Japan ranging from 1.8 to 6.4 mg/L whereas the concentration of arsenic in New Zealand water could reach up to 8.5 mg/L [2, 29, 30]. In extreme cases, analysis from well drinking water in Jessore, Bangladesh, revealed that the levels of arsenic could reach up until 225 mg/L [31]. On the other hand, the concentration of arsenic in plants is solely depending on the amount of arsenic that the plant is being exposed to where the concentration of arsenic could range from less than 0.01 μg/g (dried weight) in the uncontaminated area to around 5 μg/g (dried weight) in the contaminated area [2]. Unlike plant, the concentration of arsenic in marine organisms and mammals has a wide range of variations ranging from 0.005 to 0.3 mg/kg in some crustaceans and molluscs, 0.54 μg/g in fish, over 100 μg/g in some shellfish, and less than 0.3 μg/g in humans and domestic animals [2]. Presence of humic acid in the shallow subsurface could affect the mobility of arsenic since humic acid could interact with aqueous arsenic for the formation of stable colloidal complexes that might play a prominent role in the enhancement of arsenic mobility. Furthermore, the combination of humic acid together with ferric hydroxide surface will lead to the formation of stable complexes that would compete with arsenic for its adsorption sites [32].

      2. Usage of Arsenic

      The first usage of arsenic in medicine could be dated around 2500 years ago where it was mainly consumed for the improvement of breathing problems as well as to give freshness, beauty, and plumpness figures in women [2]. Arsenic in the form of arsenical salvarsan (arsenic containing drug) was the initial antimicrobial agent used in the treatment of infectious diseases such as syphilis and sleeping sickness in 1908 [3]. This drug was specifically developed by Sahachiro Hata under the guidance of Paul Ehrlich in 1908 where they named the drug as arsphenamine no. 606 [33]. Meanwhile, arsenic in the form of arsenic trioxide (As2O3) is one of the most common forms of arsenic, which is often used in manufacturing and agriculture industry and for medical purposes such as in the treatment of acute promyelocytic leukemia [34]. Arsenic trioxide is also proven to be useful in criminal homicides due to its characteristic, which is tasteless, colorless, highly toxic, and soluble in water [2, 4]. The high usage of arsenic trioxide in suicide cases had made it to be often referred as the “inheritance powder” in the 18th century [4].

      During the 1970s, arsenic was mainly used in agriculture industry in the form of insecticide’s component in order to get rid of the insects [2, 13, 35]. Arsenic was also used as cotton desiccants and wood preservatives in United States [2]. The usage of arsenic as the cotton desiccant was introduced around year 1956 and was widely used due to its effectiveness and affordable price [36]. Besides that, arsenic was also being used in ceramic and glass industry, pharmaceutical industry, and food additives as well as pigments in paint [13, 34]. Meanwhile, arsenic in the form of 4-aminoben-zenearsenic acid (p-arsenilic acid, p-ASA) has been used as animals food additive for feeding of boiler chickens [37].

      3. Toxicity of Arsenic

      It has been noticed that the extensive usage of arsenic in the industrial and agrochemical applications is of few causes of groundwater and sediment arsenic contamination in the environment [6, 38] in which effects are much smaller compared to the natural causes [39]. The presence of arsenic in soil and water has become an increasing problem in many countries around the world, including Bangladesh, India, Chile, and Taiwan [2, 40, 41], and natural geological source is one of the main causes of contamination [34]. Consumption of drinking water that has been contaminated by hazardous level of arsenic will lead to a wide range of diseases such as arsenic dermatosis, lung cancer, liver cancer, uterus cancer, skin cancer and occurrence of skin, and bladder and hepatocellular carcinoma that will result in slow and painful death [1, 2, 41–43]. In Southwestern Taiwan, the human consumption of artesian well waters which contains high concentration of arsenic has also led to Blackfoot disease, which is an endemic peripheral vascular disease in that area [40]. In China, up to the year 2012, 19 provinces had been found to have As concentration in drinking water exceeding the standard level (0.05 mg/L). Inner Mongolia, Xinjiang, and Shanxi Provinces are historical well-known “hotspots” of geogenic As-contaminated drinking water [44].

      Deltaic plain contaminated groundwater of Ganges-Meghna-Brahmaputra rivers in Bangladesh and West Bengal had resulted in an alarming environmental problem as this water is often consumed by people who live in that area [6, 45]. The presence of aqueous arsenic is mainly due to rock weathering as well as sediment deposition and downstream transport of rich mineral arsenic that was originally present in Himalayas [4]. Massive constructions of wells which are meant to supply an improved quality of water with waterborne pathogens free to the people living in this area had created another problem as the ground water in that area was arsenic contaminated [4]. In Nepal, arsenic (As) contamination was a major issue in water supply drinking systems especially in high density population such as Terai districts. The local inhabitants still use hand tube and dug wells (with hand held pumps that are bored at shallow to medium depth) for their daily water requirements [46]. The results of the analysis on 25,058 samples tested in 20 districts, published in the report of arsenic in Nepal, demonstrated that 23% of the samples were containing 10–50 μg/L of As, and 8% of the samples were containing more than 50 μg/L of As. Recent status from over 737,009 samples tested has shown that 7.9% and 2.3% were contaminated by 10–50 μg/L and >50 μg/L of As, respectively [46]. Other places reporting the ground water arsenic contamination include south West Coast of Taiwan Antofagasta in Chile six areas of Region Lagunera located in the central part of North Mexico Monte Quemado Cordoba province in Argentina Millard County in Utah, United States Nova Scotia in Canada and Inner Mongolia, Qinghai, Jilin, Shanxi, Xinjiang Uygur A.R., Ningxia, Liaoning, and Henan provinces in China [2].

      Accidental ingestion of pesticides or insecticides containing arsenic will also result in an acute arsenic poisoning which sometimes could lead to mortality when 100 mg to 300 mg of doses were being consumed [1]. The symptoms of acute arsenic poisoning are vomiting, abdominal pain, diarrhea, and cramping, which will then cause renal failure, haematological abnormalities such as leukemia and anemia, pulmonary oedema, and respiratory failure, and it could further lead to shock, coma, and death [1, 2, 34]. In another study, Lai et al. [47] reported that the consumption of water contaminated with arsenic will increase the risk of diabetes mellitus by twofold. In US, prevalence of diabetes increased among people having urine arsenic concentrations in more than 20% of the general population [48]. Arsenic contamination from industrial sources has also led to skin manifestation of chronic arsenic poisoning, which affected 19.9% of the human populations living in Ron Phibun, Thailand [2]. On the other hand, arsenic poisoning caused by ingestion of food (especially seafood product) and beverages contaminated by arsenic has been reported in Japan, England, Germany, and China [2]. In Campinas, Brazil, 116 samples of seafood (used for sashimi making) from Japanese restaurants have been evaluated for the presence of As [49]. Several samples were found with percentage above the maximum limit permitted by European regulations including 90% tuna, 48% salmon, 31% mullet, and 100% octopus. It was concluded that the octopus was the sashimi which most contributed to arsenic. In other case, the arsenic concentration in rice was found to be high in Bangladesh [50].

      Phosphate fertilization is suggested to lower the arsenate uptake in plants because both compounds enter the rice via the same transporter. However, there are arguments in certain cases because under flooding conditions, As is present as arsenite, which cannot compete with phosphate furthermore, phosphate increases As mobility because it competes with arsenate for the adsorption site on Fe-oxides/hydroxides [51]. Presence of over 1.0 μg/g arsenic concentration in hair, 20 to 130 μg/g in nails, and over 100 μg per day in urine is an indication of arsenic poisoning [2]. Significant correlation was also observed with levels in human urine, toenail, and hair samples [31]. A meta-analysis assessing the effects of exposure to arsenic suggests that 50% increment of arsenic levels in urine would be associated with 0.4 decrement in the intelligence quotient (IQ) of children aged 5–15 years [52]. Arsenic uptake is adventitious because arsenate and arsenite are chemically similar to the required nutrients [53]. At neutral pH, the trivalent forms of these metalloids are structurally similar to glycerol, and hence they can enter cells through aquaporins [54].

      4. Technologies/Methods for the Removal of Arsenic from Environment

      According to World Health Organisation (WHO) standard set in the year 1993, the maximum limit of arsenic contamination in drinking water is 10 μg/L or 10 ppb. [1]. This limit was later adopted by European Union in the year 1998 (council directive 98/83/EC), transposed by Portuguese legislation by Law Decree (DL) number 236/2001 [1, 55]. In the year 2006, United States has also adopted the WHO standard for lowering the federal drinking water standard for maximum limit of arsenic from 50 μg/L to 10 μg/L [8]. Technologies for removing arsenic from the environment should meet several basic technical criteria that include robustness, no other side effect on the environment, and the ability to sustain water supply systems for long terms and meet the quality requirement of physical chemical, and microbiological approaches [1]. Currently, there are many methods for removing arsenic from the soil contaminated with arsenic, which could be divided into three categories, including physical, chemical, and biological approaches [14].

      In the physical approaches, the concentration of arsenic in soil could be reduced by mixture of both contaminated and uncontaminated soils together that will lead to an acceptable level of arsenic dilution [14]. Soil washing is another method which is grouped under physical approaches whereby arsenic contaminated soil will be washed with different concentration of chemicals such as sulfuric acid, nitric acid, phosphoric acid, and hydrogen bromide [14]. The choice of chemicals used for extractant and high cost have often restricted the usage of soil washing into a smaller-scale operations as it is the disadvantages of using soil washing method [14]. Meanwhile, cement can immobilise soluble arsenites and has been successfully used to stabilise As-rich sludges which may be suitable for treating sludges generated from precipitative removal units [15]. Furthermore, the disposal of water treatment wastes containing As, with a particular emphasis on stabilisation/solidification (S/S) technologies, has been assessed for their appropriateness in treating As containing wastes. In this process, brine resulting from the regeneration of activated alumina filters is likely to accelerate cement hydration. Furthermore, additives (surfactants, cosolvents, etc.) have also been used to enhance the efficiencies of soil flushing using aqueous solutions as water solubility is the controlling mechanism of contaminant dissolution. The usage of surfactant alone gives about 80–85% of efficiencies in laboratory experiments. Studies indicated that when soil flushing is applied in the field, efficiency can vary from 0% to almost 100%. It often gives moderate efficiencies by using only one product (surfactant, cosolvent, and cyclodextrin). On the other hand, the use of more complex methods with polymer injection leads to higher efficiencies [16].

      The current available chemical remediation approaches mainly involving methods such as adsorption by using specific media, immobilization, modified coagulation along with filtration, precipitations, immobilizations, and complexation reactions [1, 14]. The coagulation along with filtration method for removing arsenic from contaminated sources is quite economic but often displayed lower efficiencies (<90%) [1]. The formation of stable phases, for example, insoluble FeAsO4 (and hydrous species of this compound such as scorodite, FeAsO4

      2H2O), is beneficial for the stabilization procedure [17]. Furthermore, the use of selective stabilizing amendments is a challenging task as the majority of polluted sites are contaminated with multiple metal(loid)s. Nanosized oxides and Fe(0) (particle size of 1 to 100 nm) are another possible enhancement for the stabilization method [17]. Natural nanoparticulate oxides are important scavengers of contaminants in soils [56] and due to their reactive and relatively large specific surface area (tens to hundreds m 2 /g), engineered oxide nanoparticles are promising materials for the remediation of soils contaminated with inorganic pollutants [18, 19]. It is reported that chemical remediation gained popularity because of its high success rate, but it could be expensive when someone would like to remediate a large area [14]. In contrast, biological remediation or bioremediation of soils contaminated with either inorganic or organic arsenic present in pesticides and hydrocarbons have been widely accepted in some places [14]. Even though bioremediation suffers several limitations, these approaches have been gaining interest for the remediation of metal(loid) contaminated soils due to their cost effectiveness [14]. Basically, bioremediation technology could be divided into subcategories: intrinsic bioremediation and engineered bioremediation [14]. Intrinsic bioremediation is generally referred to as the degradation of arsenic by naturally occurring microorganisms without intervention by human, and this method is more suitable for remediation of soil with a low level of contaminants [14]. Engineered bioremediation often relies on intervention of human for optimizing the environment conditions to promote the proliferation and activity of microorganisms that lived in that area. As a result, the usage of engineered bioremediation method is more favorable in the highly contaminated area [14].

      Mechanism for arsenic detoxification can be divided into four which known as uptake of As(V) in the form of arsenate by phosphate transporters, uptake of As(III) in the form of arsenite by aquaglyceroporins, reduction of As(V) to As(III) by arsenate reductases, and extrusion or sequestration of As(III) [57]. AQPs have been shown to facilitate diffusion of arsenic [53, 54]. The microbial oxidation of As in Altiplano basins (rivers in northern Chile) was demonstrated by Leiva et al. [20]. The oxidation of As (As(III) to As(V)) is a critical transformation [58] because it favors the immobilization of As in the solid phase. As(III) was actively oxidized by a microbial consortium, leading to a significant decrease in the dissolved As concentrations and a corresponding increase in the sediment’s As concentration downstream of the hydrothermal source. In situ oxidation experiments demonstrated that the As oxidation required a biological activity, and microbiological molecular analysis had confirmed the presence of As(III)-oxidizing groups (aro A-like genes) in the system. In addition, the pH measurements and solid phase analysis strongly suggest that As removal mechanism must involve adsorption or coprecipitation with Fe-oxyhydroxides. Taken together, these results indicated that the microorganism-mediated As oxidation contributed to the attenuation of As concentrations and the stabilization of As in the solid phase, therefore controlling the amount of As transported downstream [20]. Since most of the cases of arsenic poisoning are due to the consumption of water contaminated by arsenic, the process of cleaning up or reducing arsenic concentration in water becomes very important. Methods used in reducing arsenic levels in water are primarily divided into (i) physiochemical methods, which include filtration or coagulation sedimentation, osmosis or electrodialysis, adsorptions, and chemical precipitations and (ii) biological methods such as phytoremediation by using aquatic plants or microbial detoxification of arsenic [14].

      Generally, two approaches are mainly employed in the phytoremediation method. The first approach uses “free-floating plants such as water hyacinth” that could adsorb metal(loid)s and the plants would be removed from the pond once the equilibrium state is achieved [14]. The second approach uses aquatic rooted plants (i.e., Agrostis sp., Pteris vittata, Pteris cretica, and others) to remove arsenic from bed filters and from water [14, 21–23]. Yang et al. [23] stated that the addition of arsenate reducing bacteria will promote the growth of P. vittata in soil. Two important processes in the removal of arsenic from water by microorganisms are biosorption and biomethylation [14]. It is reported that biomethylation (by As(III) S-adenosylmethionine methyltransferase) is the reliable biological process for removing arsenic from aquatic media [14].

      Recently, the arsenite (As(III)) S-adenosylmethionine methyltransferase (ArsM) gene has been inserted into the chromosome of Pseudomonas putida KT2440 for potential bioremediation of environmental arsenic [59]. The first structure of As(III) S-adenosylmethionine methyltransferase by X-ray crystallography was described by [60]. In this enzyme, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508 [61]. Substitution of any of the three led to the loss of As(III) methylation [61]. The relationship between the arsenic and S-adenosylmethionine binding sites to a final resolution of

      1.6 Å. As(III) binding causes little change in conformation, but binding of SAM reorients helix α4 and a loop (residues 49–80) towards the As(III) binding domain, positioning the methyl group to be transfer to the metalloid [60].

      5. Arsenic Resistant Microorganisms

      Studies of bacterial growth at high arsenic-phosphorus ratios demonstrated that high arsenic concentrations can be tolerated relatively and that it can be involved in vital functions in the cell [62]. Corynebacterium glutamicum survives arsenic stress with two different classes of arsenate reductases. Cg-ArsC1 and Cg-ArsC2 are the single-cysteine monomeric enzymes coupled to the mycothiol/mycoredoxin redox pathway using a mycothiol transferase mechanism, while Cg-ArsC1’ is a three-cysteine containing homodimer that uses a reduction mechanism linked to the thioredoxin pathway [63]. The presence of naturally occurring arsenate and arsenite in water and soil environment which could enter the cells by the phosphate-transport system has given pressure for microorganisms to maintain their arsenic detoxification systems for surviving purposes. One of the commonest forms of arsenic resistance in microorganisms is by detoxification operons, which are encoded on genomes or plasmids [64].

      Most of the detoxification operons consist of three genes, which are known as arsC (reduction of arsenate to arsenite), arsR (transcriptional repressor), and arsB (can also be a subunit of the ArsAB As(III)-translocating ATPase, an ATP-driven efflux pump) [3, 53, 64]. Moreover, some detoxification operons also contain two additional genes (arsD-metallochaperone and arsA-ATPase) [3, 64]. The ArsD metallochaperone binds cytosolic As(III) and transfers it to the ArsA subunit of the efflux pump [53]. In normal process, arsenate that enters the cell will be reduced to arsenite by ArsC gene before it is transported out of the cell by ArsB gene [10, 64]. As a result, a more toxic form of arsenic will be introduced into the environment. In another study, Villegas-Torres et al. [65] indicated that the arsenic resistance ability in Bacillus sphaericus could be due to the presence of arsC gene, which could be horizontally transferred between microorganisms isolated from Columbian oil polluted soil that contain high arsenic levels.

      The other reduction of arsenate to arsenite by microorganisms is via dissimilatory reduction mechanism that could be carried out in facultative anaerobe or strict anaerobe condition with the arsenate acting as the terminal electron acceptor [24]. These microorganisms have the ability to oxidize inorganic (sulfide and hydrogen) and organic (e.g., formate, aromatics, and lactase acetate) as an electron donor which will lead to the production of arsenite, and they were normally named dissimilatory arsenate-respiring prokaryotes (DARPs) [4].

      Two other families of arsenate reductase are known as thioredoxin (Trx) clade and Arr2p arsenate reductase. It is reported that Trx clade is linked with arsC arsenate reductase gene while Arr2p is related to different class of larger protein tyrosine phosphatases [66]. Zargar et al. [67] reported that ArxA (arsenite oxidase) enzymes, which are present in Alkalilimnicola ehrlichii MLHE-1 strain (a chemoliautotroph bacteria) could couple with arsenite oxidation as well as nitrate reduction. Different types of bacteria with the ability of resisting arsenic are Rhodococcus, Arthrobacter, Acinetobacter, Agrobacterium, Staphylococcus, Escherichia coli, Thiobacillus, Achromobacter, Alcaligene, Pseudomonas, Microbacterium oxydans, Ochrobactrum anthropi, Cupriavidus, Desulfomicrobium, Cyanobacteria, Sulfurospirillum, Wolinella, Citrobacter, Agrobacterium, an arsenic reducing bacteria from Flavobacterium-Cytophaga group, Scopulariopsis koningii, Fomitopsis pinicola, Penicillium gladioli, Fusarium oxysporum meloni, Fucus gardneri, Bosea sp., Psychrobacter sp., Polyphysa peniculus, Methanobacterium, Bradyrhizobium, Rhodobium, Sinorhizobium, and Clostridium [13, 21, 53, 68–71].

      Liao et al. [69] reported that 11 arsenic reducing bacteria strains from seven different genera (i.e., Pseudomonas, Psychrobacter, Citrobacter, Bacillus, Bosea, Vibrio, and Enterobacter) were isolated from environmental groundwater samples collected from well AG1 in Southern Yunlin County, west-central Taiwan. In Liao et al. [69] report, they indicated that diverse community of microorganisms holds a significant impact in the biotransformation of arsenic that is present in the aquifer, and these communities of bacteria are well adapted to high arsenic concentrations that are present in the water. In another study, Mumford et al. [72] reported that Alkaliphilus oremlandii and ferum reducing bacteria such as Geobacter species were present in arsenic rich groundwater beneath a site-specific site (C6) on Crosswicks Creek, New Jersey. Other bacteria with the ability of reducing arsenate to arsenite are Sulfurospirillum barnesii and Sulfurospirillum arsenophilum from the ε-proteobacteria as well as Pyrobaculum arsenaticum from Thermoproteales order and Chrysiogenes arsenatis [4, 10, 73, 74]. Afkar [10] reported that the reduction of arsenate to arsenite by S. barnesii strain SeS-3 is associated with the membrane cell where this resistance mechanism is encoded by a single operon that consists of arsenite ion-inducible repressor. Besides that, Afkar [10] also indicated that S. barnesii strain SeS-3 reduced arsenate to arsenite under anaerobic condition using arsenate as terminal electron acceptors while lactate as the carbon source.

      In another study, Youssef et al. [75] reported that both Neisseria mucosa and Rahnella aquatilis are able to reduce arsenate and selenate. In their study, both N. mucosa and R. aquatilis were grown in a neutral pH medium (pH 7) containing five mM sodium arsenates where the sodium lactate acts as an electron donor while N. mucosa and R. aquatilis act as the electron acceptor organisms. Although both N. mucosa and R. aquatilis strains studied are able to grow at higher pH medium (pH10), their growth rate decreased drastically (reduction of 43% in N. mucosa and 67.2% in R. aquatilis) and has been observed [75]. Meanwhile, archaebacterium Sulfolobus acidocaldarius strain BC, Alcaligenes faecalis, Shewanella algae, β-proteobacteria strain UPLAs1, Alcaligenes faecalis, Comamonas terrae sp. nov, some heterotrophic bacteria (Herminiimonas arsenicoxydans), and chemoliautotrophic bacteria are reported to have the ability to oxidize arsenite to a less toxic arsenate [4, 14, 25–28]. In this case, arsenite will often serve as an electron donor for reducing the nitrate or oxygen that will produce energy in order to fix carbon dioxide [26]. Two genes, aoxA and aoxB encoding for arsenite oxidase played an essential role in the oxidation of arsenite into arsenate [27]. The insertion of mini-Tn5::lacZ2 transposon in aoxA or aoxB gene will stop to arsenite oxidation process [27].

      In another review by Silver and Phung [12], they indicated that both asoA and asoB genes which encoded for large molybdopterin-containing and small Rieske (2Fe-2S) cluster the subunit of oxidation of arsenite in Alcaligenes faecalis. They identified that the upstreams of asoB consist of 15 genes while the downstreams of asoA consist of six genes, which are involved in arsenic resistance and metabolisms [12]. Besides bacteria, certain species of algae such as Fucus gardneri and Chlorella vulgaris are also known to have the ability to accumulate arsenic [76, 77].

      Table 1 shows the advantages and disadvantages of physical, chemical, and biological methods for the removal of arsenic compounds. Physical method exhibits the simplest choice, but it was however limited to small scale operations. Chemical method had gained popularity by its high success rate however, the remediation area can be exposed to other types of chemical contaminants. The usage of biological and phytoremediation methods might be the most practical methods for a small area but more research needs to be carried out especially in methylations, reduction, and oxidation using microorganisms for more effective method to remove the arsenic compound as they have a high potential application in the future.

      Previously, bacterial biosensors (whole-cell) were being used to detect inorganic arsenic [78]. Biosensor technology was widely studied by using potentiometry, amperometry, and conductometry [79–81]. Only a few studies were carried out based on capacitometry [82] especially by using DNA and antibodies [83, 84]. Previously, capacitive sensor using enzyme was introduced for toxin detection [82]. A biosensor selective for the trivalent organoarsenicals methyl arsenate and phenyl arsenite over inorganic arsenite was reported by Chen et al. [78] which may be useful for detecting degradation of arsenic-containing herbicides and growth promoters. A surface plasmon resonance biosensor for the study of trivalent arsenic was also reported by Liu et al. [85]. This biosensor indicates that the 3D hydrogel-nanoparticle coated sensors exhibited a higher sensitivity than that of the 2D AuNPs decorated sensors. It was shown that binding of As(III) into ArsA was greatly facilitated by the presence of magnesium ion and ATP. For future research, biosensor based on capacitometry using enzymes [82] such as As(III) S-adenosylmethionine methyltransferase for arsenic detection remains interesting to be explored.

      6. Conclusion

      Arsenic is a metalloid that causes harm to humans and environments. However, certain species of prokaryotes have the abilities to use arsenic either through oxidation or reduction process for energy conservation and growth purposes. It is important to remove and reduce this pollutant from the environment through different approaches such as physical, chemical, and biological. The use of bioremediation to remove and mobilize arsenic from contaminated soils and aquifers could be an effective and economic way since a wide range of microorganisms have been found to be successfully degrading this pollutant from the environment.

      Conflict of Interests

      The authors declare that there is no conflict of interests regarding the publication of this paper.

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      Copyright

      Copyright © 2014 K. T. Lim et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


      4 Answers 4

      This is actually an interesting question! Let me answer both the parts separately, taking the example of Listerine ® mouthwash.

      Is an antiseptic's inability to eliminate 100% of all germs due to its "chemistry", or is it due to physical factors?

      In most of the cases, this is due to the physical factors. Clearly, your mouth is not a flat surface. It has many depressions and elevations. And these irregularities are the perfect hotels for all kinds of pathogens. Also, it is difficult for most chemicals to reach those spots and stay there for long enough so that they can act on the pathogens. Thus, most of these chemicals would be unable to kill 100% pathogens because of their inability to reach all of them. Also, in rare cases, it might also be due to chemical factors(!) Yes, I'm talking about antibiotic resistance. And in that case, it would be practically impossible to kill 100% pathogens, no matter whether they're hidden or exposed.

      Do antiseptics/mouthwashes/handwashes even kill 99.9% of all germs in the first place? Or is it (as I strongly suspect) an example of marketing fraud?

      For that, lets first see what Listerine ® contains. As given on its website, its main ingredients are (I'm simply copying what they write on their page, followed by checking whether its true):

      Eucalyptol – with antibacterial properties, this eucalyptus-derived essential oil works as an anti-fungal agent within the mouth.

      Methol – this natural oil as germ-killing abilities to help halt the growth of bacteria.

      Thymol – this powerful oil is derived from the ajowan herb, and helps decrease the risk of gum disease.

      Methyl Salicylate – for minty freshness from morning til night, the flavouring agent in this essential oil is, well… essential!

      We just need to know whether these ingredients really work or not (why? We'll talk about this later on) and I'll skip methyl salicylate for this (they don't even claim that it is antimicrobial). Beginning with eucalyptol, it has been shown that oil of Eucalyptus globulus (of which eucalyptol is a component) has antimicrobial properties against Escherichia coli and Staphylococcus aureus (see Bachir et al, 2012). It has also been shown to possess antifungal properties (Safaei-Ghomi et al, 2010). Coming to methol, it has also been shown to have antibacterial properties against various Staphylococcus and Lactobacillus species (Freires et al, 2015). Finally, there have been lots of studies about the antimicrobial properties of thymol. You can check out the Wikipedia page for information.

      So, where is the percentage? The point is, the exact percentage depends on a lot of factors. When an enterprise, such as Listerine ® , claims that their product has been shown to be 99.9% (or any number) efficient against bacteria, they need to cite the particular study through which they claim this number. But they can not, in any case, be definite that their product will be 99.9% effective everytime. How effective a product is also depends on the conditions under which it is tested. Mostly, these tests are performed on a petri dish in a laboratory, something very different from your mouth. Thus, although they can claim that their product is scientifically proven to be 99.9% effective, they cannot claim it to be 99.9% effective when you use it. Again, they cannot claim 100% effectiveness because this makes them liable (saying our product has been shown to be 100% effective requires them to show that even a single microbe did not survive on the petri dish they used for experiment). Also, this gives them a way to escape in case anybody complaints about their product not being effective (since they caught infection even after using their product). In such situation, they can easily say that their product is not 100% efficient!


      Microbial Degradation of Petroleum Hydrocarbon Contaminants: An Overview

      One of the major environmental problems today is hydrocarbon contamination resulting from the activities related to the petrochemical industry. Accidental releases of petroleum products are of particular concern in the environment. Hydrocarbon components have been known to belong to the family of carcinogens and neurotoxic organic pollutants. Currently accepted disposal methods of incineration or burial insecure landfills can become prohibitively expensive when amounts of contaminants are large. Mechanical and chemical methods generally used to remove hydrocarbons from contaminated sites have limited effectiveness and can be expensive. Bioremediation is the promising technology for the treatment of these contaminated sites since it is cost-effective and will lead to complete mineralization. Bioremediation functions basically on biodegradation, which may refer to complete mineralization of organic contaminants into carbon dioxide, water, inorganic compounds, and cell protein or transformation of complex organic contaminants to other simpler organic compounds by biological agents like microorganisms. Many indigenous microorganisms in water and soil are capable of degrading hydrocarbon contaminants. This paper presents an updated overview of petroleum hydrocarbon degradation by microorganisms under different ecosystems.

      1. Introduction

      Petroleum-based products are the major source of energy for industry and daily life. Leaks and accidental spills occur regularly during the exploration, production, refining, transport, and storage of petroleum and petroleum products. The amount of natural crude oil seepage was estimated to be 600,000 metric tons per year with a range of uncertainty of 200,000 metric tons per year [1]. Release of hydrocarbons into the environment whether accidentally or due to human activities is a main cause of water and soil pollution [2]. Soil contamination with hydrocarbons causes extensive damage of local system since accumulation of pollutants in animals and plant tissue may cause death or mutations [3]. The technology commonly used for the soil remediation includes mechanical, burying, evaporation, dispersion, and washing. However, these technologies are expensive and can lead to incomplete decomposition of contaminants.

      The process of bioremediation, defined as the use of microorganisms to detoxify or remove pollutants owing to their diverse metabolic capabilities is an evolving method for the removal and degradation of many environmental pollutants including the products of petroleum industry [4]. In addition, bioremediation technology is believed to be noninvasive and relatively cost-effective [5]. Biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be removed from the environment [6] and is cheaper than other remediation technologies [7].

      The success of oil spill bioremediation depends on one’s ability to establish and maintain conditions that favor enhanced oil biodegradation rates in the contaminated environment. Numerous scientific review articles have covered various factors that influence the rate of oil biodegradation [7–12]. One important requirement is the presence of microorganisms with the appropriate metabolic capabilities. If these microorganisms are present, then optimal rates of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate concentrations of nutrients and oxygen are present and that the pH is between 6 and 9. The physical and chemical characteristics of the oil and oil surface area are also important determinants of bioremediation success. There are the two main approaches to oil spill bioremediation: (a) bioaugmentation, in which known oil-degrading bacteria are added to supplement the existing microbial population, and (b) biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition of nutrients or other growth-limiting cosubstrates.

      The success of bioremediation efforts in the cleanup of the oil tanker Exxon Valdez oil spill of 1989 [13] in Prince William Sound and the Gulf of Alaska created tremendous interest in the potential of biodegradation and bioremediation technology. Most existing studies have concentrated on evaluating the factors affecting oil bioremediation or testing favored products and methods through laboratory studies [14]. Only limited numbers of pilot scale and field trials have provided the most convincing demonstrations of this technology which have been reported in the peer-reviewed literature [15–18]. The scope of current understanding of oil bioremediation is also limited because the emphasis of most of these field studies and reviews has been given on the evaluation of bioremediation technology for dealing with large-scale oil spills on marine shorelines.

      This paper provides an updated information on microbial degradation of petroleum hydrocarbon contaminants towards the better understanding in bioremediation challenges.

      2. Microbial Degradation of Petroleum Hydrocarbons

      Biodegradation of petroleum hydrocarbons is a complex process that depends on the nature and on the amount of the hydrocarbons present. Petroleum hydrocarbons can be divided into four classes: the saturates, the aromatics, the asphaltenes (phenols, fatty acids, ketones, esters, and porphyrins), and the resins (pyridines, quinolines, carbazoles, sulfoxides, and amides) [19]. Different factors influencing hydrocarbon degradation have been reported by Cooney et al. [20]. One of the important factors that limit biodegradation of oil pollutants in the environment is their limited availability to microorganisms. Petroleum hydrocarbon compounds bind to soil components, and they are difficult to be removed or degraded [21]. Hydrocarbons differ in their susceptibility to microbial attack. The susceptibility of hydrocarbons to microbial degradation can be generally ranked as follows: linear alkanes

      branched alkanes small aromatics cyclic alkanes [6, 22]. Some compounds, such as the high molecular weight polycyclic aromatic hydrocarbons (PAHs), may not be degraded at all [23].

      Microbial degradation is the major and ultimate natural mechanism by which one can cleanup the petroleum hydrocarbon pollutants from the environment [24–26]. The recognition of biodegraded petroleum-derived aromatic hydrocarbons in marine sediments was reported by Jones et al. [27]. They studied the extensive biodegradation of alkyl aromatics in marine sediments which occurred prior to detectable biodegradation of n-alkane profile of the crude oil and the microorganisms, namely, Arthrobacter, Burkholderia, Mycobacterium, Pseudomonas, Sphingomonas, and Rhodococcus were found to be involved for alkylaromatic degradation. Microbial degradation of petroleum hydrocarbons in a polluted tropical stream in Lagos, Nigeria was reported by Adebusoye et al. [28]. Nine bacterial strains, namely, Pseudomonas fluorescens, P. aeruginosa, Bacillus subtilis, Bacillus sp., Alcaligenes sp., Acinetobacter lwoffi, Flavobacterium sp., Micrococcus roseus, and Corynebacterium sp. were isolated from the polluted stream which could degrade crude oil.

      Hydrocarbons in the environment are biodegraded primarily by bacteria, yeast, and fungi. The reported efficiency of biodegradation ranged from 6% [29] to 82% [30] for soil fungi, 0.13% [29] to 50% [30] for soil bacteria, and 0.003% [31] to 100% [32] for marine bacteria. Many scientists reported that mixed populations with overall broad enzymatic capacities are required to degrade complex mixtures of hydrocarbons such as crude oil in soil [33], fresh water [34], and marine environments [35, 36].

      Bacteria are the most active agents in petroleum degradation, and they work as primary degraders of spilled oil in environment [37, 38]. Several bacteria are even known to feed exclusively on hydrocarbons [39]. Floodgate [36] listed 25 genera of hydrocarbon degrading bacteria and 25 genera of hydrocarbon degrading fungi which were isolated from marine environment. A similar compilation by Bartha and Bossert [33] included 22 genera of bacteria and 31 genera of fungi. In earlier days, the extent to which bacteria, yeast, and filamentous fungi participate in the biodegradation of petroleum hydrocarbons was the subject of limited study, but appeared to be a function of the ecosystem and local environmental conditions [7]. Crude petroleum oil from petroleum contaminated soil from North East India was reported by Das and Mukherjee [40]. Acinetobacter sp. was found to be capable of utilizing n-alkanes of chain length C10–C40 as a sole source of carbon [41]. Bacterial genera, namely, Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia, and Mycobacterium isolated from petroleum contaminated soil proved to be the potential organisms for hydrocarbon degradation [42]. The degradation of poly-aromatic hydrocarbons by Sphingomonas was reported by Daugulis and McCracken [43].

      Fungal genera, namely, Amorphoteca, Neosartorya, Talaromyces, and Graphium and yeast genera, namely, Candida, Yarrowia, and Pichia were isolated from petroleum-contaminated soil and proved to be the potential organisms for hydrocarbon degradation [42]. Singh [44] also reported a group of terrestrial fungi, namely, Aspergillus, Cephalosporium, and Pencillium which were also found to be the potential degrader of crude oil hydrocarbons. The yeast species, namely, Candida lipolytica, Rhodotorula mucilaginosa, Geotrichum sp, and Trichosporon mucoides isolated from contaminated water were noted to degrade petroleum compounds [45].

      Though algae and protozoa are the important members of the microbial community in both aquatic and terrestrial ecosystems, reports are scanty regarding their involvement in hydrocarbon biodegradation. Walker et al. [51] isolated an alga, Prototheca zopfi which was capable of utilizing crude oil and a mixed hydrocarbon substrate and exhibited extensive degradation of n-alkanes and isoalkanes as well as aromatic hydrocarbons. Cerniglia et al. [52] observed that nine cyanobacteria, five green algae, one red alga, one brown alga, and two diatoms could oxidize naphthalene. Protozoa, by contrast, had not been shown to utilize hydrocarbons.

      3. Factors Influencing Petroleum Hydrocarbon Degradation

      A number of limiting factors have been recognized to affect the biodegradation of petroleum hydrocarbons, many of which have been discussed by Brusseau [53]. The composition and inherent biodegradability of the petroleum hydrocarbon pollutant is the first and foremost important consideration when the suitability of a remediation approach is to be assessed. Among physical factors, temperature plays an important role in biodegradation of hydrocarbons by directly affecting the chemistry of the pollutants as well as affecting the physiology and diversity of the microbial flora. Atlas [54] found that at low temperatures, the viscosity of the oil increased, while the volatility of the toxic low molecular weight hydrocarbons were reduced, delaying the onset of biodegradation.

      Temperature also affects the solubility of hydrocarbons [62]. Although hydrocarbon biodegradation can occur over a wide range of temperatures, the rate of biodegradation generally decreases with the decreasing temperature. Figure 1 shows that highest degradation rates that generally occur in the range 30–40

      C in soil environments, 20–30 C in some freshwater environments and 15–20 C in marine environments [33, 34]. Venosa and Zhu [63] reported that ambient temperature of the environment affected both the properties of spilled oil and the activity of the microorganisms. Significant biodegradation of hydrocarbons have been reported in psychrophilic environments in temperate regions [64, 65].


      Nutrients are very important ingredients for successful biodegradation of hydrocarbon pollutants especially nitrogen, phosphorus, and in some cases iron [34]. Some of these nutrients could become limiting factor thus affecting the biodegradation processes. Atlas [35] reported that when a major oil spill occurred in marine and freshwater environments, the supply of carbon was significantly increased and the availability of nitrogen and phosphorus generally became the limiting factor for oil degradation. In marine environments, it was found to be more pronounced due to low levels of nitrogen and phosphorous in seawater [36]. Freshwater wetlands are typically considered to be nutrient deficient due to heavy demands of nutrients by the plants [66]. Therefore, additions of nutrients were necessary to enhance the biodegradation of oil pollutant [67, 68]. On the other hand, excessive nutrient concentrations can also inhibit the biodegradation activity [69]. Several authors have reported the negative effects of high NPK levels on the biodegradation of hydrocarbons [70, 71] especially on aromatics [72]. The effectiveness of fertilizers for the crude oil bioremediation in subarctic intertidal sediments was studied by Pelletier et al. [64]. Use of poultry manure as organic fertilizer in contaminated soil was also reported [73], and biodegradation was found to be enhanced in the presence of poultry manure alone. Maki et al. [74] reported that photo-oxidation increased the biodegradability of petroleum hydrocarbon by increasing its bioavailability and thus enhancing microbial activities.

      4. Mechanism of Petroleum Hydrocarbon Degradation

      The most rapid and complete degradation of the majority of organic pollutants is brought about under aerobic conditions. Figure 2 shows the main principle of aerobic degradation of hydrocarbons [75]. The initial intracellular attack of organic pollutants is an oxidative process and the activation as well as incorporation of oxygen is the enzymatic key reaction catalyzed by oxygenases and peroxidases. Peripheral degradation pathways convert organic pollutants step by step into intermediates of the central intermediary metabolism, for example, the tricarboxylic acid cycle. Biosynthesis of cell biomass occurs from the central precursor metabolites, for example, acetyl-CoA, succinate, pyruvate. Sugars required for various biosyntheses and growth are synthesized by gluconeogenesis.


      The degradation of petroleum hydrocarbons can be mediated by specific enzyme system. Figure 3 shows the initial attack on xenobiotics by oxygenases [75]. Other mechanisms involved are (1) attachment of microbial cells to the substrates and (2) production of biosurfactants [76]. The uptake mechanism linked to the attachment of cell to oil droplet is still unknown but production of biosurfactants has been well studied.


      5. Enzymes Participating in Degradation of Hydrocarbons

      Cytochrome P450 alkane hydroxylases constitute a super family of ubiquitous Heme-thiolate Monooxygenases which play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds [77]. Depending on the chain length, enzyme systems are required to introduce oxygen in the substrate to initiate biodegradation (Table 1). Higher eukaryotes generally contain several different P450 families that consist of large number of individual P450 forms that may contribute as an ensemble of isoforms to the metabolic conversion of given substrate. In microorganisms such P450 multiplicity can only be found in few species [78]. Cytochrome P450 enzyme systems was found to be involved in biodegradation of petroleum hydrocarbons (Table 1). The capability of several yeast species to use n-alkanes and other aliphatic hydrocarbons as a sole source of carbon and energy is mediated by the existence of multiple microsomal Cytochrome P450 forms. These cytochrome P450 enzymes had been isolated from yeast species such as Candida maltosa, Candida tropicalis, and Candida apicola [79]. The diversity of alkaneoxygenase systems in prokaryotes and eukaryotes that are actively participating in the degradation of alkanes under aerobic conditions like Cytochrome P450 enzymes, integral membrane di-iron alkane hydroxylases (e.g., alkB), soluble di-iron methane monooxygenases, and membrane-bound copper containing methane monooxygenases have been discussed by Van Beilen and Funhoff [80].

      6. Uptake of Hydrocarbons by Biosurfactants

      Biosurfactants are heterogeneous group of surface active chemical compounds produced by a wide variety of microorganisms [57, 58, 60, 81–83]. Surfactants enhance solubilization and removal of contaminants [84, 85]. Biodegradation is also enhanced by surfactants due to increased bioavailability of pollutants [86]. Bioremediation of oil sludge using biosurfactants has been reported by Cameotra and Singh [87]. Microbial consortium consisting of two isolates of Pseudomonas aeruginosa and one isolate Rhodococcus erythropolis from soil contaminated with oily sludge was used in this study. The consortium was able to degrade 90% of hydrocarbons in 6 weeks in liquid culture. The ability of the consortium to degrade sludge hydrocarbons was tested in two separate field trials. In addition, the effect of two additives (a nutrient mixture and a crude biosurfactant preparation on the efficiency of the process was also assessed. The biosurfactant used was produced by a consortium member and was identified as being a mixture of 11 rhamnolipid congeners. The consortium degraded 91% of the hydrocarbon content of soil contaminated with 1% (v/v) crude oil sludge in 5 weeks. Separate use of any one additive along with the consortium brought about a 91–95% depletion of the hydrocarbon content in 4 weeks, with the crude biosurfactant preparation being a more effective enhancer of degradation. However, more than 98% hydrocarbon depletion was obtained when both additives were added together with the consortium. The data substantiated the use of a crude biosurfactant for hydrocarbon remediation.

      Pseudomonads are the best known bacteria capable of utilizing hydrocarbons as carbon and energy sources and producing biosurfactants [37, 87–89]. Among Pseudomonads, P. aeruginosa is widely studied for the production of glycolipid type biosurfactants. However, glycolipid type biosurfactants are also reported from some other species like P. putida and P. chlororaphis. Biosurfactants increase the oil surface area and that amount of oil is actually available for bacteria to utilize it [90]. Table 2 summarizes the recent reports on biosurfactant production by different microorganisms. Biosurfactants can act as emulsifying agents by decreasing the surface tension and forming micelles. The microdroplets encapsulated in the hydrophobic microbial cell surface are taken inside and degraded. Figure 4 demonstrates the involvement of biosurfactant (rhamnolipids) produced by Pseudomonas sp. and the mechanism of formation of micelles in the uptake of hydrocarbons [75].


      Involvement of biosurfactant (rhamnolipid) produced by Pseudomonas sp in the uptake of hydrocarbons.

      7. Biodegradation of Petroleum Hydrocarbons by Immobilized Cells

      Immobilized cells have been used and studied for the bioremediation of numerous toxic chemicals. Immobilization not only simplifies separation and recovery of immobilized cells but also makes the application reusable which reduces the overall cost. Wilsey and Bradely [91] used free suspension and immobilized Pseudomonas sp. to degrade petrol in an aqueous system. The study indicated that immobilization resulted in a combination of increased contact between cell and hydrocarbon droplets and enhanced level of rhamnolipids production. Rhamnolipids caused greater dispersion of water-insoluble n-alkanes in the aqueous phase due to their amphipathic properties and the molecules consist of hydrophilic and hydrophobic moieties reduced the interfacial tension of oil-water systems. This resulted in higher interaction of cells with solubilized hydrocarbon droplets much smaller than the cells and rapid uptake of hydrocarbon in to the cells. Diaz et al. [92] reported that immobilization of bacterial cells enhanced the biodegradation rate of crude oil compared to free living cells in a wide range of culture salinity. Immobilization can be done in batch mode as well as continuous mode. Packed bed reactors are commonly used in continuous mode to degrade hydrocarbons. Cunningham et al. [93] used polyvinyl alcohol (PVA) cryogelation as an entrapment matrix and microorganisms indigenous to the site. They constructed laboratory biopiles to compare immobilised bioaugmentation with liquid culture bioaugmentation and biostimulation. Immobilised systems were found to be the most successful in terms of percentage removal of diesel after 32 days.

      Rahman et al. [94] conducted an experiment to study the capacity of immobilized bacteria in alginate beads to degrade hydrocarbons. The results showed that there was no decline in the biodegradation activity of the microbial consortium on the repeated use. It was concluded that immobilization of cells are a promising application in the bioremediation of hydrocarbon contaminated site.

      8. Commercially Available Bioremediation Agents

      Microbiological cultures, enzyme additives, or nutrient additives that significantly increase the rate of biodegradation to mitigate the effects of the discharge were defied as bioremediation agents by U.S.EPA [95]. Bioremediation agents are classified as bioaugmentation agents and biostimulation agents based on the two main approaches to oil spill bioremediation. Numerous bioremediation products have been proposed and promoted by their vendors, especially during early 1990s, when bioremediation was popularized as “the ultimate solution” to oil spills [96].

      The U.S. EPA compiled a list of 15 bioremediation agents [95, 97] as a part of the National Oil and Hazardous Substances Pollution Contingency Plan (NCP) Product Schedule, which was required by the Clean Water Act, the Oil Pollution Act of 1990, and the National Contingency Plan (NCP) as shown in Table 3. But the list was modified, and the number of bioremediation agents was reduced to nine.

      Studies showed that bioremediation products may be effective in the laboratory but significantly less so in the field [14, 17, 18, 98]. This is because laboratory studies cannot always simulate complicated real world conditions such as spatial heterogeneity, biological interactions, climatic effects, and nutrient mass transport limitations. Therefore, field studies and applications are the ultimate tests or the most convincing demonstration of the effectiveness of bioremediation products.

      Compared to microbial products, very few nutrient additives have been developed and marketed specifically as commercial bioremediation agents for oil spill cleanup. It is probably because common fertilizers are inexpensive, readily available, and have been shown effective if used properly. However, due to the limitations of common fertilizers (e.g., being rapidly washed out due to tide and wave action), several organic nutrient products, such as oleophilic nutrient products, have recently been evaluated and marketed as bioremediation agents. Four agents, namely, Inipol EAP22, Oil Spill Eater II (OSE II), BIOREN 1, and BIOREN 2, listed on the NCP Product Schedule have also been put into this category.

      Inipol EAP22 (Societe, CECA S.A., France) is listed on the NCP Product Schedule as a nutrient additive and probably the most well-known bioremediation agent for oil spill cleanup due to its use in Prince William Sound, Alaska. This nutrient product is a microemulsion-containing urea as a nitrogen source, sodium laureth phosphate as a phosphorus source, 2-butoxy-1-ethanol as a surfactant, and oleic acid to give the material its hydrophobicity. The claimed advantages of Inipol EAP22 include (1) preventing the formation of water-in-oil emulsions by reducing the oil viscosity and interfacial tension (2) providing controlled release of nitrogen and phosphorus for oil biodegradation (3) exhibiting no toxicity to flora and fauna and good biodegradability [99].

      Oil Spill Eater II (Oil Spill Eater International, Corp.) is another nutrient product listed on the NCP Schedule [97]. This product is listed as a nutrient/enzyme additive and consists of “nitrogen, phosphorus, readily available carbon, and vitamins for quick colonization of naturally occurring bacteria”. A field demonstration was carried out at a bioventing site in a Marine Corps Air Ground Combat Center (MCAGCC) in California to investigate the efficacy of OSEII for enhancing hydrocarbon biodegradation in a fuel-contaminated vadose zone [106].

      Researchers from European EUREKA BIOREN program conducted a field trial in an estuary environment to evaluate the effectiveness of two bioremediation products (BIOREN 1 and 2) [114, 115]. The two nutrient products were derived from fish meals in a granular form with urea and super phosphate as nitrogen and phosphorus sources and proteinaceous material as the carbon source. The major difference between the two formulations was that BIOREN 1 contained a biosurfactant. The results showed that the presence of biosurfactant in BIOREN 1 was the most active ingredient which contributed to the increase in oil degradation rates whereas BIOREN 2 (without biosurfactant) was not effective in that respect. The biosurfactant could have contributed to greater bioavailability of hydrocarbons to microbial attack.

      9. Phytoremediation

      Phytoremediation is an emerging technology that uses plants to manage a wide variety of environmental pollution problems, including the cleanup of soils and groundwater contaminated with hydrocarbons and other hazardous substances. The different mechanisms, namely, hydraulic control, phytovolatilization, rhizoremediation, and phytotransformation. could be utilized for the remediation of a wide variety of contaminants.

      Phytoremediation can be cost-effective (a) for large sites with shallow residual levels of contamination by organic, nutrient, or metal pollutants, where contamination does not pose an imminent danger and only “polishing treatment” is required (b) where vegetation is used as a final cap and closure of the site [116].

      Advantages of using phytoremediation include cost-effectiveness, aesthetic advantages, and long-term applicability (Table 4). Furthermore, the use of phytoremediation as a secondary or polishing in situ treatment step minimizes land disturbance and eliminates transportation and liability costs associated with offsite treatment and disposal.

      Research and application of phytoremediation for the treatment of petroleum hydrocarbon contamination over the past fifteen years have provided much useful information that can be used to design effective remediation systems and drive further improvement and innovation. Phytoremediation could be applied for the remediation of numerous contaminated sites. However, not much is known about contaminant fate and transformation pathways, including the identity of metabolites (Table 4). Little data exists on contaminant removal rates and efficiencies directly attributable to plants under field conditions.

      The potential use of phytoremediation at a site contaminated with hydrocarbons was investigated. The Alabama Department of Environmental Management granted a site, which involved about 1500 cubic yards of soil of which 70% of the baseline samples contained over 100 ppm of total petroleum hydrocarbon (TPH). After 1 year of vegetative cover, approximately 83% of the samples were found to contain less than 10-ppm TPH. Removal of total petroleum hydrocarbon (TPH) at several field sites contaminated with crude oil, diesel fuel, or petroleum refinery wastes, at initial TPH concentrations of 1,700 to 16,000 mg/kg were also investigated [117, 118]. Plant growth was found to vary depending upon the species. Presence of some species led to greater TPH disappearance than with other species or in unvegetated soil. Among tropical plants tested for use in Pacific Islands, three coastal trees, kou (Cordia subcordata), milo (Thespesia populnea), and kiawe (Prosopis pallida) and the native shrub beach naupaka

      tolerated field conditions and facilitated cleanup of soils contaminated with diesel fuel [119]. Grasses were often planted with trees at sites with organic contaminants as the primary remediation method. Tremendous amount of fine roots in the surface soil was found to be effective at binding and transforming hydrophobic contaminants such as TPH, BTEX, and PAHs. Grasses were often planted between rows of trees to provide soil stabilization and protection against wind-blown dust that could move contaminants offsite. Legumes such as alfalfa (Medicago sativa), alsike clover (Trifolium hybridum), and peas (Pisum sp.) could be used to restore nitrogen to poor soils. Fescue (Vulpia myuros), rye (Elymus sp.), clover (Trifolium sp.), and reed canary grass (Phalaris arundinacea) were used successfully at several sites, especially contaminated with petrochemical wastes. Once harvested, the grasses could be disposed off as compost or burned.

      Microbial degradation in the rhizosphere might be the most significant mechanism for removal of diesel range organics in vegetated contaminated soils [120]. This occurs because contaminants such as PAHs are highly hydrophobic, and their sorption to soil decreases their bioavailability for plant uptake and phytotransformation.

      10. Genetically Modified Bacteria

      Applications for genetically engineered microorganisms (GEMs) in bioremediation have received a great deal of attention to improve the degradation of hazardous wastes under laboratory conditions. There are reports on the degradation of environmental pollutants by different bacteria. Table 5 shows some examples of the relevant use of genetic engineering technology to improve bioremediation of hydrocarbon contaminants using bacteria. The genetically engineered bacteria showed higher degradative capacity. However, ecological and environmental concerns and regulatory constraints are major obstacles for testing GEM in the field. These problems must be solved before GEM can provide an effective clean-up process at lower cost.

      The use of genetically engineered bacteria was applied to bioremediation process monitoring, strain monitoring, stress response, end-point analysis, and toxicity assessment. Examples of these applications are listed in Table 6. The range of tested contaminants included chlorinated compounds, aromatic hydrocarbons, and nonpolar toxicants. The combination of microbiological and ecological knowledge, biochemical mechanisms, and field engineering designs are essential elements for successful in situ bioremediation using genetically modified bacteria.

      11. Conclusion

      Cleaning up of petroleum hydrocarbons in the subsurface environment is a real world problem. A better understanding of the mechanism of biodegradation has a high ecological significance that depends on the indigenous microorganisms to transform or mineralize the organic contaminants. Microbial degradation process aids the elimination of spilled oil from the environment after critical removal of large amounts of the oil by various physical and chemical methods. This is possible because microorganisms have enzyme systems to degrade and utilize different hydrocarbons as a source of carbon and energy.

      The use of genetically modified (GM) bacteria represents a research frontier with broad implications. The potential benefits of using genetically modified bacteria are significant. But the need for GM bacteria may be questionable for many cases, considering that indigenous species often perform adequately but we do not tap the full potential of wild species due to our limited understanding of various phytoremediation mechanisms, including the regulation of enzyme systems that degrade pollutants.

      Therefore, based on the present review, it may be concluded that microbial degradation can be considered as a key component in the cleanup strategy for petroleum hydrocarbon remediation.

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      Copyright

      Copyright © 2011 Nilanjana Das and Preethy Chandran. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


      Endospore Development

      The process of forming an endospore is complex. The model organism used to study endospore formation is Bacillus subtilis. Endospore development requires several hours to complete. Key morphological changes in the process have been used as markers to define stages of development. As a cell begins the process of forming an endospore, it divides asymmetrically (Stage II). This results in the creation of two compartments, the larger mother cell and the smaller forespore. These two cells have different developmental fates. Intercellular communication systems coordinate cell-specific gene expression through the sequential activation of specialized sigma factors in each of the cells. Next (Stage III), the peptidoglycan in the septum is degraded and the forespore is engulfed by the mother cell, forming a cell within a cell. The activities of the mother cell and forespore lead to the synthesis of the endospore-specific compounds, formation of the cortex and deposition of the coat (Stages IV+V). This is followed by the final dehydration and maturation of the endospore (Stages VI+VII). Finally, the mother cell is destroyed in a programmed cell death, and the endospore is released into the environment. The endospore will remain dormant until it senses the return of more favorable conditions. [A sigma factor is a small protein that directs RNA polymerase to specific cites on DNA to initiate gene expression.]


      Electrical hazards

      Electrical hazards are potentially life threatening and found much too frequently. First, equip all electrical power outlets in wet locations with ground-fault circuit interrupters, or GFCIs, to prevent accidental electrocutions. GFCIs are designed to &ldquotrip&rdquo and break the circuit when a small amount of current begins flowing to ground. Wet locations usually include outlets within six feet of a sink, faucet, or other water source and outlets located outdoors or in areas that get washed down routinely. Specific GFCI outlets can be used individually, or GFCIs can be installed in the electrical panel to protect entire circuits.

      Another very common electrical hazard is improper use of flexible extension cords. Do not use these as a substitute for permanent wiring. The cord insulation should be in good condition and continue into the plug ends. Never repair cracks, breaks, cuts, or tears with tape. Either discard the extension cord or shorten it by installing a new plug end. Take care not to run extension cords through doors or windows where they can become pinched or cut. And always be aware of potential tripping hazards when using them. Use only grounded equipment and tools and never remove the grounding pin from the plug ends. Also, do not use extension cords in a series&mdashjust get the right length of cord for the job.

      The use of hanging pendants and electrical outlets are widespread in research lab facilities to help keep cords off of floors and out of the way. Check electrical pendants for proper strain relief and type of box used. The box should be totally closed and without any holes. If it contains knockouts or holes for mounting, it is not the right type for a hanging pendant.

      As a final check for possible electrical hazards, look over your lighting. Protect all lights within seven feet of the floor to guard against accidental breakage. Slip plastic protective tubes over florescent bulbs prior to mounting or install screens onto the fixtures.


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