ECOLOGICAL MICROBIOLOGY - M. I. Chernyavskaya - 2016

TOPIC 5. THE USE OF MICROORGANISMS IN SOLVING ENVIRONMENTAL PROBLEMS

Due to their inexhaustible metabolic potential, microorganisms are utilized by humans for A wide variety of purposes, including the resolution of environmental problems. Ecobiotechnology is a branch of science and Applied Biotechnology that investigates the theoretical and practical aspects of employing living organisms for environmental protection.

One of the most vital areas of ecobiotechnology is bioremediation—the cleanup of environmental pollution of natural and anthropogenic origin using biological agents. Microorganisms were first employed for wastewater Treatment from domestic waste as early as the 19th century. Already at the beginning of the 20th century, aerobic wastewater treatment systems utilizing activated sludge and forced aeration began to be applied, which significantly increased the speed and efficiency of bioremediation processes.

Currently, microorganisms are used to treat wastewater, exhaust gases, soils, and Water bodies contaminated As a result of human activity. Aeration tanks, biological filters, and anaerobic Digestion of organic waste in digesters and bioreactors of other designs are employed for wastewater treatment.

Biological treatment processes involve a complex biological association consisting not only of Bacteria, but also of unicellular eukaryotic organisms—Fungi, Protozoa (amebae, flagellates, and Ciliates), microscopic animals (rotifers, nematodes, water mites), etc. During biological treatment, this complex association forms as activated sludge or biofilm. Activated sludge consists of dark brown flocs up to hundreds of microns in size, comprising 70% living microorganisms and 30% inorganic particles. Microorganisms are attached to a solid carrier, together forming zoogloea—a Symbiosis of microbial populations covered by a common mucous sheath. Biofilm is a slimy growth of living microorganisms, 1 to 3 mm thick, covering the material of the filter bed in treatment facilities.

An aeration tank is an open Structure through which aerated wastewater and activated sludge suspension are passed. Aeration tanks belong to homogeneous bioreactors. A typical design of an aeration tank is a reinforced concrete airtight vessel of rectangular cross-section, connected to a settling tank. The aeration tank is divided by longitudinal baffles into several channels (3 – 4). The biotreatment process in an aeration tank consists of two stages:

1) interaction of settled wastewater containing suspended particles and organic matter with air and activated sludge particles in the aeration tank for a certain period of time (from 4 to 24 hours or more, depending on the type of wastewater, effluent quality requirements, etc.). The oxidation of wastewater organic matter by activated sludge microorganisms is carried out in two stages. In The First stage, microorganisms adsorb the pollutants of the wastewater; In the second stage, they oxidize them and restore their oxidizing capacity. During treatment, biomass growth of activated sludge occurs, leading to its "Aging" and a decrease in biocatalytic activity. Therefore, most of the sludge is removed from the system, while a portion is returned to the Reactor;

2) Separation of water and activated sludge particles in the secondary clarifier.

A biofilter is the most common type of fixed-film bioreactor used for the treatment of wastewater and waste gases. In biofilters, wastewater or polluted air is passed through a layer of coarse-grained material (filter bed) covered with a thin bacterial film, which facilitates intensive biological purification processes. A distinctive feature of the packing, or filter bed, is a high specific surface area for Microbial growth AND high porosity. Porosity provides the necessary gas-dynamic Properties of the bed and promotes the passage of air and liquid through it. As wastewater percolates through the filter bed material, a series of sequential processes takes place:

1) contact with the biofilm developing On the surface of the filter material particles;

2) sorption of organic substances by The surface of microbial Cells;

3) oxidation of wastewater substances in the course of microbial METABOLISM.

Continuous growth and sloughing off of the biofilm occur within the biofilter. The dead biofilm is washed away by the flow of treated water and carried out of the biofilter. The purified water enters the clarifier, is freed from biofilm particles therein, and is subsequently discharged into a water body.

In addition to aerobic wastewater treatment Methods, anaerobic methods exist, the undisputed advantage of which is a high degree of carbon conversion of pollutants with a relatively low biomass yield. The process of anaerobic digestion of wastewater organic waste yields such a valuable product as biogas. Anaerobic treatment processes take place in septic tanks and anaerobic digesters, with the settled sludge rather than the wastewater itself undergoing digestion. Biodegradation of pollutants in septic tanks proceeds on The basis of a complex microbial association and includes hydrolytic processes involving acidogenic and heteroacetogenic bacteria, as well as methanogenesis involving methanogens. Septic tanks are often used to digest sludge from primary clarifiers (which are part of aeration tanks). In this process, the volume of sludge decreases significantly, and the number of pathogenic microorganisms is reduced. The digested sludge is then disposed of or buried.

The purification of waste gases can be carried out in trickling-bed bioreactors. Destructor microorganisms in such bioreactors are immobilized on artificial carriers (e.g., polyamide fibers). An important condition for the efficient functioning of this type of bioreactor is maintaining humidity at a constant optimal level, which is achieved by uniformly irrigating the microbial carrier.

To clean up contaminated water bodies and soils, biopreparations based on destructor microorganisms are utilized. Microorganisms are introduced into the soil or water body as a suspension, or they can be immobilized on various carriers, most commonly of natural origin (peat, sawdust, husk). Frequently, to enhance the efficiency of treatment processes, biopreparations are used in combination with mineral fertilizers.

Crude oil and petroleum products rank first among soil and water pollutants. Their unwanted release into the environment occurs during oil extraction, transportation, and refining. Crude oil is a complex mixture consisting of more than 1,000 Organic compounds of various classes (aliphatic, mono- and polycyclic aromatic, alicyclic Hydrocarbons) and inorganic impurities.

Microorganisms capable of utilizing petroleum are very widespread and can be isolated from any type of soil and water. However, when large amounts of oil enter the soil or a water body, depression of the native microbiota and a sharp decline in its Abundance occur, which is why self-purification processes proceed extremely slowly. The restoration of the microbiota begins with a gradual increase in the number of oil degraders (hydrocarbon-oxidizing microorganisms). In environments with chronic petroleum pollution, a noticeable numerical predominance of this group of microorganisms is observed.

The ability to use petroleum as a carbon and energy source is characteristic not of isolated specialized forms, but of many groups of microorganisms. However, it is difficult to imagine an Organism capable of utilizing all components of petroleum. Each destructor microorganism is characterized by a specific spectrum of utilized compounds found in petroleum. Oil degraders are most commonly found among representatives of bacterial genera such as Rhodococcus, Pseudomonas, Acinetobacter, Arthrobacter, Bacillus, Cytophaga, Clostridium, Corynebacterium, Flavobacterium, Methanobacterium, Micrococcus, Mycobacterium, Nocardia, mycelial fungi such as Aspergillus, Penicillium, Mucor, Fusarium, Trichoderma, and Yeasts such as Candida, Endomyces, Rhodotorula, Saccharomyces, Torulopsis.

Among the adaptations developed by oil-degrading bacteria, their ability to produce biosurfactants (Surfactants) should be noted. Since petroleum components are predominantly hydrophobic, their penetration into The Cell is hindered. Different groups of microorganisms solve this problem in various ways: some produce surfactants into the external environment, thereby increasing the solubility of petroleum hydrocarbons and their bioavailability (e.g., bacteria of the genera Bacillus and Pseudomonas); others produce cell-bound surfactants that increase the Hydrophobicity of the cell surface (Rhodococcus and other nocardioform bacteria).

Within the cell, hydrocarbons undergo enzymatic Cleavage by mono- and Dioxygenases. The genes encoding these Enzymes are organized in clusters and often have plasmid localization. For instance, in bacteria of the genus Pseudomonas, Plasmids such as OCT (determining alkane utilization), TOL (determining monoaromatic hydrocarbon utilization), and NAH (determining polyaromatic hydrocarbon utilization) have been described.

Control Questions

1. Define THE CONCEPT OF "ecobiotechnology".

2. What are activated sludge and biofilms?

3. What is the design and operating principle of an aeration tank?

4. What is the design and operating principle of a biofilter?

5. What are the advantages of anaerobic digestion of wastewater organic waste over aerobic treatment methods?

6. Why are natural carriers predominantly used when remediating soils and water bodies from pollution using immobilized microorganisms?

7. Give Examples of petroleum-oxidizing microorganisms.

8. How can the taxonomic diversity of petroleum-oxidizing microorganisms be explained?

Class="center">Laboratory Work 7. Microbiological Degradation of Petroleum

Objective: to evaluate the microbiological degradation of petroleum by representatives of various bacterial genera at different temperatures.

Materials and Equipment: PDB, M9 liquid mineral medium, petroleum, sterile test tubes, 1 — 2 ml and 5 — 10 ml pipettes, 20 — 200 µl pipette, 20 — 200 µl pipette tips, spirit lamp, shaker, 25 and 37 °C incubators.

Procedure

1. Cultures of petroleum-degrading microorganisms of various genera (Bacillus, Rhodococcus, Pseudomonas, etc.) grown in PDB are added in an amount of 0.1 ml to test tubes containing 4 ml of M9 liquid mineral medium (in duplicate). The M9 mineral medium without bacterial culture is used as a control.

2. Petroleum (1 %) is added to the test tubes as the sole carbon source.

3. One series of test tubes, including the control, is incubated with aeration at 25 °C, and the second series at 37 °C for 7 — 14 days, recording changes compared to the control daily. The observation results are entered into the table (Table 9).

Table 9. Microbial Degradation of Petroleum