MICROBIOLOGY - M.H. Serhiichuk - 2008

Class="center">Chapter 12. HUMAN USE OF MICROORGANISMS

Environmental Bioremediation

Bioremediation refers to any process involving microorganisms, Fungi, plants, or their Enzymes aimed at restoring polluted environments to their natural state.

Aerobic and anaerobic microorganisms are employed to treat municipal and industrial wastewater, groundwater, and contaminated soils, while agricultural waste Processing yields biogas.

Wastewater and groundwater Treatment. The foundation of biological wastewater treatment lies in the METABOLIC ACTIVITY OF soil, aquatic, or specially selected microorganisms. Microorganisms degrade organic pollutants, utilizing them in their METABOLISM as nutrient sources.

Depending on their origin, wastewaters are classified into: atmospheric (stormwater), municipal (domestic-fecal), and industrial.

Atmospheric wastewater is generated by precipitation (rain, snow) and directly enters Water bodies and groundwater.

Domestic wastewater originates from food waste and human physiological activities. The primary pollutants in these waters are naturally occurring Organic compounds. Domestic wastewater is non-toxic, and its pollutants are readily biodegradable.

Industrial wastewater is produced through The Use of water in manufacturing, specifically in primary production processes, equipment cooling, and auxiliary shop operations. Generally, industrial wastewaters are characterized by high concentrations of organic and inorganic pollutants; organic pollutants typically act as toxic xenobiotics, and the contamination is poorly biodegradable or non-biodegradable. These waters exhibit high acidity or alkalinity and can have elevated temperatures.

A xenobiotic (from the Greek *xenos* – foreign, *bios* – life) refers to chemical compounds that are foreign, atypical, and unnatural to living organisms and the environment, having been artificially synthesized by humans. Because molecules with such structures did not previously exist in nature, microorganisms generally lack the corresponding metabolic pathways and enzymes for their utilization. Most xenobiotics resist biological degradation and are thus persistent compounds. Xenobiotics frequently exhibit toxic, mutagenic, and carcinogenic properties.

Wastewater treatment Methods are categorized into physical, physical-chemical, and biological (Fig. 12.9).

Fig. 12.9. Methods for treating domestic and industrial wastewater

Despite their widespread application, physical and physical-chemical wastewater treatment methods have significant drawbacks. They are expensive, energy-intensive, and require additional Reagents. Furthermore, physical-chemical water treatment is frequently accompanied by secondary soil or air pollution.

Biological methods utilize natural water purification processes rooted in The activity of soil, aquatic, or specially selected microorganisms. Microorganisms degrade organic pollutants, incorporating them into their metabolism as nutrient sources. Destruction (degradation, mineralization) is the complete breakdown (oxidation and decarboxylation) of a chemical compound into CO₂ and H₂O. Certain xenobiotics cannot be completely broken down by microorganisms into end products; instead, their molecules are merely transformed. Transformation refers to the simplification of a molecule's Structure and its partial mineralization.

Biological wastewater treatment methods are subdivided into soil-based and industrial methods. Soil-based methods involve the construction of primitive facilities, bunds, and dams, including irrigation fields, filtration fields, filter wells, sand-gravel filters, and stabilization ponds. Wastewater is purified in these systems through a combination of biological processes (putrefaction, bio-oxidation) and physical processes (evaporation, filtration, freezing). The biological processes are driven by soil microorganisms.

The general drawbacks of soil-based methods include: treatment facilities occupy large areas, possess low capacity and efficiency, and operate primarily during the warm season. The processes occurring within them are virtually uncontrollable. Soil-based methods are unsuitable for treating industrial wastewater, and when applied to domestic sewage, they can serve as sources for the dissemination of pathogenic Bacteria and helminth eggs. Moreover, a portion of the pollutants from these primitive treatment facilities can infiltrate open water bodies and groundwater.

Industrial methods involve the use of sophisticated treatment plant designs, multi-stage technological schemes, and complex microbial consortia. Treatment can be carried out under either aerobic or anaerobic conditions.

Aerobic biological treatment facilities (aeration tanks, biofilters, rotating biological contactors) purify wastewater through the metabolic activity of aerobic microorganisms operating in a planktonic state or as biofilms.

Aeration tanks (aerotanks) are aerobic biological treatment facilities. The first aeration tank was developed in England in 1914. Typically, these are reinforced concrete channels, 4–5 m deep and 3–11 m wide (Fig. 12.10).

Fig. 12.10. General view of an aeration tank (top) and a radial sedimentation tank (bottom)

A crucial and essential prerequisite for the operation of aeration tanks is the continuous oxygenation of the water, i.e., aeration.

Water pollutants in aeration tanks are degraded through the activity of activated sludge. Activated sludge is a complex biocenosis consisting of bacteria, fungi, Algae, Protozoa, worms, and certain Arthropods. It appears as brown flocs and forms in the aeration tank under specific conditions. Bacteria play the primary role in wastewater purification, driving the biochemical transformation of organic and Inorganic Compounds. Compared to other members of the biocenosis, bacteria are the most resistant to the Toxic effects of pollutants and adapt quite rapidly to changing wastewater compositions and oxygen fluctuations. Bacteria also serve as a food source for aquatic organisms (hydrobionts).

Microorganisms of activated sludge:

- bacteria of the genera Nitrobacter, Nitrosomonas, Pseudomonas, Bacillus, Arthrobacter, Achromobacter, Flavobacterium, Alcaligenes, Corynebacterium, Micrococcus, Nocardia, Sarcina, Mycobacterium, Zoogloea ramigera (Fig. 12.11);

Fig. 12.11. Activated sludge bacteria

(a, b - Zoogloea ramigera, c, d - Microthrix parvicella, e - Nostocoida limicola, f - Sphaerotilus natans)

- fungi represented by the genera Mucor, Rhizopus, Aspergillus, Penicillium, Fusarium, Trichoderma;

- protozoa: Sarcodina, Flagellata, and Ciliata (Fig. 12.12);

Fig. 12.12. Representatives of various protozoan classes inhabiting activated sludge:

Sarcodina: a - Pelomyxa palustris; b - Centropyxis aculeata; c - Amoeba limax;

Flagellata: d - Bodo pubinus; e - Diplosiga socialis; Ciliata: f - Euplotes charon; g - Colpidium colpoda; h - Epistylis plicatilis;

i - Vorticella convallaria

- worms: Annelids (classes Oligochaeta, Polychaeta), pseudocoelomates (class Nematoda), and rotifers (class Rotatoria). Among rotifers, Philodina roseola, Cathypna luna, Notommata ansata, Monostyla lunaris, Adineta sp., Rotaria rotatoria, and others are most frequently found in activated sludge. Rotifers are quite sensitive to environmental changes, making many of them reliable indicator organisms.

The primary technological value of activated sludge lies in its ability to form large, relatively heavy conglomerates—flocs—which settle fairly easily and quickly in secondary clarifiers, separating from the purified water. The main role in floc formation belongs to the bacterium Zoogloea ramigera. It is a capsular, Gram-negative, monotrichous, heterotrophic, aerobic rod. It synthesizes an extracellular, Cellulose-like polymer composed of N-acetylglucosamine and N-acetylfucosamine in a 1 : 2 ratio.

Hydrobionts feed on activated sludge bacteria, acting as natural cleaners, and by consuming finely dispersed Suspensions, they also help clarify the water. They maintain biological equilibrium within this ecological niche.

Aeration tanks are widely used for the treatment of domestic wastewater and certain industrial effluents that do not contain high concentrations of xenobiotics.

Biological filters (biofilters) are systems designed so that wastewater is purified by passing through a carrier coated with a biological film. A classic biofilter is a circular, polygonal, or rectangular reinforced concrete tank with a false bottom that holds the filter media (carrier) (Fig. 12.13).

Fig. 12.13. Biofilter: general view (top) and structural diagram (bottom)

The filter media can be bulky (gravel, expanded clay) or structured/flat (slate sheets, plastic panels, synthetic fabrics, woven threads and fibers in the form of brush-like elements or cilia). These carriers allow for the concentration of a significant microbial biomass within the treatment facility, which accelerates The oxidation of wastewater contaminants.

Air is supplied to the biofilter either naturally through ventilation openings in the housing or forced via a compressor. The microbiota in a biofilter is better supplied with oxygen than in an aeration tank, as it consumes oxygen In both dissolved and gaseous forms.

Biofilters can be used to treat wastewater or air contaminated with volatile organic compounds. When treating wastewater, the water enters the top of the biofilter through spray nozzles, while air is supplied from the bottom for aeration. The treated water is recirculated through the system using a pump. Wastewater recirculation increases its contact time with the

biofilm, thereby enhancing treatment efficiency. When a biofilter is used for waste gas treatment, the polluted air is introduced from the bottom, while the biofilm is irrigated from above with nutrient-rich water.

The purification process in a biofilter is carried out by microorganisms that develop as a biofilm on the carrier media (Fig. 12.14).

Fig. 12.14. Appearance of a biofilm developing on a biofilter carrier

Immobilized biomass has several significant advantages over planktonic (free-floating) biomass:

- biomass immobilization allows for its high concentration within a treatment facility;

- biofilms are less sensitive to toxic substances and abrupt Changes in the physicochemical parameters of wastewater (pH, salt concentration, etc.). A biofilter biofilm is heterogeneous, consisting of multiple microorganism species adapted to a specific wastewater composition. Nutrients and oxygen reach the biofilm microbiota in varying concentrations. Aerobic species thrive On the surface where oxygen is abundant, whereas facultative and obligate anaerobes reside in the deeper layers where oxygen concentration is lower. Surface microorganisms are exposed to more concentrated wastewater, thus receiving better Nutrition and exhibiting higher resistance to its toxic effects. Deep microorganisms typically consume the metabolites of surface species and lower concentrations of water pollutants.

Biofilters are successfully used to treat toxic wastewater containing xenobiotics.

Rotating biological contactors are semi-submerged metal or plastic disks, on the surfaces of which a microbial biofilm intensively develops on various types of carriers. The disks are attached to a slowly rotating shaft. The biofilm is periodically submerged in water and then exposed to the air as it rotates upward.

Anaerobic biological treatment facilities (methanogenesis tanks, anaerobic bioreactors) purify wastewater through the metabolic activity of anaerobic microorganisms present in a planktonic state or as biofilms.

Methanogenesis tanks (digesters) are airtight, closed structures in which the transformation of organic matter is driven by the activity of a complex community of anaerobic microorganisms (Fig. 12.15).

Fig. 12.15. General view and Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF a methanogenesis tank

Traditionally, methanogenesis tanks are used for the Digestion of primary settling tank raw sludge and excess activated sludge from aeration tanks. Sludge and activated sludge are loaded into the digester and held without oxygen access for 10–30 days under mesophilic (30–35 ⁰С) or thermophilic (50–55 ⁰С) conditions; thus, this technological process is batch-operated. The anaerobic microbial community of the digester—anaerobic sludge—carries out a complex transformation of organic compounds, which includes four stages:

1. The stage of Enzymatic Hydrolysis of complex polymer molecules (Polysaccharides, Proteins, Lipids) into simple ones (sugars, organic acids, glycerol, Amino Acids) via extracellular enzymes produced by cellulolytic, pectolytic, xylanolytic, amylolytic, and proteolytic bacteria.

2. The acidogenesis stage is characterized by the accumulation of short-chain organic acids due to the activity of primary fermentative bacteria: 20% of organic matter is converted into acetate, 15% into propionate, and 65% into other volatile Fatty acids, alcohols, CO₂, and H₂. Intensive progression of this stage can lead to a sharp drop in medium pH and inhibition of subsequent stages.

3. The acetogenesis stage. Organic acids and alcohols are converted into acetate by syntrophic bacteria (secondary fermenters).

4. The methanogenesis stage is characterized by biogas production. Its main component, methane, is released as the final metabolite of methanogenic archaea growing on a mixture of CO₂ and H₂, methanol, methylamines, and acetate. The second constituent of biogas, CO₂, accumulates primarily As a result of acetate methanogenesis.

Methanogenesis tanks and similar anaerobic reactors are used not only for the disposal of complex organic waste but also for biogas generation. Frequently, these two tasks are performed simultaneously.

Biogas is a mixture of gases produced during the biodegradation of organic Materials under anaerobic conditions. It consists of 50–70% methane, 30–40% carbon dioxide, and trace amounts of other gases.

Upon completion of methane Fermentation, digested sludge (undigested residue) and anaerobic microbial biomass remain in the Reactor. After biogas extraction, the digested sludge is removed from the reactor as a byproduct. It is a stabilized, pathogen-free material that can be utilized as agricultural fertilizer.

Currently, more than 60 varieties of biogas technologies are in use and under development. The basic technological scheme for biogas production consists of several units:

1. A homogenizer for mixing various raw materials, grinding them, and, if necessary, diluting them with water.

2. A methanogenesis tank (anaerobic reactor) – an air- and water-tight chamber where methane fermentation takes place.

3. A gas holder – a storage vessel for biogas.

4. Units for desulfurization (removal of H₂S) and gas drying (if required).

5. Biogas supply systems for space heating or electricity generation.

6. A system for removing digested sludge from the reactor.

For anaerobic biological wastewater treatment, anaerobic bacterial biomass can be used in the form of a biofilm immobilized on an inert carrier. The design of such a bioreactor somewhat resembles a biofilter submerged in wastewater. Wastewater is fed from the bottom, flows through the biofilm at a certain velocity, and discharges at the top of the treatment plant; unlike methane tanks, this is a continuous technological process (Fig. 12.16).

Fig. 12.16. Anaerobic continuous bioreactor with biomass immobilized on a carrier

Such treatment facilities are used to purify wastewater from fermentation plants, as well as the chemical and pharmaceutical industries.

Water treatment technologies based on granulated anaerobic sludge date back to the 1970s. At that time, Dutch scientist Dr. Lettinga noticed that in an upflow anaerobic sludge blanket reactor under certain conditions, anaerobic sludge forms aggregates in the form of granules 0.5–2 mm in diameter.

In the absence of any additional inert carrier, the water flow creates selective conditions under which only those Cells capable of adhering to each other are retained and proliferate in the bioreactor. Over time, these aggregates develop into compact, granule-like biofilms.

Granulated anaerobic sludge is used for wastewater treatment in distilleries, sugar refineries, breweries, starch production plants, the pulp and paper industry, and various Branches of the food processing industry.

The UASB reactor was proposed by Lettinga in 1980. According to his design, wastewater flows upward. The lower part of the reactor hosts

anaerobic processes, while the upper section houses a three-phase separator where phase Separation occurs: the solid phase (granules return downward), the liquid phase (treated water exits the reactor), and the gaseous phase (biogas is removed from the reactor). Wastewater flows through the granulated sludge bed and is purified. The granules fill with biogas and float to the surface; once the biogas is released, they settle back to the bottom of the reactor.

The EGSB reactor features an expanded granulated sludge bed, which allows for a higher upflow velocity of water. High flow velocities make it possible to treat wastewater containing suspended solids (as particulates do not have time to settle to the bottom). In addition, this facility provides water recirculation, enabling the treatment of toxic effluents (toxic wastewater is diluted with already purified water before entering the bioreactor).

The traditional and most widespread technological scheme for municipal (domestic sewage) wastewater treatment worldwide (Fig. 12.17) includes three stages:

1. Mechanical treatment.

1.1. In the screening facility, water passes through screens that trap coarse mechanical impurities. These residues are hauled away to an incinerator.

1.2. Sand settles out in grit chambers, is separated from the water, and is hauled away.

1.3. In primary settling tanks, insoluble impurities are separated from the water by gravity, settle to the bottom, and are pumped into methane tanks. Fats and oils collect on The surface of the settling tank, are skimmed by special devices into a hopper, and are likewise directed into methane tanks.

2. Biological treatment.

2.1. In aeration tanks, soluble organic pollutants in the water undergo aerobic oxidation by activated sludge.

2.2. In secondary settling tanks, activated sludge settles and separates from the water. Part of it is recycled back into the aeration tanks, while the excess is sent to methane tanks or hauled to sludge drying beds.

2.3. In methane tanks, anaerobic digestion of raw sludge and fats from the primary settling tanks, as well as excess activated sludge biomass from the secondary settling tanks, takes place.

3. Physico-chemical advanced water treatment involves disinfection, i.e., the elimination of pathogenic microorganisms. This can be achieved through chlorination, and sometimes UV irradiation or ozonation. For complete and safe advanced treatment, the water can be treated with coagulants and flocculants, settled, and filtered through sand and activated carbon.

Fig. 12.17. Technological scheme of domestic sewage wastewater treatment:

1 - screening facility; 2 - grit chamber; 3 - sand removal area;

4 - primary settling tank; 5 - methane tank; 6 - aeration tank; 7 - secondary settling tank;

8 - physico-chemical advanced water treatment

Soil and groundwater bioremediation. Soil bioremediation can be carried out in two ways:

- by adding bioelements (nitrogen and phosphorus), water, and oxygen to the contaminated soil, which helps stimulate soil microbial activity and, consequently, enhances pollutant degradation;

- by introducing specially selected or genetically modified high-activity degrader strains into the contaminated soil. Soil bioremediation is typically conducted in bioreactors—vessels or containers where pollutant biodegradation takes place under controlled conditions. The design of a bioreactor depends on the type of pollutant, the COMPOSITION OF THE contaminated soil, and the cost of the technology. There are two MAIN TYPES OF bioreactors:

Solid-phase bioreactors (Fig. 12.18) are used for cases of shallow soil contamination. Remediation is carried out by native soil microorganisms. A small amount of liquid is supplied to the bioreactor through a specialized water-spraying system or via natural precipitation. Nutrients are also added to the soil. The required oxygen level is maintained by physically turning or mixing the soil. A system of pipes may be installed in the soil to extract volatile emissions.

Fig. 12.18. Diagram of soil bioremediation in a solid-phase reactor

Slurry reactors are more efficient for remediating soils heavily contaminated with complex pollutants. In these systems, soil is mixed with a large volume of water, microorganisms, and nutrients to form a soil slurry.

Groundwater bioremediation is performed using immobilized biofilm reactors under aerobic or anaerobic conditions. Groundwater is first pumped to the surface, passed through the bioreactor, and then returned underground.