Important Microorganisms in Wastewater Bacteria and fungi

Bacteria are the most important and the largest components of the microbial community in all biological wastewater treatment processes. Depending on the biological process and pH, the number concentration of bacteria is different, with activated sludge (aggregates of healthy aerobic bacteria living in colonial structures called flocs) having the largest number concentration of bacteria. Bacteria range in size from approximately 0.5 to 5 ^m and take one of four major shapes: sphere (cocci), straight rod, curved rod (vibrio), and spiral (spirilla). They appear singly, in pairs, in packets, or in chains. Bacteria are classified into two major groups: heterotroph (which use organic matters as both energy and carbon sources for synthesis) and autotroph (which use inorganic matters for energy source and CO2 for carbon source). The heterotrophs can be further subdivided into three categories: aerobic (using free oxygen for decomposing organic matters), anaerobic (using no free oxygen for decomposing organic matters), and facultative (thriving both in aerobic and in anaerobic environments).

Aerobic bacteria require free dissolved oxygen to decompose organic materials (Equation 2.2):

aerobes

This microbial reaction is autocatalytic, meaning that the bacteria that function as catalysts are also produced by the reaction. Aerobic bacteria are predominant in biological wastewater treatment processes such as activated sludge and trickling filters and other biological processes that utilize free oxygen for their biochemistry. Aerobic bioconversion of organic matters is a biochemically efficient and rapid process that produces resulting products with highly oxidized compounds such as CO2 and water.

The metabolism of aerobic bacteria is much higher than that of anaerobic bacteria. This augmentation means that 90% fewer organisms for aerobic metabolism are needed compared to the anaerobic process, or that treatment is accomplished in 90% less time. This provides a number of advantages, including a higher percentage of organic removal. Aerobic bacteria living in flocs are kept in suspension by the mechanical action used to introduce oxygen into the wastewater. This mechanical action exposes the floc to the organic matters while biological treatment takes place. Following digestion, a gravity clarifier separates and settles out the floc.

Anaerobic bacteria live and reproduce in the absence of free oxygen. They utilize compounds such as sulfates and nitrates for energy, and their metabolism is substantially reduced. In order to remove a given amount of organic matters in an anaerobic environment, the organic matters must be exposed to a significantly higher quantity of bacteria and/or engaged for a much longer period of time. A representative use for anaerobic bacteria would be in a septic tank. The slower metabolism of anaerobic bacteria requires that the wastewater be held several days in order to achieve even a nominal 50% reduction in organic matters. The advantage of using the anaerobic process is that mechanical equipment is not required. Anaerobic bacteria release hydrogen sulfide as well as methane gas, both of which can create hazardous conditions. The following reactions represent the anaerobic transformation by anaerobes common in wastewater treatment (Equations 2.3 and 2.4):

anaerobes

Organics + NO3 b Anaerobes + CO2 + N2 + energy which utilizes bounded oxygen in nitrate, or anaerobes

Organics + SO2-4 b Anaerobes + CO2 + H2S + energy which utilizes bounded oxygen in sulphate. Anaerobic bacterial activities are primarily founded in the digestion of sludge and wastewater lagoons. Anaerobic processes are normally biochemically inefficient and generally slow and produce complex end products some of which emit an obnoxious smell. In food and agricultural wastewater treatment, proteins are often degraded anaerobically into amino acids and CO2 (like aerobic degradation), H2, alcohols, organic acids, methane, hydrogen sulphide, phenol, and indol.

Most of the bacteria that absorb the organic matters in a wastewater treatment system are facultative in nature. The nature of individual facultative bacteria is dependent upon the environment in which they live. Usually, facultative bacteria such as E. coli will be anaerobic unless there is some type of mechanical or biochemical process used to add oxygen to the wastewater. When bacteria are in the process of being transferred from one environment to the other, the metamorphosis from anaerobic to aerobic state (and vice versa) takes place within a couple of hours. Common bacteria found in biological wastewater treatment processes are listed in Table 2.1.

Using glucose as the organic substance and formula C5H7O2N to represent the composition of microorganisms, the basic organic bioconversion brought about by aerobes in biological wastewater treatment plants may be represented by the following (Equation 2.5):

C6H12O6 + 0.5NH+4 b C5H7O2N + 3.5CO2 + 5H2O + 0.5H+

Many studies on cell compositions have revealed that bacteria are composed of 80% water and 20% dry matter; approximately 90% of the dry matter in bacteria is organic. An approximate formula of C5H7O2N is often used in expressing the biochemical reaction; however, the formulation C60H87O23N12P may also be used when phosphorus is considered. The inorganic compounds in cells are about 50% phosphorus, 15% sulphur, 11% sodium, 9% calcium, 8% magnesium, 6% potassium, and 1% iron. All these inorganic elements are required for microbial growth, and since all of them are derived from the environment, shortage of any of

Table 2.1. Common organisms encountered in biological wastewater treatment (excluding flies).

Species

Genre

Process Involved

Achromobacter

Bacteria

Biofilters and activated sludge

Acinetobacter

Bacteria

Biological phosphorous removal

Alcaligenes

Bacteria

Biofilters, activated sludge, and sludge

digester

Bloodworm

Metazoa

Biofilters and treated sludge

Chironomus

Metazoa

Stabilization ponds and sludge

Crustacea

Metazoa

Stabilization ponds and activated sludge

Daphnia

Metazoa

Activated sludge and ponds

Desulfovibrio

Bacteria

Sludge digesters

Flavobacterium

Bacteria

Activated sludge, biofilters, sludge digester

GAO

Bacteria

Biological phosphorus removal

Geotrichum

Fungus

Activated sludge and biofilters

Gordonia

Bacteria

Activated sludge

Micrococcus

Bacteria

Activated sludge and biofilters

Microtrix

Bacteria

Activated sludge

Nitrobacter

Bacteria

Nitrification

Nitrosomonas

Bacteria

Nitrification

PAO

Bacteria

Biological phosphorus removal

Pseudomonas

Bacteria

Denitrification

Rotifera

Metazoa

Activated sludge

Sphaerotilus

Bacteria

Activated sludge

natans

Tubifex

Metazoa

Biofilters

Vorticella

Protozoa

All aerobic processes and ponds

Zoogloea

Bacteria

Activated sludge and biofilters

ramigera

these elements would result in stunted growth or altering growth path. The pH of the environment is also important in microbial activities. Most bacteria cannot tolerate pH levels above 9.5 or below 4.0. The optimum pH value range of optimal growth for bacteria lies between 6.5 and 7.5.

Fungi are a group of microscopic nonphotosynthetic plants including yeasts and molds. Yeasts are widely used in the food industry for brewing and baking, and molds are filamentous fungi that bear a resemblance to higher plants in structure with branched, fractallike growths. Fungi tend to compete disadvantageously with bacteria for nutrients, so their numbers are low except when the pH is low because the acidic condition favors growth of fungi. Fungi such as molds are nuisances in many biological wastewater processes because of their filamentous nature, which interferes with floc settling in flocculation and sedimentation basins.

The majority of filamentous organisms are bacteria, although some of them are classified as algae, fungi, or other life forms. There are a number of types of filamentous bacteria that proliferate in the activated sludge process. Filamentous organisms perform several different roles in the process, some of which are beneficial and some of which are detrimental. When filamentous organisms are in low concentrations in the process, they serve to strengthen the floc particles. This effect reduces the amount of shearing in the mechanical action of the aeration tank and allows the floc particles to increase in size. Larger floc particles are more readily settled in a clarifier. Larger floc particles settling in the clarifier also tend to accumulate smaller particulates (surface adsorption) as they settle, producing an even higher-quality effluent. Conversely, if the filamentous organisms reach too high a concentration, they can extend dramatically from the floc particles and tie one floc particle to another (in-terfloc bridging) or even form a filamentous mat of extra large size. Due to the increased surface area without a corresponding increase in mass, the activated sludge will not settle well. This results in less solids separation and may cause a washout of solid material from the system. In addition, air bubbles can become trapped in the mat and cause it to float, resulting in a floating scum mat. Due to the high surface area of the filamentous bacteria, once they reach an excess concentration, they can absorb a higher percentage of the organic matters and inhibit the growth of more desirable organisms.

Algae

Algae are photosynthetic eukaryotes that inhabit all water bodies. There are only two situations where algae are involved in wastewater: trickling filters and stabilization bonds. Only stabilization ponds utilize algae to treat wastewater. The distinct feature of algae is that they use photosynthesis to produce energy via chlorophyll (which causes green color in green plants). The major group of algae is green algae that are found in aquatic environments. Blue-green algae are prokaryotes.

42 Food and Agricultural Wastewater Utilization and Treatment Protozoa and metazoa

In a wastewater treatment system, the next higher life form above bacteria is protozoa. These single-celled animals perform three significant roles in the activated sludge process: floc formation, cropping of bacteria, and removal of suspended material. Protozoa are also indicators of biomass health and effluent quality. Because protozoa are much larger in size than individual bacteria, identification and characterization is readily performed. Four major groups of protozoa have been identified: Mastigo-phora, Sarcodina, Sporazoz, and Ciliata. Ciliatae are the largest and most important protozoa in biological wastewater treatment, where they feed on bacteria and aid in both bioflocculation and clarification. Metazoans are very similar to protozoa except that they are usually multicelled animals. Macro-invertebrates such as nematodes and rotifers are typically found only in a well-developed biomass. The presence of protozoa and meta-zoans and the relative abundance of certain species can be a predictor of operational changes within a wastewater treatment plant. In this way, a wastewater treatment plant operator is able to make adjustments based on observations of changes in the protozoan and metazoan population in order to minimize negative operational effects.

Role of microorganisms in biological wastewater treatment

The role of microorganisms in wastewater treatment varies with the specific biological process and the environment the microorganisms are in. In the activated sludge process, which is operated often as a BOD reducer, the flocs that characterize the essence of the activated sludge process comprise microorganisms, organic matters, inorganic colloidal materials, and larger particulates. The structures of flocs provide certain advantages because they serve as not only colonies for BOD removal agents such as bacteria but also traps for soluble and insoluble BOD where they are readily hydrolyzed by extracellular enzymes prior to being absorbed and metabolized by microorganisms. Another important function for activated sludge is its significant role in promoting good settlement in the secondary sedimentation tanks or basins.

In trickling filters, the role of microorganisms in wastewater treatment is played out in the slime layer (called the biofilm) that adheres to the surface of the supporting media also known as the filter media. A trickling filter is a biological wastewater treatment system that consists of a circu lar bed of coarse stones and plastics that are continuously subject to a trickling flow of wastewater from an overhead, rotating distributor. The bacteria in the wastewater attach themselves to the bedding materials where the organic matters break down. Slime-producing bacteria, such as Z. ramigera, often initiate the formation of and the thickening of the biofilm. However, many other organisms contribute to further colonization of the biofilm of a multiple-layered structure with an outer layer of, often, fungi and the removal of BOD from the passing wastewater. This is, at least in terms of composition of the biomass of the biofilm, in contrast to that of activated sludge, where the existence and role of algae and fungi are insignificant. The design of trickling filters also exposes the process to limitations of mass transfers of O2, soluble organic matters, and metabolized substances. This is a more severe issue when the thickness of the biofilm is considerable enough to affect the biodegradation of organic matters.

Anaerobic digestion is a slower biodegradation process of organic matters by anaerobic and facultative bacteria and is usually carried out in a continuously stirred tank reactor (CSTR) in order to suspend the insoluble organic materials. The reactor usually has a residence time of several days and is fed with a slurry of solids. The end products from the reactor usually are solids (a less amount than the feed), CO2, and CH4. Two groups of bacteria are involved; one group comprised of nonmethanogenic bacteria converts organic matters to simpler compounds such as organic acids, carbon dioxide, and hydrogen, and the second group, named meth-anogenic bacteria, transforms the metabolized products into methane. The interdependence between these two groups of bacteria fosters a delicate relationship that could be easily out of sync with some changes in general environmental parameters such as pH. The fragile alliance of the bacteria in these systems contributes to the difficulty in operations of anaerobic digesters.

The role of a wastewater stabilization pond, a condensed ecosystem of nature, on the other hand, is more complex, despite its simplistic appearance and operational logistics. It has diverse species of biota and incorporates several complete nutrient recycles: carbon, nitrogen, and sulphur. Depending on the primary purposes of the ponds, they can be divided into three basic groups: anaerobic, facultative, and maturation. The microorganisms inhabited in these communities vary considerably in terms of dominant species in the ponds. In general, the higher the BOD is, the lower the diversity of species in these ponds would be.

44 Food and Agricultural Wastewater Utilization and Treatment Microbial Metabolism

Microbial energy generation

Microorganisms consume energy for their growth, reproduction, and maintenance (tasks such as motility, transport of materials in and out of the cell, and synthesis of new cell materials). The energy is derived from either physical source (light) or chemical source (breakdown of substrates). It is converted into biologically utilizable energy by microbial metabolism and stored inside the microorganism in a chemical form as a compound called adenosine 5'-triphosphate (ATP). It consists of an adenosine molecule that is linked to three inorganic phosphate molecules by phosphoryl bonds. These bonds are the energy source for microbial activities since the formation of these bonds requires a large amount of energy and the hydrolysis of the bonds releases energy that can be utilized microbiologically. The production of ATP is through the reaction between adenosine 5'-diphosphate (ADP) and inorganic phosphate resulting in a new phosphoryl bond in the ATP. Once the ATP is formed, it can be stored in the cell and used as needed by hydrolyzing the phosphoryl bond. Two types of phosphorylation reactions form ATP: substrate-level and oxida-tive. The substrate-level phosphorylation is particularly important for a certain bacterial growth that is devoid of free oxygen. The anaerobic microbes synthesize ATP exclusively with one of six inorganic phosphate compounds (substrates) in an enzymatic reaction. The other source of the microbial energy generation can be viewed as a biological redox half-reaction of NAD(P)H and NADH with nicotinamide adenine dinucleotide (NAD+) and its phosphorylated product of NADP+ as acceptors for electrons. The oxidation of NADH and NAD(P)H releases energy to synthesize ATP. For example, oxidation of one mole of NAD(P)H helps yield three mole of ATP.

Uptake of substrates into microbial cell

Microbial growth requires the substrates in the wastewater to be brought inside the cell for utilization. Not all organic particulates or soluble solids can penetrate through the rigid, hydrophobic cell wall of a bacterium. Only small hydrophobic molecules can permeate the cell membrane unassisted. Some species of microorganisms have the ability to secrete enzymes outside the cell, hydrolyzing the larger molecules into smaller sol uble molecules that can enter the cell. Any form of close contact between substrates and the cells enhances the enzymatic breakdown, as in the cases in the trickling filters and activated sludge processes.

If the concentration of the substrate molecules is higher than the concentration across the cell wall and inside the cell, the small hydrophobic substrate molecules can permeate the cell wall of a microorganism via molecular diffusion mechanism. The mechanism of this diffusional mass transfer is similar to the mass transfer of molecules across a synthetic membrane. The majority of substrate transport, however, relies on a more active form of mass transfer that requires energy. As described previously, ATP contains energy-rich phosphoryl bonds that can be broken by hydrolysis and a large amount of energy released. Hence, ATP hydrolysis is an exergonic reaction. However, the release of energy from hydrolysis is not in the form of heat. It is rather used to drive the coupled biological reactions that need energy to complete (these reactions are called endergonic reactions). A portion of ATP energy is used for the active transport of substrates. This type of active mass transport of substrates requires a group of carrier enzymes called permeases (a word combining permeate and the suffix ases for enzymes). Permeases are substrate-selective, and therefore the uptakes of substrates in wastewater often are restricted by the amount of permeases present. The permease-assisted substrate transport overcomes the limit associated with the requirement of concentration gradient across the cell wall of molecular diffusion. It is not unusual to have internal substrate concentration inside the bacterial cell up to a thousandfold higher than the level in the wastewater. This is also the reason why the microorganisms can live in low-BOD environments such as rivers and oceans.

Oxidation of organic and inorganic substrates

Organic matters in wastewater are not directly oxidized to CO2 and H2O because there is no energy conservation mechanism accommodating the release of large amounts of energy resulting from oxidations with CO2 and H2O as the end products. Rather, they are oxidized in small steps. This typically involves transfer of an electron from the substrate being oxidized to some acceptor molecule that will be reduced as a result. The major electron acceptors (sometimes also called hydrogen acceptors due to the fact that for every electron removal there is a simultaneous loss of proton and the net result is loss of a hydrogen atom) in microbial cells are two carrier molecules known as pyridine nucleotides: NAD and NADP. When they undergo redox reactions, the energy released from oxidation of NAD and NADP helps synthesize ATP. Microorganisms that obtain their reducing equivalents necessary for energy generation from oxidation of organic matters are called organotrophs (including photoorganotrophs, which derive energy from sunlight for photosynthesis and chemoorganotrophs, which generate energy from oxidation of organic compounds).

Many microorganisms are also able to oxidize inorganic materials. These microbes are termed lithotrophs. Bacteria that obtain their energy from the oxidation of inorganic compounds coupled with the energy release to ATP synthesis by means of electron transport chain are called chemolithotrophs. Those lithotrophs that derive their energy directly from sunlight are also known as photolithotrophs. There are many potential inorganic energy sources that include H2, NH3, metal ions (e.g., Fe2+), and sulphur for biochemical reactions in the wastewater treatment.

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