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State-of-the-Art Application of

Biotechnology in

Wastewater Treatment


by Wilfredo I. Jose, Ph.D.


Even before the discovery of the existence of microorganisms, man has produced fermented food and feeds. The same degrading action of the microorganisms has been used in human wastes, which is naturally biodegradable. But for large scale treatment, man had to design artificial processes emulating the degrading activities of naturally occurring microorganisms. Microbiologists, engineers and chemists worked hand in hand in accomplishing this objective Now we know that for biodegradable materials other than human wastes, the process is also valid. These biological waste treatment schemes belong to one of the largest field of application within biotechnology.
Usually biotechnology aims to synthesize products by using specially isolated pure strains of microorganisms under optimal conditions. Defined compositions of nutrient media (carbon source, energy source, nitrogen, sulfur, phosphorus, trace minerals and vitamins) ':ire used. The growth and yields of biomass and products under proper cultural conditions are related to the properties of the pure strain. The production of antibiotic, a valuable product, is an example of this development in the field of traditional biotechnology.

Waste treatment biotechnology differs substantially in comparison to traditional processes. first, no profit-oriented and commercial products are produced except cleaner effluent for a better environment which soon will be invaluable. Second the waste materials are often mixed. finally, pure culture cannot be used and one can only rely on naturally present mixed population or enrichment of the mixed cultures if possible. Therefore, the scale of economics is quite different.

Industrial microbiologists have recognized the usefulness of microorganisms in waste treatment since 1914, with the development of' a very versatile biological process unit known as the activated sludge process. This process is dependent on a mixed culture of naturally occurring microorganisms, each with the ability to degrade a component of the waste fraction and able to biologically coexists together. A recent advancement due to research and development was improvement of the biological floc formation and reduction of sludge output by inoculating said process system with specially adapted and cultured microorganisms. At present, cultures that are able to degrade specific priority toxicants and undesirable compounds in waste streams are already available. With the advent of genetic engineering, particular microorganisms can be designed and tailored to degrade specific types of wastes. However, releasing these genetically-engineered microorganisms might affect the ecosystem adversely like the chemical waste nightmare that man has created. Learning from this, man has now developed extensive researches on the after effects of these strains once it is applied on a large scale.

Today, the state of the art process is the supplementation of existing bacterial population with bacterial strains that are capable of higher rates of reduction or utilizing strains that are capable of degrading compounds that has previously been considered non-biodegradable. This paper considers the poorly degradable substances found in industrial waste streams that are now amenable to microbial degradation


Aerobic metabolism consists of two processes: first, the electron transfer from organic substrates to oxygen -- as source of energy for the cell, and secondly, the addition of oxygen to the organic substrate -- prepares the substrate for further metabolism. The degradation of xenobiotics and difficultly degradable compounds are important to the second part. In aromatic compounds, the ring cleavage is dependent in oxygen. The availability of molecular oxygen for reaction depends on several enzymes. Researches on the anaerobic degradation of organic compounds are limited but the process will be important and attractive in the near future.


In nature, numerous microorganisms utilize hydrocarbons for growth and energy source. The microbes oxidizes the terminal methyl group in the aliphatic hydrocarbons. The hydrocarbon becomes a fatty acid. In general, each species can degrade limited kinds of hydrocarbons. for example, Methanomonasmethanooxidans can attack only methane, while Nocardia Paraffinicum and some species of Pseudomonas can utilize several hydrocarbons, not all necessarily found in petroleum.

Since benzenoid structures are the most common organic compounds in nature, microorganisms attack them fairly well. However, aromatic polycyclics and those with uncommon substituents (e.g. polychlorinated biphenyls) are difficult to degrade. The degradation of aromatic compounds start with ring cleavage. Examples of microbe that carry this attack are Pseudomonas putida and P. ovali.


Halogenated compounds find uses as solvents, aerosol propellants, lead scavengers, nematocides and fumigants, among others. Pseudomonas species and Xantobacter autophicus are able to degrade these compounds. Halogenated aromatic compounds used as solvents, lubricants, intermediates in synthesis, insulators, plasticizers, etc. are degraded via halocatechal formation or dehalogenation before ring cleavage. Examples of these microorganisms are Pseudomonas and Athrobacter species.


Nitroaromatic compounds, used in the manufacture of dyes, drugs, pesticides, explosives and industrial solvents are toxic. As the simpler compounds are completely biodegradable more complex nitroaromatics such as 2, 4, 6-trinitrotoluene are not degraded. In aerobic conditions, polymerization may take place, while in anaerobic condition, transformation to amines may take place.


Polychlorinated biphenyls used in transformer oils, capacitor dielectrics, and heat transformer liquids are toxic to animals and man. Acintobacter and Alcaligenes species are capable of transforming many PCB's. PCB's containing more than four chlorines are resistant to degradation.


Xenobiotics and pollutants are discharged to the environment through point sources or in a dispersed manner through consumers and end-product users. With proper desimination of information regarding pollution of these consumed wastes and with proper control of by institution of efficient waste collection and treatment systems, the often times complex physical and chemical properties of the waste fraction can be controlled. Thus, the use of these microorganisms in the degradation of these pollutants can be optimized.


Experiments with pure cultures in single substrates form the basis of collective knowledge of the biosynthetic pathways of poorly degradable compounds in microorganisms. Once a pure culture is available, the development of the biotechnological processes is possible. To isolate microorganisms with biodegradable potential, microbiologists use the technique of enrichment culture. The procedure is to allow the microorganisms with potential to grow in a medium with the poorly degradable compound as the growth limiting source of an essential nutrient. Only those microorganisms that can degrade that substance will grow.
A series of subcultures allows one to evaluate the success of enrichment. Sewage, where many microorganisms come in contact with xenobiotics, is the usual source for the enrichment of bacteria with degradative capabilities. However, isolates from natural environments where the compounds of interest are found is usually successful. This includes samples from industrial production lines, pesticide treated soils, waste dumps, or industrial waste treatment plants.


Fermentation procedures are usually conventional, starting with the slant inoculum preparation and sequential seeding to fermentors as large as 10,000 liters. Although mixed cultures are cultivated, sterile conditions are maintained to guard against contamination with Salmonella, Staphylococcus and Streptococcus. The cultural conditions maintained produces microorganisms that are repressed and conditioned to their final environment. Centrifugation or filtration are the methods used for cell concentration. Spore formers are air dried and non-spore formers are freeze dried. The cultures are then blended with additives before final packaging.


For significant advances in the microbial treatment of wastes, the identification of organic chemicals which resists degradation in conventional waste treatment plants is necessary. Upon identification, their degradation in existing plant treatment plants can now be improved or appropriate specialized technologies for their biodegradation can be achieved. Among the organic chemicals in the EPA list of priority pollutants are pesticides and metabolites, phenolic compounds, halogenated aliphatics, aromatics, nitroaromatics, chloroaromatics, polychlorinated biphenyls, phthalate esters, polycyclic aromatic hydrocarbons, and nitrosamines. This EPA list is useful in defining research direction for the improvement of wastewater treatment systems.


Biomass consists of cellulose, hemicellulose and lignin. Lignin acts as the cementing material in lignocellulosic materials and protect the structure from microbial degradation. The biodegradation of lignin is important because of its increasing number of industrial uses. Lignosulfonates, which are more resistant to biodegradation than lignin, are the waste products in the sulfite process' for pulp and paper manufacture. White rot fungi can degrade lignosulfonates. Other fungi and mixed microbes promote precipitation via poliplerization.


Commercial detergents contain 10% to 20% surfactants for cleaning purposes. Anionic surfactants is not biodegradable. A substituted linear alkyl benzene sulfonate is more biodegradable but tends to accumulate in sewage systems. Cultures of adapted microorganisms degrade alkyl sulfates and alkyl sulfonates quite rapidly.


At present, 3500 dyes are in use (out of 40,000 dyes and pigments with 7,000 different chemical structures). Therefore we cannot make generalizations of their biodegradability. The textile and dyestuff industries are responsible for the entry of dyes into the environment, although in small amounts. A model for biodegradation is the action of a Pteudomon M species on azo dyes to produce biomass, CO2, H2O and NH3. In actual situations, we can use mixed cultures of adapted microorganisms.


Highly efficient and long lasting, early synthetic organic pesticides were useful but accumulates in the ecosphere. More degradable or metabolizable types have been developed. Organochlorines such as DOT can be mineralized by Pseuomonas aeroginosa although very slowly. Some pure or mixed cultures can act on organophosphorus insecticides. Other cultures can utilize s-triazines as growth nutrient.


Because of their high molecular weights, plastics are extremely resistant to microbial attack. Some bacteria attack only polymers with low molecular weights. Acinobacter. and Monaella species attack polybutadiene with a degree of polymerization of 43, while Pseudomonas species degrade polystyrene with a degree of polymerization of 3. Plasticizers in plastics are more prone to microbial attack. These substances are degraded by microorganisms from soil and sewage.


Researches on biodegradation of xenobiotics and pollutants are accumulating. The results of the microbial, biological, and genetic studies will eventually improve the practical application of the treatment processes on the industrial scale. With higher degree of sophistication being attained, more and more specific microbial strains for the biodegradation of particular wastes will be utilized. Ideally, waste treatment plants using microorganisms should emulate industrial fermentors.

However, aseptic condition is not possible and the system is confronted with varying composition, temperature, and volume. With constantly changing toxic loads, the microorganisms may be harmed. Intermittent feeding might washout desirable strains. However, whatever problems are encountered, several microbial processes are now successful. Special mixed cultures of mutant bacteria for specific type of wastes are now available commercially. They are now more efficient in that they consume less energy than conventional schemes. Biological processes are now increasingly more attractive, efficient and most of all, economical.


With genetic engineering, specific or multiple activities of microbial cultures is now a reality. This is leading to new approaches in waste treatment. New microbial species that have been genetically altered and which could not be found in nature can be patented. The accumulation of research and studies as well as the results of the current state of the art application of biotechnology is waste treatment will lead to more efficient systems.


Gasner, L., "Miroorganisms for Waste Treatment" in Microbial Technology, Vol. 2 H.J. Peppler and D. Periman, eds. Academic Press, N.Y., 1979, pp. 211-22.

Leisinger, T. and W. 8runner, "Poorly Degradable Substances", in Biotechnology, Vol. 8, H.J. Rehm and G. Reed, eds., VCH Weinheim, 1986, pp. 475-513.

Ball C., Ed., Genetics and Breeding of Industrial Microorganisms, CRC Press, Boca Raton, 1984.

Brown, L.R., "Oil Degrading Microorganisms", Chemical Engineering Progress, Vol. B3, No. 10, October, 1987, pp. 35-40.

Scientific American, Vol. 245, No.3, September, 19B1.

Murray Moo-Young Ed., Comprehensive Biotechnology, Vol. 4, Pergamon Pr Oxford, 19B5.



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