Fighting against POLLUTION to Save Environment
Experimentation on bio degradation of detergents by OECD-301 B Method [Modified Strum Method]:
J. Microb. World: 2 (1) pp.49-58, 2000.
D.B. Boralkar1 and Vinita M. Dhupkar2
1. Central Pollution Control Board, Parivesh Bhawan, East Arjun Nagar, Delhi - 110 032.
2. Central Laboratory, Maharashtra Pollution Control Board CIDCO Bhawan, Belapur CBD, New Mumbai-400614

Increased domestic use of detergents has created a lot of problems in the sewer system. Linear alkyl benzene sulphonates (LAS) have replaced the branched alkyl benzene sulphonates due to their grater biodegradability. Alpha-olefin sulphonates (AOS) have also been introduced. Besides above active matters, commercially available detergents contain large number of other additions, largely inorganics viz. phosphates. Some premier detergents now contain enzymes (detergent alkylates), bleaching/ oxidising agents. These represent increased pollutional load on the eco-system.

The Biodegradation of a detergent is not a constant but a variable dependent on its formulation, molecular properties and the environment in which it is found. The biodegradability scores are also dependent on the test system used including the environment.

The OECD-301 B (modified sturm test) deals with total carbon content in a detergent formulation. Test organisms are inoculated into a chemically defined microbial growth medium containing the test sample. CO2 evolved is measured and compared with the theoretical amount of carbon dioxide, to indicate degree of biodegradation.

In India, the OECD-301 B test was first carried out by the Industrial Toxicology Reserch Centre (ITRC), Lucknow, in 1980 to test biodegradability of linear alkyl benzyne sulphonates (LAS). At the Maharashtra pollution Control Board's Central Laboratory, Mumbai, experiments were carried out, in 1993-94 , to study biodegradability of detergents. This included both detergent raw materials and products based on LAS and AOS.


Linear alkyl benzene sulphonates (LAS) are the major active matters in commercially availabble detergents. Their degradation pathway and the bacterial response for the degradation are discussed below.

Possible metabolic pathways by which micro-organisms degrade and mineralise LAS have been described. However, knowledge of the pathways is still incomplete because LAS does not have a single unique molecular structure. It consists of at least four homologous (C10 to C13), each of which is present as four or m ore isomers depending on the position of the sulphopheny1 group on alkyl chain (1).

In the degradative pathway the alkyl chain is first attacked by woxidation of one terminal C atom, forming a sulphonyl alkonoic acid via - CH2OH and - CHO. The C atom attacked is the one farthest from the phenyl group.

The carboxylic acid formed is then converted by B-oxidation to another sulphophenyl mono alkanoic acid containing two fewer C atoms than the original molecule, producing acetic acid or derivative in the process. It was thought that a-oxidation also occurs to form a sulphophenyl mono alkanoic acid containing only one fewer C atom. However, this is a very rare reaction. The process of B-oxidation continues producing mono carboxylic acids of lower molecular weight.

At this state, tlie ring is cleaved by meta breakdown of catechol.

The next step is desulfonation. Confirmation is not yet available on the timing of desulfonation, i.e. before or after opening of the ring.

Final product is S04, produced either by enzymatic auto-oxidation of the released sulphite or by catalytic oxidation by metals, such as cobalt (Fig. 1) (2). Metabolic intermediates are formed during degradation of LAS isomers by Pseudomonas sp. as follows (2):

2 PSP - C10 3 PSP - Butyric acid 5 - PSP-heaxnoic acid
2 PSP - C11 4 PSP - Valeric acid -
 2 PSP - C12 3 PSP - Butyric acid 5 - PSP-heaxnoic acid
2 PSP - C13 4 PSP - Valeric acid 5 - PSP-heptanoic acid
3 PSP - C11 3 PSP - Valeric acid 5 - PSP-heptanoic acid
3 PSP - C12 4 PSP - Hexaonoic acid -
3 PSP - C13 3 PSP - Valeric acid 5 - PSP-heptanoic acid
 4 PSP - C13 4 PSP - Valeric acid 5 - PSP-heptanoic acid
PSP = Para sulpho phenyl

Bacteria Degrading LAS
Many bacteria reported to degrade LAS, are commonly found in the environment, for example Pseudomonas, Aerobacter and Alacaligenes. These species together with Micrococcus, Flavobacetrium and Paracolobactrum give better results of biodegradation.

The degradation of LAS by mixed population has been investigated. Nine species of bacteria were identified - five of genera Peudomonas two each of Acromobacter and Acinetobacter. Bacteria were isolated from activated sludge and rivers and some species of Pseudomonas and Alcaligenes could degrade the alkyl chain of LAS but not he benzene ring, while one strain could not degrade LAS but could degrade ring of the intermediates (2).

Researchers have reported that the primary degradability of homologues of LAS in general increases as the chain length increases. Greater the distance between sulphonic group and the terminal methyl group on the alkyl chain, faster is the degradation. This is known as the "Distance Principle".