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  • APPLIED MICROBIOLOGY, Jan. 1968, p. 62-68 Vol. 16, No I Copyright © 1968 American Society for Microbiology Prinited in U.S.A.

    Activated-Sludge Nitrification in the Presence of Linear and Branched-Chain Alkyl

    Benzene Sulfonates CHARLES R. BAILLOD AND W. C. BOYLE

    Department of Civil Engineering, The University of Wisconsini, Madison, Wisconsin 53706

    Received for publication 18 July 1967

    The effects of biodegradable linear alkyl benzene sulfonate and branched-chain alkyl benzene sulfonate detergents on activated-sludge nitrification were investi- gated by administering a synthetic waste containing up to 23 mg of each detergent per liter to eight bench-scale, batch, activated-sludge units. It was found that both detergents tended to promote complete oxidation of ammonia to nitrate, whereas control units produced approximately equal amounts of nitrite and nitrate. Various hypotheses are offered to explain the phenomenon.

    Within recent years, biodegradable linear alkyl benzene sulfonates (LAS) have replaced the older branched-chain alkyl benzene sulfonates (ABS) in nearly all household detergent formu- lations. It is apparent that these surfactants reach biological waste treatment plants where they come into contact with the biota of the plant. Small concentrations of certain organic substances are known to exert inhibitory effects on nitrification (3, 6, 14, 17), and surface-active agents have been reported to produce physio- logical effects on bacteria (6). The purpose of this study was to investigate the effects of anionic detergents on nitrification occurring in the ac- tivated-sludge process. Particular attention was devoted to determining what, if any, differences exist between the effects of LAS and ABS on activated-sludge nitrification.

    In fresh sewage, most nitrogen is in the form of organic nitrogen (protein, urea, and amino acids) with some ammonia. During biological stabilization of the waste, protein proteolysis and deamination produces ammonia, and urea is hydrolyzed to form additional amounts of am- monia. Ammonia may be utilized in bacterial cell synthesis, or it may be oxidized to nitrite. Downing et al. (2) indicated that, in heavily loaded activated-sludge plants, the rate of sludge syn- thesis is high, and the relatively slow-growing nitrifying bacteria are literally "squeezed out" by the heterotrophic sludge mass; consequently, nearly all of the ammonia is utilized in sludge synthesis. On the other hand, in lightly loaded plants the net rate of synthesis is low, and the slow-growing nitrifiers can become established in

    the sludge, resulting in the conversion of am- monia to nitrite. The nitrite is usually rapidly oxidized to nitrate; in the absence of dissolved oxygen, nitrite may be denitrified to nitrogen gas. Denitrification has been proposed as an efficient means of removing nitrogen in waste treatment (9).

    Nitrification may or may not be desirable, de- pending on the biota and dissolved-oxygen levels of the receiving stream. Nitrate tends to encourage algal blooms, whereas ammonia nitrogen serves as a nitrogen source for many bacteria. Nitrate may also act as a hydrogen acceptor through its reduction to nitrite. Consequently, an effluent containing 10 mg of nitrate nitrogen per liter could supply an oxygenation capacity equivalent to 11.4 mg of dissolved oxygen per liter through nitrate reduction. Furthermore, nitrification can cause a significant oxygen depletion when it takes place in receiving waters. Thus, the advantages and disadvantages of nitrification in waste treat- ment take on a quantitative significance only when judged in relation to the problems likely to be encountered in a particular receiving stream.

    Nitrification is defined as the biological oxida- tion of ammonia to nitrite and the subsequent conversion of nitrite to nitrate according to the stoichiometric equations:

    (1) 2NH4+ + 302 - 2NO2- + 2H20 + 4H+

    (2) 2NO2-+ 02 - 2NO3- These oxidations are believed to be carried out primarily by genera of the bacterial family Nitro- bacteriaceae, notably Nitrosomonas and Nitro-


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    bacter. In view of the well-documented ability of certain heterotrophs to produce nitrite (4, 5, 7) and the reported ability of certain fungi to pro- duce both nitrite and nitrate (13, 15), it is possible that at least a portion of the nitrification taking place in activated sludge could be due to or- ganisms other than the Nitrobacteriaceae. Since no isolation nor identification of nitrifying or- ganisms was attempted in this work, organisms which produce nitrite will be referred to collec- tively as the nitrite-forming group; those which produce nitrate will be referred to as the nitrate- forming group.


    Eight 2-liter Erlenmeyer flasks were operated as bench-scale, batch, activated-sludge units. Aeration was accomplished by passing unmetered air through cylindrical diffuser stones at rates high enough to keep the mixed-liquor solids suspended. With this aeration procedure, the mixed-liquor dissolved oxygen levels were maintained between 8 and 9 mg/liter. Each unit was inoculated with 200 ml of return sludge obtained from the Nine Springs Sewage Treat- ment Plant, Madison, Wis. A substrate was then added and aeration was begun. Later, in order to supply a more adequate inoculum of nitrifying organ- isms, each unit was inoculated with 100 ml of a highly nitrified, unsettled, trickling-filter effluent (also from the Nine Springs Plant). The volumetric operating parameters for each

    batch unit were maintained as follows: total volume under aeration, 1,430 ml; volume of substrate added, 760 ml; volume of return sludge, 670 ml; and interval between feedings, 24 hr. At the end of the aeration period, the sludge was allowed to settle for approxi- mately 30 min, and the supernatant fluid was siphoned off. Fresh substrate (760 ml) was added to each unit and aeration was resumed. To minimize foaming, a coating of Dow-Corning antifoam emulsion was applied to the necks of all units. Throughout the entire study, the mixed-liquor temperature ranged from 24.5 to 27 C. The substrate consisted of nonfat dry milk solids,

    with ammonium phosphate, phosphate buffer, and tap water added to provide trace metals. A detailed composition and analysis of the substrate without added detergents is as follows. Composition: nonfat dry milk solids, 500 mg/liter; (NH4)jHPO4, 10 mg/liter as N; K2,HPO4, 348 mg/liter; tap water, 14% (v/v); and distilled water, 86% (v/v). Analysis: pH, 7.85; chemical oxygen demand (COD), 500 mg/liter; biochemical oxygen demand (BOD), 330 mg/liter; organic nitrogen, 28 mg/liter as N; am- monia nitrogen, 10 mg/liter as N; nitrite, 0.0 mg/liter; nitrate, 0.0 mg/liter; alkalinity, 200 mg/liter as CaCO3; and methylene blue active substance (MBAS), 0.1 mg/liter.

    The two types of anionic detergents used were standard reagents of known composition and per- centage of methylene blue active substance, obtained from the Assoc. of American Soap and Glycerine

    Producers, Inc. The composition of the reagents was as follows. ABS reagent: ABS, 54.8%; Na2SO4, 40.3%; free oil, 0.5%; and Na2CO3, 0.7%. Equivalent weight, 348. LAS reagent: sodium dodecyl benzene sulfonate, 42.1%; NaCl, 1.0%; alcohol insolubles (mainly Na2SO4 with some sodium tripolyphosphate), 50.5%; and unsulfonated oils, 0.6%.

    In the case of ABS, "alkyl" merely signifies a randomly branched hydrocarbon chain averaging 12 carbon atoms in total length, whereas LAS denotes a homogeneous compound with a linear 12-carbon alkyl side-chain. Standard solutions of 4,000 mg of MBAS per liter were prepared for each reagent and were added to the substrate in order to obtain the desired concentrations of detergents.

    Chemical analyses were performed according to the procedures outlined in Standard Methods for the Examination of Water and Wastewater (American Public Health Assoc., Inc., New York, 1960). Nitrate was determined by the brucine procedure, whereas ammonia was determined by both direct nesslerization and titration. Detergents remaining in the effluent were determined according to the methylene blue pro- cedure proposed by Longwell and Maniece (11).

    For a period of 2 months, the eight experimental units were operated on a substrate free of detergents. During the first month, the substrate composition and volumetric loading parameters were varied in order to develop a substrate composition and de- tention time which were both realistic and conducive to nitrification. At the end of the first month, the mixed liquor was homogenized and was redispensed to the units. During the second month, all units were operated identically on the detergent-free substrate described previously. Sludge was wasted occasionally during this period in order to maintain the mixed- liquor suspended solids in the neighborhood of 1,500 to 2,000 mg/liter. After the acclimation period, the batch-activated sludge units were subjected to three concentrations of both ABS and LAS detergents. The type and concentration of detergent fed to each unit are shown in Table 1.


    The initial-shock effects of the detergents were observed by sampling each unit at intervals during

    TABLE 1. ABS and LAS concentrations present in feed solutions

    Unit Type of detergent (mg/liter)

    1 LAS 6.6 2 LAS 13.8 3 LAS 23.0 4 ABS 6.6 5 ABS 13.8 6 ABS 23.0 7 Control 0.0 8 Control 0.0

    a MBAS = methylene blue active substance.

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