Graham F. White

Microbial cleavage of nitrate esters : defusing the environment

SUMMARY: The products of condensing organic hydroxyl groups (ROH) with the mineral acids (hydrochloric, nitric, phosphoric and sulphuric) collectively constitute a major part of the output of the synthetic organic chemical industry, with a wide diversity of applications including surfactants, pesticides, herbicides, dyes, methylating agents, explosives and pharmaceuticals. Compounds containing similar or related structures also occur as natural products. Phosphate esters are, of course, exceptional for their ubiquity in the living world, ranging from the simple intermediary metabolites of glycolysis, through phospholipids, to the backbone of DNA. Sulphate esters too are abundant (Dodgson et al., 1982) and naturally occurring halogenated compounds are also being detected in increasing numbers (for examples, see Strunz, 1984; Engvild, 1986; Neidleman & Geigert, 1986; Harper & Hamilton, 1988). It is therefore no surprise that living organisms have evolved phosphatase (Boyer, 1971), sulphatase (Dodgson & Rose, 1975; Dodgson et al., 1982) and dehalogenase (Neilson, 1990; Hardman, 1991) enzyme systems for initiating biodegradation of such compounds by the removal of the mineral moiety. In marked contrast, we are unable to find any examples of naturally occurring nitrate esters, so that the introduction of such compounds into the environment during their industrial production and usage constitutes a true xenobiotic challenge to microorganisms. This raises intriguing questions about microbial capability for biotransformation/biodegradation of nitrate esters, not only from an academic viewpoint but also because of the wide industrial usage of these compounds and their likely impact on the environment.

SDS-degrading bacteria attach to riverine sediment in response to the surfactant or its primary biodegradation product dodecan-1-ol

A laboratory-scale river microcosm was used to investigate the effect of the anionic surfactant sodium dodecyl sulphate (SDS) on the attachment of five Pseudomonas strains to natural river-sediment surfaces. Three of the Pseudomonas strains were chosen for their known ability to express alkylsulphatase enzymes capable of hydrolysing SDS, and the other two for their lack of such enzymes. One strain from each category was isolated from the indigenous bacterial population present in the river sediment used; other isolates were from soil or sewage. The alkylsulphatase phenotypes were confirmed by gel zymography of cell extracts. Addition of SDS to mixed suspensions of river sediment with any one of the biodegradation-competent strains stimulated the attachment of bacteria to the sediment particles. In contrast, the attachment of biodegradation-incompetent strains was weak and, moreover, was unaffected by SDS. The SDS-stimulated attachment for competent organisms coincided with rapid biodegradation of the surfactant. The primary intermediate of SDS biodegradation, dodecan-1-ol, accumulated transiently, and the numbers of attached bacteria correlated closely with the amount of dodecan-1-ol present. Direct addition of dodecan-1-ol also stimulated attachment but the effect was more immediate compared with SDS, when there was a lag period of approximately 2 h. To account for these observations, a model is proposed in which SDS stimulates the attachment of biodegradation-competent bacteria through its conversion to dodecan-1-ol, and it is hypothesized that the observed reversibility of the attachment is due to the subsequent removal of dodecan-1-ol by further bacterial metabolism.

Complete Denitration of Nitroglycerin by Bacteria Isolated from a Washwater Soakaway

ABSTRACT Four axenic bacterial species capable of biodegrading nitroglycerin (glycerol trinitrate [GTN]) were isolated from soil samples taken from a washwater soakaway at a disused GTN manufacturing plant. The isolates were identified by 16S rRNA gene sequence homology asPseudomonas putida, an Arthrobacter species, aKlebsiella species, and a Rhodococcus species. Each of the isolates utilized GTN as its sole nitrogen source and removed nitro groups sequentially from GTN to produce glycerol dinitrates and mononitrates (GMN), with the exception of theArthrobacter strain, which achieved removal of only the first nitro group within the time course of the experiment. TheKlebsiella strain exhibited a distinct preference for removal of the central nitro group from GTN, while the other five strains exhibited no such regioselectivity. All strains which removed a second nitro group from glycerol 1,2-dinitrate showed regiospecific removal of the end nitro group, thereby producing glycerol 2-mononitrate. Most significant was the finding that theRhodococcus species was capable of removing the final nitro group from GMN and thus achieved complete biodegradation of GTN. Such complete denitration of GTN has previously been shown only in mixed bacterial populations and in cultures of Penicillium corylophilum Dierckx supplemented with an additional carbon and nitrogen source. Hence, to the best of our knowledge, this is the first report of a microorganism that can achieve complete denitration of GTN.

Novel Alkylsulfatases Required for Biodegradation of the Branched Primary Alkyl Sulfate Surfactant 2-Butyloctyl Sulfate

ABSTRACT Recent reports show that contrary to common perception, branched alkyl sulfate surfactants are readily biodegradable in standard biodegradability tests. We report here the isolation of bacteria capable of biodegrading 2-butyloctyl sulfate and the identification of novel enzymes that initiate the process. Enrichment culturing from activated sewage sludge yielded several strains capable of growth on 2-butyloctyl sulfate. Of these, two were selected for further study and identified as members of the genus Pseudomonas. Strain AE-A was able to utilize either sodium dodecyl sulfate (SDS) or 2-butyloctyl sulfate as a carbon and energy source for growth, but strain AE-D utilized only the latter. Depending on growth conditions, strain AE-A produced up to three alkylsulfatases, as shown by polyacrylamide gel electrophoresis zymography. Growth on either SDS or 2-butyloctyl sulfate or in nutrient broth produced an apparently constitutive, nonspecific primary alkylsulfatase, AP1, weakly active on SDS and on 2-butyloctyl sulfate. Growth on 2-butyloctyl sulfate produced a second enzyme, AP2, active on 2-butyloctyl sulfate but not on SDS, and growth on SDS produced a third enzyme, AP3, active on SDS but not on 2-butyloctyl sulfate. In contrast, strain AE-D, when grown on 2-butyloctyl sulfate (no growth on SDS), produced a single enzyme, DP1, active on 2-butyloctyl sulfate but not on SDS. DP1 was not produced in broth cultures. DP1 was induced when residual 2-butyloctyl sulfate was present in the growth medium, but the enzyme disappeared when the substrate was exhausted. Gas chromatographic analysis of products of incubating 2-butyloctyl sulfate with DP1 in gels revealed the formation of 2-butyloctanol, showing the enzyme to be a true sulfatase. In contrast, Pseudomonas sp. strain C12B, well known for its ability to degrade linear SDS, was unable to grow on 2-butyloctyl sulfate, and its alkylsulfatases responsible for initiating the degradation of SDS by releasing the parent alcohol exhibited no hydrolytic activity on 2-butyloctyl sulfate. DP1 and the analogous AP2 are thus new alkylsulfatase enzymes with novel specificity toward 2-butyloctyl sulfate.

A Comparative Study of the Biodegradation of the Surfactant Sodium Dodecyltriethoxy Sulphate by Four Detergent-degrading Bacteria

The 35S-labelled metabolites produced during biodegradation of sodium dodecyltriethoxy [35S]sulphate (SDTES) by four bacterial isolates were identified and quantified. All four isolates used ether-cleavage as the predominant primary degradation pathway. In two of the organisms, the etherase system (responsible for approx. 60-70% of primary biodegradation) liberated mono-, di- and triethylene glycol monosulphates in substantial proportions, the last two esters undergoing some further oxidation to acetic acid 2-(ethoxy sulphate) and acetic acid 2-(diethoxy sulphate), respectively. For these isolates, liberation of SO4(2-) directly from SDTES was also significant (30-40%) and the organisms were shown to contain alkyl sulphatases active towards SDTES. For the remaining two isolates, etherase action was even more important (responsible for greater than 80% of primary biodegradation) and was restricted almost totally to the alkyl-ether bond to generate mainly triethylene glycol sulphate, some of which was further oxidized. Very small amounts of diethylene glycol monosulphate were also produced, but its mono-homologue, and the oxidation products of both these esters, were absent. Small amounts of inorganic sulphate (approx. 10%) were liberated by these isolates and one of them also produced compounds tentatively identified as intermediates of omega-/beta-oxidation.

Glycolytic Breakdown of Sulfoquinovose in Bacteria: a Missing Link in the Sulfur Cycle

ABSTRACT Sulfoquinovose (6-deoxy-6-sulfo-d-glucopyranose), formed by the hydrolysis of the plant sulfolipid, is a major component of the biological sulfur cycle. However, pathways for its catabolism are poorly delineated. We examined the hypothesis that mineralization of sulfoquinovose to inorganic sulfate is initiated by reactions of the glycolytic and/or Entner-Doudoroff pathways in bacteria. Metabolites of [U-13C]sulfoquinovose were identified by 13C-nuclear magnetic resonance (NMR) in strains of Klebsiella and Agrobacterium previously isolated for their ability to utilize sulfoquinovose as a sole source of carbon and energy for growth, and cell extracts were analyzed for enzymes diagnostic for the respective pathways. Klebsiella sp. strain ABR11 grew rapidly on sulfoquinovose, with major accumulations of sulfopropandiol (2,3-dihydroxypropanesulfonate) but no detectable release of sulfate. Later, when sulfoquinovose was exhausted and growth was very slow, sulfopropandiol disappeared and inorganic sulfate and small amounts of sulfolactate (2-hydroxy-3-sulfopropionate) were formed. In Agrobacterium sp. strain ABR2, growth and sulfoquinovose disappearance were again coincident, though slower than that in Klebsiella sp. Release of sulfate was still late but was faster than that in Klebsiella sp., and no metabolites were detected by 13C-NMR. Extracts of both strains grown on sulfoquinovose contained phosphofructokinase activities that remained unchanged when fructose 6-phosphate was replaced in the assay mixture with either glucose 6-phosphate or sulfoquinovose. The results were consistent with the operation of the Embden-Meyerhoff-Parnas (glycolysis) pathway for catabolism of sulfoquinovose. Extracts of Klebsiella but not Agrobacterium also contained an NAD+-dependent sulfoquinovose dehydrogenase activity, indicating that the Entner-Doudoroff pathway might also contribute to catabolism of sulfoquinovose.

Ether-bond scission in the biodegradation of alcohol ethoxylate nonionic surfactants by Pseudomonas sp. strain SC25A.

Pseudomonas sp. strain SC25A, previously isolated for its ability to grow on alcohol ethoxylates (PEG dodecyl ethers) as sole source of carbon and energy, was shown to be capable of growth on the dodecyl ethers of mono-, di, tri- and octaethylene glycols. Comparative growth yields for this series of alcohol ethoxylate nonionic surfactants indicated that, whereas all of the carbon of monoethylene glycol dodecyl ether (MEGDE) was assimilable, only the alkyl chains were assimilated from the higher ethoxamers. These results are interpreted in terms of a primary biodegradation mechanism in which the scission of the dodecyl-ether bond is the first step. In the case of MEGDE this step separates the dodecyl chain from a C2 fragment, both of which are readily assimilable; for the higher ethoxamers, the assimilable dodecyl chain is accompanied by an ether-containing PEG derivative which would require further rounds of either scission before assimilation. Whole cells and cell extracts converted [1-14C]MEGDE initially and very rapidly to radiolabelled dodecanol. Disappearance of [14C]dodecaol was accompanied by production of [14C]dodecanal. [14C]Dodecanoic acid was present at relatively low concentrations throughout the incubation periods. [14C]Dodecan-1, 12-dioic acid was produced in significant quantities (up to 25% radiolabel), and the onset of its production coincided with the peak concentration of dodecanal, the disappearance of which mirrored the appearance of the dioic acid. Under anaerobic conditions in the presence of cell extracts, dodecanol (55% of radiolabel) and dodecanal (22%) accumulated rapidly from MEGDE, but there was little subsequent conversion to mono- or dicarboxylic acids. These results are interpreted in terms of a pathway initiated by dodecyl-ether cleavage to produce dodecanol, which is subsequently oxidized to dodecanal and dodecanoic acid. The formation of dodecan-1, 12-dioic acid, probably from dodecanal, may represent a means of harbouring carbon under non-growing conditions.

Immobilization of the surfactant-degrading bacterium Pseudomonas C12B in polyacrylamide gel. III. Biodegradation specificity for raw surfactants and industrial wastes

The surfactant-degrading bacterium Pseudomonas C12B was immobilized in polyacrylamide gel beads and the specificity of the immobilized cells towards various surfactants was examined. Profiles of activity towards primary alkyl sulfates of chain lengths from C6 to C14 were very similar for free and immobilized cells and reflected the known specificities of the alkylsulfatase enzymes that initiate the degradation. Initial rates of surfactant disappearance were in the order alkyl sulfates > linear alkyl benzene sulfonates > alkyl ethoxy sulfates ⪢ alkane sulfonates. Sulfate esters, but not sulfonates, were totally degraded within 48h. Immobilized cells also removed surfactant from solutions containing raw alkyl ethoxy sulfate mixtures (used to formulate shampoos), diluted whole-shampoo formulations, and mixed balance-tank effluent containing waste shampoo, hair dyes, and permanent wave formulations from a hair products factory. Repeated exposure to these wastes in the presence of added basal salts showed that the immobilized cells retained at least 60% and 28% of their activity after 6-day and 13-day operating periods, respectively. Balance-tank effluent, while initially producing the lowest activity, also provided the greatest stability even in the absence of added basal salts. Broad substrate specificity for sulfated surfactants, coupled with good biocatalyst stability, suggested that this system offered considerable potential for on-site treatment of surfactant wastes.

Multiple interactions in riverine biofilms - surfactant adsorption, bacterial attachment and biodegradation

Many organic pollutants, especially synthetic surfactants, adsorb onto solid surfaces in natural and engineered aquatic environments. Biofilm bacteria on such surfaces make major contributions to microbial heterotrophic activity and biodegradation of organic pollutants. This paper reviews evidence for multiple interactions between surfactants, biodegradative bacteria, and sediment-liquid interfaces. Biodegradable surfactants e.g. SDS, added to a river-water microcosm were rapidly adsorb to sediment surface and stimulated the indigenous bacteria to attach to the sediment particles. Recalcitrant surfactants and non-surfactant organic nutrients did not stimulate attachment Attachment of bacteria was maximal when biodegradation was fastest, and was reversed when biodegradation was complete. Dodecanol, the primary product of SDS-biodegradation, markedly stimulated attachment. When SDS was added to suspensions containing sediment and either known degraders or known non-degraders, only the degraders became attached, and attachment accelerated surfactant biodegradation to dodecanol. These cyclical cooperative interactions have implications for the design of biodegradability-tests, the impact of surfactant adjuvants on biodegradability of herbicides/pesticides formulated with surfactants, and the role of surfactants used to accelerate bioremediation of hydrocarbon-polluted soils.

Bacterial biodegradation of glycerol trinitrate

Abstract Glycerol trinitrate (GTN) is widely used as a high explosive and as a potent vasodilator in the treatment of heart diseases including angina pectoris. Its sensitivity to detonation by mechanical shock, and its pharmacological potency, while critical to its utility, also constitute problems for handling and treating wastes arising from the production and use of the compound. This paper describes bacterial biodegradation of GTN, which may offer a means of removal of GTN from waste streams and bioremediation of contaminated land sites. Pseudomonas sp. strain RI-NG1 was isolated from river sediment by enrichment culture on minimal salts/glycerol/GTN medium. Batch culture experiments established that this isolate utilised GTN as its sole source of nitrogen. Analysis of the growth medium using HPLC showed the presence of glycerol dinitrates and glycerol mononitrates. Isomeric glycerol 1,2-dinitrate and glycerol 1,3-dinitrate were produced simultaneously and concomitantly with the disappearance of GTN, with significant regioselectivity for the production of the 1,3-isomer. After GTN disappearance was complete, the dinitrates were further degraded to glycerol 1- and 2-mononitrates. Preliminary experiments indicated that the nitro group was liberated as nitrite, not nitrate. The bacterial biodegradation of GTN thus shows parallels with GTN metabolism in eukaryotic (mammalian and fungal) systems.