Dehalogenation in marine sediments containing natural sources of halophenols

Halophenols such as 2,4-dibromophenol (DBP) occur naturally in some marine sediments, as a consequence of various animal and algal activities. In an earlier study, DBP was observed in the burrow microenvironment of the hemichordate Saccoglossus kowalewskii. At the concentrations found in the burrow lining, aerobic respiration appeared to be inhibited significantly relative to anaerobic catabolism. This effect, as well as factors contributing to the degradation of DBP, has been documented further here. Results from the addition of radiolabeled DBP to oxic and anoxic sediment slurries and growth experiments with aerobic and anaerobic enrichments suggested that aerobes did not significantly metabolize DBP and that concentrations likely to be encountered on the inner surfaces of the burrow wall were inhibitory. In contrast, only minimal inhibition of growth occurred for anaerobes exposed to 1 mM DBP; in addition, DBP was substantially degraded in both enrichments and sediments under anaerobic conditions. Dehalogenation with the consequent production of phenol appeared to initiate anaerobic degradation. Sulfate-reducing bacteria did not dehalogenate DBP but appeared to degrade phenol. Decreased bacterial numbers and marked differences in the concentration and chemical speciation of iron in sediments from S. kowalewskii burrows may be attributed to toxic effects of DBP on aerobic bacteria.     

Isolation, characterization, and polyaromatic hydrocarbon degradation potential of aerobic bacteria from marine macrofaunal burrow sediments and description of Lutibacterium anuloederans gen. nov., sp. nov., and Cycloclasticus spirillensus sp. nov.

Two new polyaromatic hydrocarbon-degrading marine bacteria have been isolated from burrow wall sediments of benthic macrofauna by using enrichments on phenanthrene. Strain LC8 (from a polychaete) and strain M4-6 (from a mollusc) are aerobic and gram negative and require sodium chloride (>1%) for growth. Both strains can use 2- and 3-ring polycyclic aromatic hydrocarbons as their sole carbon and energy sources, but they are nutritionally versatile. Physiological and phylogenetic analyses based on 16S ribosomal DNA sequences suggest that strain M4-6 belongs to the genus Cycloclasticus and represents a new species, Cycloclasticus spirillensus sp. nov. Strain LC8 appears to represent a new genus and species, Lutibacterium anuloederans gen. nov., sp. nov., within the Sphingomonadaceae. However, when inoculated into sediment slurries with or without exogenous phenanthrene, only L. anuloederans appeared to sustain a significant phenanthrene uptake potential throughout a 35-day incubation. In addition, only L. anuloederans appeared to enhance phenanthrene degradation in heavily contaminated sediment from Little Mystic Cove, Boston Harbor, Boston, Mass.     

Biodegradation of Phenol: Mechanisms and Applications

Phenol, or hydroxybenzene, is both a synthetically and naturally produced aromatic compound. Microorganisms capable of degrading phenol are common and include both aerobes and anaerobes. Many aerobic phenol-degrading microorganisms have been isolated and the pathways for the aerobic degradation of phenol are now firmly established. The first steps include oxygenation of phenol by phenol hydroxylase enzymes to form catechol, followed by ring cleavage adjacent to or in between the two hydroxyl groups of catechol. Phenol hydroxylases ranging from simple flavoprotein monooxygenases to multicomponent hydroxylases, as well as the genes coding for these enzymes, have been described for a number of aerobic phenol-degrading microorganisms. Phenol can also be degraded in the absence of oxygen. Our knowledge of this process is less advanced than that of the aerobic process, and only a few anaerobic phenol-degrading bacteria have been isolated to date. Convincing evidence from both pure culture studies with the denitrifying organism Thauera aromatica K172 and with two Clostridium species, as well as from mixed culture studies, indicates that the first step in anaerobic phenol degradation is carboxylation in the para-position to form 4-hydroxybenzoate. Following para-carboxylation, thioesterification of 4-hydroxybenzoate to co-enzyme A allows subsequent ring reduction, hydration, and fission. Para-carboxylation appears to be involved in the anaerobic degradation of a number of aromatic compounds. Numerous practical applications exist for microbial phenol degradation. These include the exploitation of indigenous anaerobic phenol-degrading bacteria in the in situ bioremediation of creosote-contaminated subsurface environments, and the use of phenol as a co-substrate for indigenous aerobic phenol-degrading bacteria to enhance in situ biodegradation of chlorinated solvents.     

Degradation of 4-<Emphasis Type="Italic">n</Emphasis>-nonylphenol under nitrate reducing conditions

Nonylphenol (NP) is an endocrine disruptor present as a pollutant in river sediment. Biodegradation of NP can reduce its toxicological risk. As sediments are mainly anaerobic, degradation of linear (4-n-NP) and branched nonylphenol (tNP) was studied under methanogenic, sulphate reducing and denitrifying conditions in NP polluted river sediment. Anaerobic bioconversion was observed only for linear NP under denitrifying conditions. The microbial population involved herein was further studied by enrichment and molecular characterization. The largest change in diversity was observed between the enrichments of the third and fourth generation, and further enrichment did not affect the diversity. This implies that different microorganisms are involved in the degradation of 4-n-NP in the sediment. The major degrading bacteria were most closely related to denitrifying hexadecane degraders and linear alkyl benzene sulphonate (LAS) degraders. The molecular structures of alkanes and LAS are similar to the linear chain of 4-n-NP, this might indicate that the biodegradation of linear NP under denitrifying conditions starts at the nonyl chain. Initiation of anaerobic NP degradation was further tested using phenol as a structure analogue. Phenol was chosen instead of an aliphatic analogue, because phenol is the common structure present in all NP isomers while the structure of the aliphatic chain differs per isomer. Phenol was degraded in all cases, but did not affect the linear NP degradation under denitrifying conditions and did not initiate the degradation of tNP and linear NP under the other tested conditions.     

Anaerobic Phenol Degradation by Microorganisms of Swine Manure

Swine manure contains diverse groups of aerobic and anaerobic bacteria. An anaerobic bacterial consortium containing sulfate-reducing bacteria (SRB) and acetate-utilizing methanogenic bacteria was isolated from swine manure. This consortium used phenol as its sole source of carbon and converted it to methane and CO₂. The sulfate-reducing bacterial members of the consortium are the incomplete oxidizers, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. The methanogenic bacteria of the consortium converted the acetic acid to methane. When a methanogen inhibitor was used in the culture medium, phenol was converted to acetic acid by the SRB, but the acetic acid did not undergo further metabolism. On the other hand, when the growth of SRB in the consortium was suppressed with a specific SRB inhibitor, namely, molybdenum tetroxide, the phenol was not degraded. Thus, the metabolic activities of both the sulfate-reducing bacteria and the methanogenic bacteria were essential for complete degradation of phenol. Received: 31 January 1997 / Accepted: 7 March 1997     

Degradation of Phthalic Acids by Denitrifying, Mixed Cultures of Bacteria

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobically on all three isomers of phthalic acid. Each culture grew anaerobically on only one isomer and also grew aerobically on the same isomer. Pure cultures were isolated from the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid) enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspension experiments indicated that protocatechuate is an intermediate of aerobic catabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalic acid) could not be isolated from the enrichments, and neither could pure cultures that grew anaerobically on any of the isomers. Cell suspension experiments suggested that separate pathways exist for the aerobic and anaerobic oxidation of phthalic acids. Each enrichment culture used only one phthalic acid isomer under anaerobic conditions, but all isomers were simultaneously adapted for the anaerobic catabolism of benzoate. Cells grown anaerobically on a phthalic acid immediately attacked the isomer under anaerobic conditions, whereas there was a lag before aerobic breakdown occurred, and, for phthalic and terephthalic acids, chloramphenicol stopped aerobic adaptation but had no effect on anaerobic catabolism. This work suggests that phthalic acids are biodegradable in anaerobic environments.     

Bacteria—food or food competitors of silver carp,Hypophthalmichthys molitrix Val.?

The role of bacteria in the nutrition of silver carp was studied by in-vitro tests, which confirmed earlier results that the potentially important source of nitrogen in bacteria is not utilized by the fish. Cultures of aerobic and anaerobic bacteria, recovered from a fish pond, were incubated with digestive fluids from the gut contents and from tissues surrounding the alimentary tract (Organ of Leydig) respectively. Growth of aerobic isolates was not affected by the digestive fluids whereas growth of all anaerobes was inhibited. A susceptibility test revealed a bacteriostatic rather than bactericidal effect of the digestive fluid on the bacteria. During 42 h incubation at approximately 20°C of gut fluid, trypsin activity decreased between 14% (in fore-gut fluid) and 62% (in mid-gut fluid). The addition of aerobic bacteria (to the mid-gut fluid) or anaerobic bacteria (to the fore-gut fluid) did not accelerate the degradation of trypsin.     

Microbial degradation of hydrochlorofluorocarbons (CHCl2F and CHCl2CF3) in soils and sediments.

The ability of microorganisms to degrade trace levels of the hydrochlorofluorocarbons HCFC-21 and HCFC-123 was investigated. Methanotroph-linked oxidation of HCFC-21 was observed in aerobic soils, and anaerobic degradation of HCFC-21 occurred in freshwater and salt marsh sediments. Microbial degradation of HCFC-123 was observed in anoxic freshwater and salt marsh sediments, and the recovery of 1,1,1-trifluoro-2-chloroethane indicated the involvement of reductive dechlorination. No degradation of HCFC-123 was observed in aerobic soils. In some experiments, HCFCs were degraded at low (parts per billion) concentrations, raising the possibility that bacteria in nature remove HCFCs from the atmosphere.     

Growth of moderately halophilic bacteria isolated from sea water using phenol as the sole carbon source

Moderately halophilic bacteria utilizing phenol as the sole carbon source were isolated by selective enrichment from sea water. The isolate (Gram-negative motile rods) was identified asDeleya venusta. It grew well in the presence of up to 1600 mg/L of phenol and 8% NaCl under aerobic conditions. When the cells were treated with chloramphenicol prior to the addition of phenol they did not utilize added phenol, even after prolonged incubation. Thus, the enzymes necessary for phenol metabolism appeared to be inducible.     

Anaerobic and aerobic degradation of pyridine by a newly isolated denitrifying bacterium.

New denitrifying bacteria that could degrade pyridine under both aerobic and anaerobic conditions were isolated from industrial wastewater. The successful enrichment and isolation of these strains required selenite as a trace element. These isolates appeared to be closely related to Azoarcus species according to the results of 16S rRNA sequence analysis. An isolated strain, pF6, metabolized pyridine through the same pathway under both aerobic and anaerobic conditions. Since pyridine induced NAD-linked glutarate-dialdehyde dehydrogenase and isocitratase activities, it is likely that the mechanism of pyridine degradation in strain pF6 involves N-C-2 ring cleavage. Strain pF6 could degrade pyridine in the presence of nitrate, nitrite, and nitrous oxide as electron acceptors. In a batch culture with 6 mM nitrate, degradation of pyridine and denitrification were not sensitively affected by the redox potential, which gradually decreased from 150 to -200 mV. In a batch culture with the nitrate concentration higher than 6 mM, nitrite transiently accumulated during denitrification significantly inhibited cell growth and pyridine degradation. Growth yield on pyridine decreased slightly under denitrifying conditions from that under aerobic conditions. Furthermore, when the pyridine concentration used was above 12 mM, the specific growth rate under denitrifying conditions was higher than that under aerobic conditions. Considering these characteristics, a newly isolated denitrifying bacterium, strain pF6, has advantages over strictly aerobic bacteria in field applications.     

Chemolithotrophic sulfur bacteria in sediments,mats, and stromatolites of western Australian saline lakes

Samples of stromatolites, microbial mats, and sediments from four saline lakes (approximate seasonal salinity ranges 20–220%o) in Western Australia were used to establish enrichments for elective cultures of aerobic and anaerobic denitrifying chemolithoautotrophs that could grow with thiosulfate as sole energy source. Organisms of these types were obtained from all sources tested. Twenty‐four pure cultures were isolated, all of which were gram‐negative, rod‐shaped bacteria exhibiting a considerable diversity of metabolic capability. Isolation of these obligate and facultative sulfur‐oxidizing chemolithotrophs from the stromatolite and mat habitats indicates the possibility that these rod‐shaped bacteria contribute to the oxidative phase of the sulfur cycle in these habitats, in addition to oxidation by phototrophs or Beggiatoa. Only four of the pure cultures could grow without salt, but all 24 showed significant halophily, some tolerating 3 M NaCl. Three novel isolates of NaCl‐dependent, thiosulfate‐oxidizing, aerobic and denitrifying obligate chemolithotrophs are described. In addition, a facultatively heterotrophic halophilic strain growing either methylotrophically on methylamine or chemolithotrophically on thiosulfate aerobically or with anaerobic denitrification was found.     

Biodegradation of free phytol by bacterial communities isolated from marine sediments under aerobic and denitrifying conditions

Biodegradation of (E)-phytol [3,7,11, 15-tetramethylhexadec-2(E)-en-1-ol] by two bacterial communities isolated from recent marine sediments under aerobic and denitrifying conditions was studied at 20 degrees C. This isoprenoid alcohol is metabolized efficiently by these two bacterial communities via 6,10, 14-trimethylpentadecan-2-one and (E)-phytenic acid. The first step in both aerobic and anaerobic bacterial degradation of (E)-phytol involves the transient production of (E)-phytenal, which in turn can be abiotically converted to 6,10,14-trimethylpentadecan-2-one. Most of the isoprenoid metabolites identified in vitro could be detected in a fresh sediment core collected at the same site as the sediments used for the incubations. Since (E)-phytenal is less sensitive to abiotic degradation at the temperature of the sediments (15 degrees C), the major part of (E)-phytol appeared to be biodegraded in situ via (E)-phytenic acid. (Z)- and (E)-phytenic acids are present in particularly large quantities in the upper section of the core, and their concentrations quickly decrease with depth in the core. This degradation (which takes place without significant production of phytanic acid) is attributed to the involvement of alternating beta-decarboxymethylation and beta-oxidation reaction sequences induced by denitrifiers. Despite the low nitrate concentration of marine sediments, denitrifying bacteria seem to play a significant role in the mineralization of (E)-phytol.     

Degradation of 2-nitrodiphenylamine, a component of Otto Fuel II, by Clostridium spp

Otto Fuel II, a propellant in torpedoes, is composed of 76% 1,2 propanediol dinitrate (PGDN), 22.5% di-n-butyl sebacate, and 1.5% 2-nitrodiphenylamine (NDPA), and is largely recalcitrant to aerobic microbial degradation. Anaerobic microbial degradation of Otto Fuel II was tested by inoculating anaerobic enrichment media, containing either 2% (vol:vol) complete Otto Fuel II or 2% of a 0.02% solution of Otto Fuel II in methanol, with soil and water from sites contaminated with munitions or with landfill leachate. Anaerobic bacterial growth was completely inhibited by 2% Otto Fuel II. Two mixed bacterial enrichments developed in anaerobic media containing 2% (v/v) of a 0.02% solution of Otto Fuel II in methanol. After incubation, PGDN could not be detected in either enrichment, but was also not detectable in sterile controls, suggesting abiotic degradation of low concentrations of PGDN in reduced anaerobic medium. NDPA did not degrade in either enrichment. Similarly, complete Otto Fuel was recalcitrant to degradation by highly reducing methanogenic biomass collected from an upflow anaerobic sludge blanket bioreactor (UASB). A comparison of the degradative ability of autoclaved and viable biomass showed that low concentrations of PGDN autodegraded, however unlike the autoclaved anaerobic biomass, the viable anaerobic biomass degraded the NDPA component of Otto Fuel II. Two strains of anaerobic clostridia, strains SP3 and SPF, that caused the disappearance of NDPA at its limit of solubility in culture media, were isolated from the UASB bioreactor biomass. SP3 and SPF were shown, by comparison of 16S rDNA sequences, to be most closely related to Clostridium butyricum and Clostridium cochlearium respectively. Although NDPA was lost from cultures of both strains, metabolic end products were not identified. Neither strain could degrade NDPA unless supplied with an alternative energy source. In the culture system used, NDPA stimulated the growth of SP3 but it had no appreciable effect on the growth of SPF. Both SP3 and SPF degraded low concentrations of trinitrotoluene (TNT), without the production of detectable concentrations of aromatic amines. A possible method for the remediation of small spills of Otto Fuel II is suggested.     

Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

The effect of organic matter on sedimentary phosphorus release in an Australian reservoir

Australian reservoirs, compared to much of the world, are subjected to extreme arid and semi-arid climatic conditions where dam volumes can range from near-empty to full, often with rapid filling events. P-release, after re-flooding of desiccated sediments, can be important to water quality, and can be further influenced by dried macrophyte, exposed as water recedes and incorporated into sediments. P-release from Lake Rowlands (New South Wales, Australia) sediments was studied under different aerobic and sterile conditions with five carbon source amendments to the sediment (the macrophyte Isoetes sp. in different stages of senescence and acetate). Sedimentary P-release involved a complex array of factors modified by aerobic, biotic and abiotic processes, organic matter breakdown, iron content of sediments and turbulence. Under aerobic conditions, P-release from sterile non-amended sediments and sterile macrophyte-amended sediments was greater than from non-sterile sediments. Under anaerobic conditions, P-release was maximal from non-sterile macrophyte-amended sediments, probably via pathways involving fermentative Fe³⁺-reducing bacteria where electrons are transferred from organic matter to amorphous Fe(OOH) leading to Fe²⁺ and consequent release of P. Macrophyte addition (whether fresh or dried) enhanced P-release under anaerobic compared with aerobic conditions. P-release from acetate-amended sediments appeared to involve acetate aerobes. The re-flooding of sediments, therefore, has the potential to create conditions that are conducive to aerobic sedimentary P-release and should be taken into account in management strategies adopted for reservoirs where levels are likely to fluctuate.     

Changes in Shewanella putrefaciens CN32 Membrane Stability upon Growth in the Presence of Soluble Mn(II), V(IV), and U(VI)

In natural reducing environments, such as anoxic sediments and soils, bacteria may be exposed to high concentrations of soluble transition metals. The aim of this study was to identify physiological and biochemical adaptations of Shewanella putrefaciens CN32 membranes to soluble Mn(II), V(IV), and U(VI). We assessed responses of CN32 to these metals, in aerobic and anaerobic cultures, by means of membrane fluidity and fatty acid composition assays. During aerobic growth, all metals had a stabilizing effect on fluidity, while under anoxic conditions this was only observed for bacteria treated with U(VI). Membrane gel-to-fluid phase transition temperatures were higher under anaerobic conditions and were not affected by the metal treatments. Fatty acid desaturation demonstrated linear correlation with significant increases in membrane fluidity, despite metal treatments that did not significantly alter fatty acid chemistry. Scanning transmission X-ray microscopy (STXM) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS) at Mn 2p- and V 2p-edges revealed that both Mn(II) and V(IV) were associated with CN32 membranes, with V(IV) associating as VO²⁺ under anoxic conditions only. The results of this study indicate that the bacterial growth environment greatly impacts membrane chemistry and stability, with overall implications for in vitro as well as in situ studies. Supplemental materials are available for this article. Please go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Comparative evaluation of anaerobic bacterial communities associated with roots of submerged macrophytes growing in marine or brackish water sediments

Sediment microbial communities are important for seagrass growth and carbon cycling, however relatively few studies have addressed the composition of prokaryotic communities in seagrass bed sediments. Selective media were used enumerate culturable anaerobic bacteria associated with the roots of the seagrass, Halodule wrightii, the fresh to brackish water plant, Vallisneria americana, and the respective vegetated and unvegetated sediments. H. wrightii roots and sediments had high numbers of sulfate-reducing bacteria whereas iron-reducing bacteria appeared to have a more significant role in V. americana roots and sediments. Numbers of glucose-utilizing but not acetate-utilizing iron reducers were higher on the roots of both plants relative to the vegetated sediments indicating a difference within the iron reducing bacterial community. H. wrightii roots had lower glucose-utilizing iron reducers, and higher acetogenic bacteria than did V. americana roots suggesting different aquatic plants support different anaerobic microbial communities. Sulfur-disproportionating and sulfide-oxidizing bacteria were also cultured from the roots and sediments. These results provide evidence of the potential importance of sulfur cycle bacteria, in addition to sulfate-reducing bacteria, in seagrass bed sediments.     

Mixed chemostat cultures of obligately aerobic and fermentative or methanogenic bacteria grown under oxygen-limiting conditions

Abstract Defined mixed cultures of an obligately aerobic Pseudomonas testosteroni and anaerobic Veillonella alcalescens strain were grown under oxygen and lactate limitation in chemostats with different oxygen supply rates. The aerobic and the anaerobic bacteria were shown to coexist and to complete for common substrates over a wide range of oxygen supply rates. Under similar conditions but with formate as the major substrate chemostat enrichments gave rise to undefined mixed cultures of aerobic, fermentative and methanogenic bacteria. The relevance of these observations to natural mineralization processes is discussed.     

Intestinal microbial patterns of the common marmoset and rhesus macaque

The intestinal microflora of common marmosets and rhesus monkeys were compared by enumerating bacteria from the small and large intestines. Rhesus monkeys had a consistent microflora pattern manifest by higher concentrations of total and Gram-negative aerobic and facultatively anaerobic bacteria, as well as aerobic and anaerobic Lactobacilli, in the large intestine as compared to the small intestine. In contrast, the marmoset microflora were considerably more variable. Approximately two-thirds of the marmosets (designated group A) had an overall profile that resembled the rhesus monkeys, but they had significantly higher concentrations of Gram-negative microflora in their large intestines than the rhesus monkeys. The remaining marmosets (group B) had higher concentrations of bacteria in the small intestine as compared to the large intestine, with the large intestinal concentrations being significantly lower than in the rhesus monkeys and group A marmosets. Moreover, the marmosets did not have detectable levels of aerobic Lactobacilli, and anaerobic Lactobacilli concentrations were significantly lower than in the rhesus macaques. Although it is unknown why microflora differ across species, it is likely that evolutionary adaptations in anatomy and functioning of the gastrointestinal tract influence the concentration and types of bacteria residing as the normal intestinal microflora.