Membrane composition and successful bioaugmentation. Studies of the interactions of model thylakoid and plasma cyanobacterial and bacterial membranes with fungal membrane-lytic enzyme Lecitase ultra

Cyanobacterial/bacterial consortia are frequently inoculated to soils to increase the soil fertility and to accelerate the biodegradation of organic pollutants. Moreover, such consortia can also be successfully applied in landfills especially for the biodegradation of plastic wastes. However, the bioaugmentation techniques turn out frequently inefficient due to the competition of the indigenous microorganisms attacking directly these inoculated or secreting to their surroundings cell wall and membrane-lytic enzymes. It can be hypothesized that the resistance of the microbial membrane to the enzymatic degradation is correlated with its lipid composition. To verify this hypothesis glycolipid and phospholipid Langmuir monolayers were applied as models of thylakoid and plasma cyanobacterial and bacterial membranes. Hybrid fungal enzyme Lecitase ultra joining the activity of lipase and phospholipase A1 was applied as the model of fungal membrane-lytic enzyme. It turned out that anionic thylakoid lipids sulfoquinovosyldiacylglycerol and phosphatidylglycerols were the main targets of Lecitase ultra in the model multicomponent thylakoid membranes. The resistance of the model plasma bacterial membranes to enzymatic degradation depended significantly to their composition. The resistance increased generally when the unsaturated lipids were exchanged to their saturated counterparts. However, most resistant turned out the membranes composed of unsaturated phosphatidylamine and saturated anionic phospholipids.     

Persistent organic pollutants in model fungal membranes. Effects on the activity of phospholipases

Soils are the final sink for multiple organic pollutants emitted to the environment. Some of these chemicals which are toxic, recalcitrant and can bioaccumulate in living organism and biomagnify in trophic chains are classified persistent organic pollutants (POP). Vast areas of arable land have been polluted by POPs and the only economically possible means of decontamination is bioremediation, that is the utilization of POP-degrading microbes. Especially useful can be non-ligninolytic fungi, as their fast-growing mycelia can reach POP molecules strongly bond to soil minerals or humus fraction inaccessible to bacteria. The mobilized POP molecules are incorporated into the fungal plasma membrane where their degradation begins. The presence of POP molecules in the membranes can change their physical properties and trigger toxic effects to the cell. To avoid these phenomena fungi can quickly remodel the phospholipid composition of their membrane with employing different phospholipases and acyltransferases. However, if the presence of POP downregulates the phospholipases, toxic effects and the final death of microbial cells are highly probable. In our studies we applied multicomponent Langmuir monolayers with their composition mimicking fungal plasma membranes and studied their interactions with two different microbial phospholipases: phospholipase C (α-toxin) and phospholipase A1 (Lecitase ultra). The model membranes were doped with selected POPs that are frequently found in contaminated soils. It turned out that most of the employed POPs do not downregulate considerably the activity of phospholipases, which is a good prognostics for the application of non-ligninolytic fungi in bioremediation.     

Effects of water soluble perfluorinated pollutants on phospholipids in model soil decomposer membranes

Water soluble perfluorinated compounds (PFCs) as perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA) and their shorter chain homologues are persistent organic pollutants widely distributed in the environment. PFCs accumulate in soils and sediments and because of their toxicity endanger the decomposer organisms. PFCs are toxic to a wide spectrum of soil bacteria and their biocide activity was related with their membrane activity; however, the exact mechanism of PFCs – bacterial membrane interactions is unknown. Therefore, to shed light on these questions we applied phospholipid Langmuir monolayers as simplified models of bacterial membranes and studied their interactions with selected environmentally relevant PFCs. The mechanical properties of the monolayers were characterized by surface pressure-mean molecular area isotherms and the analysis of compression modulus. The effects of PFC on the texture of the model membranes were studied with Brewster angle microscopy, whereas their influence on molecular packing in the 2D crystal lattice was searched by the Grazing Incidence X-ray diffraction technique. The effects of PFCs on the phospholipid polar heargroup conformation were studied by PM-IRRAS spectroscopy, whereas the effectivenes of the incorporation of PFCs into the model membrane was monitored in penetration tests. It turned out that the membranes rich in phosphatidylethanolamine typical to Gram negative bacteria are much PFCs susceptible than the cardiolipin rich membranes imitating Gram positive species. Moreover, the studies indicated that the switch from eight‑carbon atom perfluorinated chains to shorter chain homologues is not necessarily environmentally benign as perfluorobutane sulfonate caused also significant structural changes in the model membranes.     

Guanidino groups greatly enhance the action of antimicrobial peptidomimetics against bacterial cytoplasmic membranes

Antimicrobial peptides or their synthetic mimics are a promising class of potential new antibiotics. Herein we assess the effect of the type of cationic side chain (i.e., guanidino vs. amino groups) on the membrane perturbing mechanism of antimicrobial α-peptide–β-peptoid chimeras. Langmuir monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) were used to model cytoplasmic membranes of both Gram-positive and Gram-negative bacteria, while lipopolysaccharide Kdo2-lipid A monolayers were mimicking the outer membrane of Gram-negative species. We report the results of the measurements using an array of techniques, including high-resolution synchrotron surface X-ray scattering, epifluorescence microscopy, and in vitro antimicrobial activity to study the molecular mechanisms of peptidomimetic interaction with bacterial membranes. We found guanidino group-containing chimeras to exhibit greater disruptive activity on DPPG monolayers than the amino group-containing analogues. However, this effect was not observed for lipopolysaccharide monolayers where the difference was negligible. Furthermore, the addition of the nitrobenzoxadiazole fluorophore did not reduce the insertion activity of these antimicrobials into both model membrane systems examined, which may be useful for future cellular localization studies.     

Evidence of aerobic utilization of di-ortho-substituted trichlorobiphenyls as growth substrates by Pseudomonas sp. SA-6 and Ralstonia sp. SA-4

Robust and effective bioremediation strategies have not yet been developed for polychlorinated biphenyl (PCB)-contaminated soils. This is in part a result of the fact that ortho - or ortho - and para -substituted congeners, frequent dead-end products of reductive dechlorination of PCB mixtures, have greatly reduced aerobic biodegradability. In this study, we report substantial evidence of utilization of diortho -substituted trichlorobiphenyls (triCBs) as growth substrates by Ralstonia sp. SA-4 and Pseudomonas sp. SA-6 in which ortho -substitution resulted in no obvious patterns of recalcitrance. These stains exhibited unusual preferences for growth on congeners chlorinated on both rings. Substrate uptake studies with benzoate-grown cells revealed that the isolates attacked the 2-chlorophenyl rings of 2,2',4- and 2,2',5-triCB. Between 71% and 93% of the initial 0.23–0.34 mM dose of congeners were transformed in less than 261 h concomitant with non-stoichiometric production of respective dichlorobenzoates and chloride ion. In enzyme assays, activity of 2,3-dihydroxybiphenyl-1,2-dioxygenase was constitutive. Additionally, these strains harboured no detectable plasmids which, coupled with exponential growth on the two triCB congeners, suggested chromosomal location of PCB degradative genes. In addition to the fact that there is a paucity of information on degradation of PCBs by tropical isolates, growth on triCBs as a sole carbon and energy source has never been demonstrated for any natural or engineered microorganisms. Such isolates may help prevent accumulation of ortho -substituted congeners in natural systems and offer the hope for development of effective bioaugmentation or sequential anaerobic–aerobic bioremediation strategies.     

Molecular diagnostics and chemical analysis for assessing biodegradation of polychlorinated biphenyls in contaminated soils

Summary The microbial populations in PCB-contaminated electric power substation capacitor bank soil (TVA soil) and from another PCB-contaminated site (New England soil) were compared to determine their potential to degrade PCB. Known biphenyl operon genes were used as gene probes in colony hybridizations and in dot blots of DNA extracted from the soil to monitor the presence of PCB-degrading organisms in the soils. The microbial populations in the two soils differed in that the population in New England soil was enriched by the addition of 1000 p.p.m. 2-chlorobiphenyl (2-CB) whereas the population in the TVA capacitor bank soil was not affected. PCB degradative activity in the New England soil was indicated by a 50% PCB disappearance (gas chromatography), accumulation of chlorobenzoates (HPLC), and¹⁴CO₂ evolution from¹⁴C-2CB. The PCB-degrading bacteria in the New England soil could be identified by their positive hybridization to thebph gene probes, their ability to produce the yellowmeta-cleavage product from 2,3-dihydroxybiphenyl (2,3-DHB), and the degradation of specific PCB congeners by individual isolates in resting cell assays. Although the TVA capacitor bank soil lacked effective PCB-degrading populations, addition of a PCB-degrading organism and 10 000 p.p.m. biphenyl resulted in a >50% reduction of PCB levels. Molecular characterization of soil microbial populations in laboratory scale treatments is expected to be valuable in the design of process monitoring and performance verification approaches for full scale bioremediation.     

Genetically modified Pseudomonas biosensing biodegraders to detect PCB and chlorobenzoate bioavailability and biodegradation in contaminated soils

Whole cell microbial biosensors offer excellent possibilities for assaying the complex nature of the bioavailable and bioaccessible fraction of pollutants in contaminated soils, which currently cannot be easily addressed. This paper describes the application and evaluation of three microbial biosensor strains designed to detect the bioavailability and biodegradation of PCBs (and end-products) in contaminated soils and sediments. Polychlorinated biphenyls (PCBs) are considered to be one of the most wide spread, hazardous and persistent pollutants. Herein we describe that there was a positive correlation between the PCB levels within the samples and the percentage of biosensor cells that were expressing their reporter gene; gfp. Immobilisation of the biosensors in calcium alginate beads allowed easy and accurate detection of the biosensor strains in contaminated soil and sludge samples. The biosensors also showed that PCB degradation activity was occurring at a much greater level in Pea inoculated planted soil compared to inoculated unplanted soil indicating rhizoremediation (the removal of pollutants by plant root associated microbes) shows considerable promise as a solution for removing organic xenobiotics from the environment.     

Microbial Degradation of Organic Pollutants in Soil in a Cold Climate

Cold-adapted microorganisms are potentially interesting for use in environmental biotechnology applications since a large part of the biosphere has low temperatures during at least parts of the year. Many studies have shown that both oil-contaminated and uncontaminated soils in the Arctic, the Antarctic and the Alps contain microbes that can degrade different hydrocarbons deriving from oils. A few studies have also been conducted on degradation of herbicides in soils at low temperatures. Furthermore, phenols and some polychlorinated biphenyl (PCB) congeners have proved to be degradable at low temperatures, using microorganisms isolated from sediments or soils. Additions of nitrogen and phosphorous to polluted soils have been shown to enhance the degradation of hydrocarbons in many cases. Bioaugmentation with hydrocarbon degrading cold-adapted microorganisms has given varying results. The inoculated microorganisms have probably been out-competed by the indigenous microorganisms in some cases. Different ways to increase the efficiency of microbial degradation of organic pollutants in soil in a cold climate is discussed.     

Degradation of anaerobic reductive dechlorinationproducts of Aroclor 1242 by four aerobic bacteria

We studied the aerobic degradation of eight PCB congeners which comprise from 70 to 85% of the anaerobic dechlorination products from Aroclor 1242, including2-, 4-, 2,4-, 2,6-, 2,2'-, 2,4'-, 2,2',4-, and2,4,4'-chlorobiphenyl (CB), and the biodegradation of their mixtures designed to simulate anaerobic dechlorination profiles M and C. StrainsComamonas testosteroni VP44 and Rhodococcus erythreus NY05 preferentially oxidizeda para-substituted ring, while Rhodococcus sp. RHA1, similar to well known strain Burkholderia sp. LB400, preferably attackedan ortho-chlorinated ring. Strains with ortho-directed attack extensively degraded2,4'- and 2,4,4'-CB into 4-chlorobenzoate, while bacteria with para-directed attack transformed these congeners mostly into potentially problematicmeta-cleavage products. The strains that preferentiallyoxidized an ortho-substituted ring readily degradedseven of the eight congeners supplied individually; only 2,6-CB was poorly degraded. Degradationof 2,2'- and 2,4,4'-CB was reduced when present in mixtures M and C. Higher efficiencies of degradation of the individual congeners and defined PCB mixtures M and C and greater production of chlorobenzoates were observed with bacteria that preferentially attackan ortho-substituted ring. PCB congeners 2,4'-, 2,2',4-, and 2,4,4'-CB canbe used to easily identify bacteria with ortho-directed attack whichare advantageous for use in the aerobic stage of the two-phase (anaerobic/aerobic)PCB bioremediation scheme.     

Microbial Reductive Dechlorination of Aroclor 1260 in Baltimore Harbor Sediment Microcosms Is Catalyzed by Three Phylotypes within the Phylum Chloroflexi

The specific dechlorination pathways for Aroclor 1260 were determined in Baltimore Harbor sediment microcosms developed with the 11 most predominant congeners from this commercial mixture and their resulting dechlorination intermediates. Most of the polychlorinated biphenyl (PCB) congeners were dechlorinated in the meta position, and the major products were tetrachlorobiphenyls with unflanked chlorines. Using PCR primers specific for the 16S rRNA genes of known PCB-dehalogenating bacteria, we detected three phylotypes within the microbial community that had the capability to dechlorinate PCB congeners present in Aroclor 1260 and identified their selective activities. Phylotype DEH10, which has a high level of sequence identity to Dehalococcoides spp., removed the double-flanked chlorine in 234-substituted congeners and exhibited a preference for para-flanked meta-chlorines when no double-flanked chlorines were available. Phylotype SF1 had similarity to the o-17/DF-1 group of PCB-dechlorinating bacteria. Phylotype SF1 dechlorinated all of the 2345-substituted congeners, mostly in the double-flanked meta position and 2356-, 236-, and 235-substituted congeners in the ortho-flanked meta position, with a few exceptions. A phylotype with 100% sequence identity to PCB-dechlorinating bacterium o-17 was responsible for an ortho and a double-flanked meta dechlorination reaction. Most of the dechlorination pathways supported the growth of all three phylotypes based on competitive PCR enumeration assays, which indicates that PCB-impacted environments have the potential to sustain populations of these PCB-dechlorinating microorganisms. The results demonstrate that the variation in dechlorination patterns of congener mixtures typically observed at different PCB impacted sites can potentially be mediated by the synergistic activities of relatively few dechlorinating species.     

Repeated application of carvone-induced bacteria to enhance biodegradation of polychlorinated biphenyls in soil

Carvone, the principal component of spearmint oil, induces biodegradation of polychlorinated biphenyls (PCB) by Arthrobacter sp. strain B1B. This study investigated the effectiveness of the repeated application of carvone-induced bacteria for bioremediation of Aroclor-1242-contaminated soil. Control treatments compared a single inoculation of carvone-induced cells, repeated applications of noninduced cells, and repeated applications of cell-free carvone/fructose medium. The results showed that repeated application of carvone-induced bacteria was the most effective treatment for mineralizing PCB, resulting in 27 ± 6% degradation of Aroclor 1242 after 9 weeks; whereas a single application of cells resulted in no significant degradation. Addition of cell-free, carvone/fructose medium resulted in 10% degradation of PCB, which suggests that this treatment stimulated biodegradation of PCB by the indigenous microflora. The di- and trichlorobiphenyls were the most readily degraded congeners. More highly chlorinated congeners, which had been previously shown to be degraded in liquid culture, were not substantially degraded in soil, indicating that low bioavailability may have limited their degradation. With the development of new technology, which permits automated in situ fermentation and delivery of degrader microorganisms, the repeated application of carvone-induced bacteria may facilitate bioremediation of PCB-contaminated soils. Received: 7 January 1998 / Received revision: 18 June 1998 / Accepted: 27 June 1998     

Root exudates and plant secondary metabolites of different plants enhance polychlorinated biphenyl degradation by rhizobacteria

Polychlorinated biphenyls (PCBs) are toxic and persistent compounds that are difficult to break down and biodegrade. Plant secondary metabolites (PSMs) on root exudates can act as inducers of the biphenyl catabolic pathway, enhancing PCB biodegradation. In this study, the authors evaluated the effect of root exudates and PSMs obtained from Avena sativa, Brachiaria decumbens, Medicago sativa, and Brassica juncea on the biodegradation of PCB 44, PCB 66, PCB 118, PCB 138, PCB 153, PCB 170, and PCB 180 by a microbial consortium isolated from the rhizosphere of plants grown on soil contaminated with Aroclor 1260. Microorganisms were identified as Pseudomonas sp. and Stenotrophomonas sp. based on their 16S rRNA sequence. The plant root exudates increased the degradation percentage of PCB 44, PCB 66, and PCB 118, which were used as carbon source by the microorganisms. Flavanone, flavone, isoflavone, 7-hydroxyflavanone, 7-hydroxyflavone, and 6-hydroxyflavone were the PSMs identified in the root exudates, which increased the degradation percentage of all seven PCB congeners; they were also used as growth substrates by microbial consortium. These results showed the importance of the interaction between plants and microorganisms for achieving the removal of persistent pollutants such as PCBs from soil.     

Plant secondary metabolite-induced shifts in bacterial community structure and degradative ability in contaminated soil

The aim of the study was to investigate how selected natural compounds (naringin, caffeic acid, and limonene) induce shifts in both bacterial community structure and degradative activity in long-term polychlorinated biphenyl (PCB)-contaminated soil and how these changes correlate with changes in chlorobiphenyl degradation capacity. In order to address this issue, we have integrated analytical methods of determining PCB degradation with pyrosequencing of 16S rRNA gene tag-encoded amplicons and DNA-stable isotope probing (SIP). Our model system was set in laboratory microcosms with PCB-contaminated soil, which was enriched for 8 weeks with the suspensions of flavonoid naringin, terpene limonene, and phenolic caffeic acid. Our results show that application of selected plant secondary metabolites resulted in bacterial community structure far different from the control one (no natural compound amendment). The community in soil treated with caffeic acid is almost solely represented by Proteobacteria, Acidobacteria, and Verrucomicrobia (together over 99 %). Treatment with naringin resulted in an enrichment of Firmicutes to the exclusion of Acidobacteria and Verrucomicrobia. SIP was applied in order to identify populations actively participating in 4-chlorobiphenyl catabolism. We observed that naringin and limonene in soil foster mainly populations of Hydrogenophaga spp., caffeic acid Burkholderia spp. and Pseudoxanthomonas spp. None of these populations were detected among 4-chlorobiphenyl utilizers in non-amended soil. Similarly, the degradation of individual PCB congeners was influenced by the addition of different plant compounds. Residual content of PCBs was lowest after treating the soil with naringin. Addition of caffeic acid resulted in comparable decrease of total PCBs with non-amended soil; however, higher substituted congeners were more degraded after caffeic acid treatment compared to all other treatments. Finally, it appears that plant secondary metabolites have a strong effect on the bacterial community structure, activity, and associated degradative ability.     

Peptoid drug discovery and optimization via surface X-ray scattering

Synthetic polymers mimicking antimicrobial peptides have drawn considerable interest as potential therapeutics. N-substituted glycines, or peptoids, are recognized by their in vivo stability and ease of synthesis. Peptoids are thought to act primarily on the negatively charged lipids that are abundant in bacterial cell membranes. A mechanistic understanding of lipid–peptoid interaction at the molecular level will provide insights for rational design and optimization of peptoids. Here, we highlight recent studies that utilize synchrotron liquid surface X-ray scattering to characterize the underlying peptoid interactions with bacterial and eukaryotic membranes. Cellular membranes are highly complex, and difficult to characterize at the molecular level. Model systems including Langmuir monolayers, are used in these studies to reduce system complexity. The general workflow of these systems and the corresponding data analysis techniques are presented alongside recent findings. These studies investigate the role of peptoid physicochemical characteristics on membrane activity. Specifically, the roles of cationic charge, conformational constraint via macrocyclization, and hydrophobicity are shown to correlate their membrane interactions to biological activities in vitro. These structure–activity relationships have led to new insights into the mechanism of action by peptoid antimicrobials, and suggest optimization strategies for future therapeutics based on peptoids.     

Phospholipid Monolayers Probed by Vibrational Sum Frequency Spectroscopy: Instability of Unsaturated Phospholipids

The surface specific technique vibrational sum frequency spectroscopy has been applied to in situ studies of the degradation of Langmuir monolayers of 1,2-diacyl-phosphocholines with various degrees of unsaturation in the aliphatic chains. To monitor the degradation of the phospholipids, the time-dependent change of the monolayer area at constant surface pressure and the sum frequency intensity of the vinyl CH stretch at the carbon-carbon double bonds were measured. The data show a rapid degradation of monolayers of phospholipids carrying unsaturated aliphatic chains compared to the stable lipids carrying fully saturated chains when exposed to the ambient laboratory air. In addition, the degradation of the phospholipids can be inhibited by purging the ambient air with nitrogen. This instability may be attributed to spontaneous degradation by oxidation mediated by various reactive species in the air. To further elucidate the process of lipid oxidation in biological membranes artificial Langmuir monolayers probed by a surface specific spectroscopic technique as in this study can serve as a model system for studying the degradation/oxidation of cell membrane constituents.     

Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic soil microorganisms.

We examined the degradation of biphenyl and the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221 by indigenous Arctic soil microorganisms to assess both the response of the soil microflora to PCB pollution and the potential of the microflora for bioremediation. In soil slurries, Arctic soil microflora and temperate-soil microflora had similar potentials to mineralize [14C]biphenyl. Mineralization began sooner and was more extensive in slurries of PCB-contaminated Arctic soils than in slurries of uncontaminated Arctic soils. The maximum mineralization rates at 30 and 7 degrees C were typically 1.2 to 1.4 and 0.52 to 1.0 mg of biphenyl g of dry soil-1 day-1, respectively. Slurries of PCB-contaminated Arctic soils degraded Aroclor 1221 more extensively at 30 degrees C (71 to 76% removal) than at 7 degrees C (14 to 40% removal). We isolated from Arctic soils organisms that were capable of psychrotolerant (growing at 7 to 30 degrees C) or psychrophilic (growing at 7 to 15 degrees C) growth on biphenyl. Two psychrotolerant isolates extensively degraded Aroclor 1221 at 7 degrees C (54 to 60% removal). The soil microflora and psychrotolerant isolates degraded all mono-, most di-, and some trichlorobiphenyl congeners. The results suggest that PCB pollution selected for biphenyl-mineralizing microorganisms in Arctic soils. While low temperatures severely limited Aroclor 1221 removal in slurries of Arctic soils, results with pure cultures suggest that more effective PCB biodegradation is possible under appropriate conditions.     

Three Stages of a Biofilm Community Developing at the Liquid-Liquid Interface between Polychlorinated Biphenyls and Water

Soil contaminated with polychlorinated biphenyls (PCB) was used as an inoculum to grow a complex biofilm community on PCB oil (Aroclor 1242) on a substratum (Permanox). The biofilm was monitored for 31 days by confocal laser scanning microscopy, community fingerprinting using single-strand conformational polymorphism (SSCP), amplicons of the 16S rRNA genes, and chemical analyses of the PCB congeners. SSCP analysis of the young biofilm revealed a rather diverse microbial community with species of the genera Herbaspirillum and Bradyrhizobium as dominant members. The biofilm developing on the PCB droplets displayed pronounced stages of PCB degradation and biofilm development not described before from pure-culture experiments. The first step was the colonization of the substratum while the PCB oil was hardly populated. When a certain density of bacteria was reached on the Permanox, the PCB was colonized, but soon the degradation of the congeners was markedly reduced and many cells were damaged, as seen by LIVE/DEAD staining. Finally, the biofilm formed aggregates and invaded the PCB oil, showing lower numbers of damaged cells than before and a dramatic increase in PCB degradation. This sequence of biofilm formation is understood as a maturation process prior to PCB oil colonization. This is followed by a thin biofilm on the PCB droplet, an aggregation process forming pockets in the PCB, and finally an invasion of the biofilm into the PCB oil. Only the mature biofilm showed degradation of pentachlorinated PCB congeners, which may be reductively dechlorinated and the resulting trichlorobiphenyls then aerobically metabolized.     

Microbial degradation of polychlorinated biphenyls in contaminated soil

Summary The potential of microbial degradation of PCB in contaminated actual site soil was investigated by incubation in percolation columns for 10 months. The addition of traces of mineral salts and nutrients resulted in a significant increase of the degradation of PCB congeners up to 5 Cl-atoms caused by the naturally occurring bacteria in the soil matrix. If only tap water was recycled the degree of PCB degradation was negligible.