Microbial, enzymatic, and immune biosensors for ecological monitoring and control of biotechnological processes

Results of the research performed at the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, on designing immunobiosensors for detection of toxic compounds and microbial cells enzyme-based biosensors for detection of hydrocarbons and alcohols, and microbial biosensors for aromatic compounds, surfactants, and biological oxygen consumption are briefed. Parameters of the mediator electrodes involving microbial cells and data on the properties of microbial biofuel cells--devices based on biosensor principle and representing alternative sources of electric energy--are given.     

Microbial,Enzymatic, and Immune Biosensors for Ecological Monitoring and Control of Biotechnological Processes

Results of the research performed at the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, on designing immunobiosensors for detection of toxic compounds and microbial cells, enzyme-based biosensors for detection of hydrocarbons and alcohols, and microbial biosensors for aromatic compounds, surfactants, and biological oxygen consumption are reviewed. Parameters of the mediator electrodes involving microbial cells and data on the properties of microbial biofuel cells—devices based on the biosensor principle and representing alternative sources of electric energy—are presented.     

Adding sodium dodecyl sulfate and Pseudomonas aeruginosa UG2 biosurfactants inhibits polycyclic aromatic hydrocarbon biodegradation in a weathered creosote-contaminated soil

  The effect of two anionic surfactants was assessed during biodegradation of 13 of the 16 USEPA priority polycyclic aromatic hydrocarbons (PAH) in a wood-preserving soil contaminated with creosote and pentacholorophenol for a period of at least 20 years. Sodium dodecyl sulfate (SDS) and biosurfactants from Pseudomonas aeruginosa UG2 were utilized at concentrations of 10, 100 and 500 μg/g soil. Because both surfactants are readily biodegradable, the microcosms received a fresh spike of surfactant every 2 weeks. Biodegradation of aged PAH residues was monitored by GC/MS for a period of 45 weeks. Results indicated that the biodegradation of the three-ring PAH was rapid and almost complete but was slowed by the addition of 100 μg/g and 500 μg/g chemical surfactant. Similarly, at the same concentrations, the two surfactants significantly decreased the biodegradation rate of the four-ring PAH. In this case, the inhibition was more pronounced with SDS. High-molecular-mass PAH (more than four rings) were not biodegraded under the test conditions. It was suggested that the preferential utilization of surfactants by PAH degraders was responsible for the inhibition observed in the biodegradation of the hydrocarbons. The high biodegradability and the inhibitory effect of these two surfactants would have a significant impact on the development of both above-ground and in situ site reclamation processes. Received: 22 February 1996 / Received revision: 31 May 1996 / Accepted: 16 June 1996     

Influence of phytogenic surfactants (quillaya saponin and soya lecithin) on bio-elimination of phenanthrene and fluoranthene by three bacteria

The influence of two phytogenic surfactants on the elimination of polycyclic aromatic hydrocarbons (PAH) was studied in shaken-batch cultures of three soil bacteria under axenic conditions. At sufficiently high concentrations, quillaya saponin and soybean lecithin solubilized phenanthrene or fluoranthene efficiently. However, complete solubilization of the PAH by lecithin only doubled the maximal rate of elimination of the two PAH compounds by Pseudomonas 0259, strain MKm (Rhizomonas ?) and Mycobacterium EMI 2. By contrast, quillaya saponin did not improve PAH bioavailability, and in strain MKm it caused significant growth lags above 2.5 g/l. Simultaneously with the elimination of the PAH the bacteria utilized the surfactants as substrates for growth. Intermediate formation of PAH metabolites was noted. The results suggest that some phytogenic surfactants might improve PAH bioavailability in rhizospheres.     

Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants.

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.     

Evaluation of substrate specificity of biosensor models based on strains degrading polycyclic aromatic compounds

Models of microbial biosensors based on 11 strains of degrading surface-active substances (SASs) and polycyclic aromatic hydrocarbons (PAHs) were studied. Substrate specificity, sensitivity, and stability of biosensor models were comparatively evaluated.     

Bacteria-degraders as the base of an amperometric biosensor for detection of anionic surfactants

Several strains belonging to genera Pseudomonas and Achromobacter and characterized by the ability to degrade anionic surfactants were tested as potential bases of microbial biosensors for surfactant detection. For each strain the substrate specificity and stability of sensor signals were studied. The total amount of the substrates tested (including carbohydrates, alcohols, aromatics, organic acids, etc.) was equal to 60; the maximal signals were observed towards the anionic surfactants. The lower limit of detection for sodium dodecyl sulfate used as a model surfactant was in the field of 1 microM for all the strains. The created microbial biosensor model can extend the practical possibilities for rapid evaluation of surfactants in water media.     

Feasibility of using prokaryote biosensors to assess acute toxicity of polycyclic aromatic hydrocarbons

The aim of this study was to assess the acute toxicity of polycyclic aromatic hydrocarbons using lux-marked bacterial biosensors. Standard solutions of phenanthrene, pyrene and benzo[a]pyrene were produced using 50 mM hydroxpropyl-β-cyclodextrin solution which contained each respective polycyclic aromatic hydrocarbon at 6.25 times the aqueous solubility limit of the compound. The polycyclic aromatic hydrocarbon solutions were incubated with each of the biosensors for 280 min and the bioluminescence monitored every 20 min. Over the incubation time period, there was no significant decrease in bioluminescence in any of the biosensors tested with the exception of Rhizobium leguminosarum biovar trifolii TA1 luxAB. In this series of incubations, there was a dramatic increase in bioluminescence in the presence of phenanthrene (2.5 times) and benzo[a]pyrene (3 times) above that of the background control (biosensor without polycyclic aromatic hydrocarbon) after 20 min. Over the next 3 h, bioluminescence decreased to that of the control. An ATP assay was carried out on the biosensors to assess if uncoupling of the oxidative phosphorylation mechanisms in the respiratory chain of the cells had occurred. However, it was found that the polycyclic aromatic hydrocarbons had no effect on the organisms indicating that there was no uncoupling. Additionally, mineralisation studies using ¹⁴C-labelled polycyclic aromatic hydrocarbons showed that the biosensors could not mineralise the compounds. This study has shown that the three polycyclic aromatic hydrocarbons tested are not acutely toxic to the prokaryotic biosensors tested, although acute toxicity has been shown in other bioassays. These results question the rationale for using prokaryote biosensors to assess the toxicity of hydrophobic chemicals, such as polycyclic aromatic hydrocarbons.     

Polycyclic aromatic hydrocarbons and heavy metals in Kostrena coastal area

The aim of this study was to determine pollution by polycyclic aromatic hydrocarbons (PAH) and heavy metals in seawater and sediment in Kostrena coastal area, as well as their toxicity using bioluminescence based tests. Total PAH concentration in seawater ranged 1.7-155.3 ng/L. The share of carcinogenetic PAH was relatively high, ranging 22-48.3%. Nickel concentrations in seawater were beyond detection limits (< 0.1 microg/L), vanadium concentrations ranged 0.66-1.96 microg/L, chrome concentrations were beyond detection limits, and copper concentrations were also beyond detection limits or extremely low (up to 0.32 microg/L). EC50 values in seawater ranged 23.80-90.90 ng/L. Correlation between total PAH concentration and toxicity of seawater showed strong connection between them (r = 0.9579). Total PAH concentration in marine sediment ranged 58.02-1116 microg/kg dry weight (d.w.). The share of carcinogenetic PAH was extremely high ranging 10-53%. Nickel concentrations in marine sediment ranged 8-24 mg/kg d.w., vanadium concentrations ranged 24-42 mg/kg d.w., chrome concentrations ranged 11-19 mg/kg d.w., and copper concentrations ranged 7-25 mg/kg d.w. EC50 values in marine sediment ranged 818-4596 microg/kg d.w. Correlation between total PAH concentration and toxicity of marine sediment showed weak connection between them (r = 0.2590). Previous studies of seawater samples from areas of the Adriatic sea under the direct influence of oil industry did not include concentrations of heavy metals, which makes our study the first to present such comprehensive results. Our results point out the need for further evaluations and following of marine environment pollution and its consequences on living organisms and marine ecosystem in whole.     

Surfactant-enhanced biodegradation of high molecular weight polycyclic aromatic hydrocarbons by stenotrophomonas maltophilia

The objectives of this study were to isolate and evaluate microorganisms with the ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) in the presence of synthetic surfactants. Stenotrophomonas maltophilia VUN 10,010, isolated from PAH-contaminated soil, utilized pyrene as a sole carbon and energy source and also degraded other high molecular weight PAHs containing up to seven benzene rings. Various synthetic surfactants were tested for their ability to improve the PAH degradation rate of strain VUN 10,010. Anionic and cationic surfactants were highly toxic to this strain, and the Tween series was used as a growth substrate. Five nonionic surfactants (Brij 35, Igepal CA-630, Triton X-100, Tergitol NP-10, and Tyloxapol) were not utilized by, and were less toxic to, strain VUN 10,010. MSR and log Km values were determined for fluoranthene, pyrene, and benzo[a]pyrene in the presence of these nonionic surfactants and their apparent solubility was increased by a minimum of 250-fold in the presence of 10 g L-1 of all surfactants. The rate of pyrene degradation by strain VUN 10,010 was enhanced by the addition of four of the nonionic surfactants (5-10 g L-1); however, 5 g L-1 Igepal CA-630 inhibited pyrene degradation and microbial growth. The specific growth rate of VUN 10,010 on pyrene was increased by 67% in the presence of 10 g L-1 Brij 35 or Tergitol NP-10. The addition of Brij 35 and Tergitol NP-10 to media containing a single high molecular weight PAH (four and five benzene rings) as the sole carbon source increased the maximum specific PAH degradation rate and decreased the lag period normally seen for PAH degradation. The addition of Tergitol NP-10 to VUN 10,010 cultures which contained a PAH mixture (three to seven benzene rings) substantially improved the overall degradation rate of each PAH and increased the specific growth rate of VUN 10,010 by 30%. Evaluation of the use of VUN 10,010 for degrading high molecular weight PAHs in leachates from surfactant-flushed, weathered, PAH-contaminated sites is warranted. Copyright 1998 John Wiley & Sons, Inc.     

Strong impact on the polycyclic aromatic hydrocarbon (PAH)-degrading community of a PAH-polluted soil but marginal effect on PAH degradation when priming with bioremediated soil dominated by mycobacteria

Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation.     

Bioremediation potential of basidiomycetes isolated from compost

The potential of a consortium of three basidiomycete mycelia isolated from compost to degrade polycyclic aromatic hydrocarbons (PAH) was first evaluated using a test based on decolorization of Poly R-478 dye. When pre-grown on straw, the consortium decolorized the dye by 83% in 7 days and generated a laccase activity of 663 IU l(-1). Its ability to degrade naphthalene was investigated in soil microcosms specially suited for this volatile PAH. The kinetic study was conducted at a maximal naphthalene concentration of 500 mg kg(-1) of soil. Naphthalene concentration, CO(2) evolution and phytotoxicity (germination index, GI%) on Lepidium sativum seeds were monitored. The naphthalene concentration decreased by about 70% in three weeks in the presence of metabolic activity, while the GI% increased indicating reduced phytotoxicity.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

Evaluation of substrate specificity of biosensor models based on strains degrading polycyclic aromatic compounds

Models of microbial biosensors based on 11 strains of degraders of surface-active substances (SASs) and polycyclic aromatic hydrocarbons (PAHs) were studied. Substrate specificity, sensitivity, and stability of biosensor models were comparatively evaluated.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 64–71.Original Russian Text Copyright © 2005 by Reshetilov, Iliasov, Fesay, Ivashchenko, Taranova, Winther-Nielsen, Emneus.     

Characterization of bacterial strains able to grow on high molecular mass residues from crude oil processing

Oil residues containing high molecular mass hydrocarbons, rich in polyaromatic compounds, are frequent end-products of crude oil processing and are poorly biodegradable. Their disposal poses an environmental problem. Through batch-enrichments from contaminated soils we have isolated and characterized seven bacterial strains that can use a residue from crude oil processing as a source of carbon and energy. The residue was a complex mixture of high molecular mass compounds, including saturated, aromatic and polycyclic aromatic hydrocarbons (PAHs). Analysis of the metabolic profiles of the strains isolated showed that they could all metabolize long-chain-length alkanes efficiently, but not PAHs. Strains degrading naphthalene, a simple PAH, did exist in the soil inocula used, but could be isolated only when enrichments were performed using pure naphthalene as the sole carbon source. All strains tested emulsified the oil residue and their ability to produce surfactants was studied.     

Strong Impact on the Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Community of a PAH-Polluted Soil but Marginal Effect on PAH Degradation when Priming with Bioremediated Soil Dominated by Mycobacteria

Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation.     

Risk assessment of polycyclic aromatic hydrocarbons (PAHs) in sediments from a mixed-use reservoir

Although reservoirs in China are of great significance, very few studies on risk assessment have been reported for reservoirs. This study investigated distribution characteristics, cancer and ecological risks, and source diagnosis of 16 priority polycyclic aromatic hydrocarbons (PAHs) in sediments from Shitou Koumen Reservoir in Jilin Province, China. A total of 12 sediment samples were collected from the reservoir in August (wet season) 2014. Total PAH concentrations in sediment samples ranged from 1294.51 ng/g to 2755.35 ng/g with a mean concentration of 1757.54 ng/g. For individual PAHs, average concentration of Nap was the highest, 800.56 ng/g, while Acy, Fla, BkF, and DahA were undetected in sediment samples. Light PAHs (2–3 rings) accounting for 74.21% was a dominant PAH compositional pattern. Pearson correlation analysis was carried out; results showed that total PAHs was strongly correlated with the highly enriched sedimentary PAHs, and pH was a major factor in controlling PAH distribution. Lifetime cancer risk was employed to assess cancer risk; results indicated that the fish-culturing area was exposed to cancer risk. The molecular diagnostic ratios of isomeric PAHs were applied to identify possible PAH sources; primary PAH sources were identified as oil-related activities, burning agricultural wastes, vehicular emissions, and industrial discharges.     

The Sonogel-Carbon materials as basis for development of enzyme biosensors for phenols and polyphenols monitoring: a detailed comparative study of three immobilization matrixes

Three amperometric biosensors based on immobilization of tyrosinase on a new Sonogel-Carbon electrode for detection of phenols and polyphenols are described. The electrode was prepared using high energy ultrasounds (HEU) directly applied to the precursors. The first biosensor was obtained by simple adsorption of the enzyme on the Sonogel-Carbon electrode. The second and the third ones, presenting sandwich configurations, were initially prepared by adsorption of the enzyme and then modification by mean of polymeric membrane such as polyethylene glycol for the second one, and the ion-exchanger Nafion in the case of the third biosensor. The optimal enzyme loading and polymer concentration, in the second layer, were found to be 285 U and 0.5%, respectively. All biosensors showed optimal activity at the following conditions: pH 7, -200 mV, and 0.02 mol l(-1) phosphate buffer. The response of the biosensors toward five simple phenols derivatives and two polyphenols were investigated. It was found that the three developed tyrosinase Sonogel-Carbon based biosensors are in satisfactory competitiveness for phenolic compounds determination with other tyrosinase based biosensors reported in the literature. The detection limit, sensitivity, and the apparent Michaelis-Menten constant K(m)(app) for the Nafion modified biosensor were, respectively, 0.064, 0.096, and 0.03 micromol, 82.5, 63.4, and 194 nA micromol(-1)l(-1), and 67.1, 54.6, and 12.1 micromol l(-1) for catechol, phenol, and 4-chloro-3-methylphenol. Hill coefficient values (around 1 for all cases), demonstrated that the immobilization method does not affect the nature of the enzyme and confirms the biocompatibility of the Sonogel-Carbon with the bioprobe. An exploratory application to real samples such as beers, river waters and tannery wastewaters showed the ability of the developed Nafion/tyrosinase/Sonogel-Carbon biosensor to retain its stable and reproducible response.     

Optimization of high-molecular-weight polycyclic aromatic hydrocarbons' degradation in a two-liquid-phase bioreactor

A microbial consortium degrading the high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) pyrene, chrysene, benzo[a]pyrene and perylene in a two-liquid-phase reactor was studied. The highest PAH-degrading activity was observed with silicone oil as the water-immiscible phase; 2,2,4,4,6,8, 8-heptamethylnonane, paraffin oil, hexadecane and corn oil were much less, or not efficient in improving PAH degradation by the consortium. Addition of surfactants (Triton X-100, Witconol SN70, Brij 35 and rhamnolipids) or Inipol EAP22 did not promote PAH biodegradation. Rhamnolipids had an inhibitory effect. Addition of salicylate, benzoate, 1-hydroxy-2-naphtoic acid or catechol did not increase the PAH-degrading activity of the consortium, but the addition of low-molecular-weight (LMW) PAHs such as naphthalene and phenanthrene did. In these conditions, the degradation rates were 27 mg l-1 d-1 for pyrene, 8.9 mg l-1 d-1 for chrysene, 1.8 mg l-1 d-1 for benzo[a]pyrene and 0.37 mg l-1 d-1 for perylene. Micro-organisms from the interface were slightly more effective in degrading PAHs than those from the aqueous phase.