A simple method for quantifying activity and survival of microorganisms involved in bioremediation processes

We have developed a substrate-induced growth response (SIGR) method for quantifying activity and population dynamics of microorganisms involved in bioremediation processes in soil and bioreactors. The biomass of organisms that can mineralize a given chemical can be estimated based on the concentration of that chemical needed to induce the growth of the standing population. Estimates of population size are obtained by using nonlinear regression techniques to fit a simple model of microbial population dynamics to biodegradation curves. Using this approach we obtain estimates of values for parameters such as initial population size and growth rate of organisms carrying out biodegradative processes. Our approach was validated by comparing model parameter estimates with independent estimates of the same parameters from the same bioremediation systems. Examples studied include pentachlorophenol degraders introduced into soil and 2,4-dinitrophenol degrading organisms in a bioreactor.     

Managed bioremediation of soil contaminated with crude oil soil chemistry and microbial ecology three years later

Analysis of samples taken from three experimental soil lysimeters demonstrated marked long-term effects of managed bioremediation on soil chemistry and on bacterial and fungal communities 3 yr after the application of crude oil or crude oil and fertilizer. The lysimeters were originally used to evaluate the short-term effectiveness of managed (application of fertilizer and water, one lysimeter) vs unmanaged bioremediation (one lysimeter) of Michigan Silurian crude oil compared to one uncontaminated control lysimeter. Three years following the original experiment, five 2-ft-long soil cores were extracted from each lysimeter, each divided into three sections, and the like sections mixed together to form composited soil samples. All subsequent chemical and microbiological analyses were performed on these nine composited samples. Substantial variation was found among the lysimeters for certain soil chemical characteristics (% moisture, pH, total Kjeldahl nitrogen [TKN], ammonia nitrogen [NH₄-N], phosphate phosphorous [PO₄-P], and sulfate [SO₄ ⁻²]). The managed lysimeter had 10% the level of total petroleum hydrocarbons (TPH-IR) found in the unmanaged lysimeter. Assessment of the microbial community was performed for heterotropic bacteria, fungi, and aromatic hydrocarbon-degrading bacteria (toluene, naphthalene, and phenanthrene) by dilution onto solid media. There was little difference in the number of heterotrophic bacteria, in contrast to counts of fungi, which were markedly higher in the contaminated lysimeters. Hydrocarbon-degrading bacteria were elevated in both oil-contaminated lysimeters. In terms of particular hydrocarbons as substrates, phenanthrene degraders were greater in number than naphthalene degraders, which outnumbered toluene degraders. Levels of sulfate-reducing bacteria seem to have been stimulated by hydrocarbon degradation.     

Cadmium recovery by a sulfate-reducing magnetotactic bacterium,Desulfovibrio magneticus RS-1, using magnetic separation

Cadmium recovery by a sulfate-reducing magnetotactic bacterium, Desulfovibrio magneticus strain RS-1, was investigated. D. magneticus precipitated >95% of cadmium at an initial concentration of 1.3 ppm in the growth medium. Electron microscopic analysis revealed that D. magneticus formed electron-dense particles on its surface when cultivated in the presence of cadmium ions (Cd²⁺). Sulfide was also found in the precipitate, and the composition ratio of sulfide/cadmium was 0.7. Sixty percent of viable RS-1 cells was recovered by a simple magnetic separation revealing the removal of 58% cadmium from the culture medium.     

Effects of slight variations in nutrient loadings on pore plugging in soil columns

The high nutrient concentrations that would exist near the nutrient injection well during the application of cometabolicin situ bioremediation may lead to the development of significant quantities of biomass at this point in the subsurface. This biomass can decrease the porosity of the soil to such an extent that nutrient injection is no longer possible. In this work, experiments were conducted using a porous media biofilm reactor, operated under constant substrate loading conditions, such that the pressure drop across the reactor was allowed to increase to maintain a constant volumetric flow rate through the reactor. Results suggest that biomass production, and hence biofilm thickness, near the injection feed port is highly sensitive to substrate loading. In addition, these variations in biofilm thickness produce dramatic differences in the pressure drop that is attained across the reactor. Use of the Kozeny-Carman equation can be used to predict that once a critical depth has been exceeded, the pressure drop across the bed will increase exponentially within biofilm depth. This result means that pressure is not a reliable indicator of the onset of pore plugging.     

Chemometric study of soil analysis data

The present paper deals with chemometric interpretation of soil analysis data collected from 31 sampling sites in the region of Kavala and Drama, Northern Greece. The determination of 16 different chemical and physicochemical characteristics is principally needed for prognosis of the land treatment and fertilizing. The study carried out indicates that the application of multivariate statistical approaches could reveal new and specific information about sampling sites. It has been found that they could be divided into four general patterns: pattern 1 contains dominantly inorganic and alkaline soil samples from semi-mountainous regions in close proximity to the seacoast; pattern 2 indicates the same soil sample type and regional location as pattern 1 but is far from the coastal line; pattern 3 includes samples from sites from the plains with organic and alkaline soils with close proximity to the coast; pattern 4 resembles pattern 3 as soil type but involves samples from sites far from the shore. Further, six latent factors were identified, conditionally named “structural”, “acidic”, “nutritional”, “salt”, “microcomponents” and “organic”. Finally, an apportioning procedure was carried out to find the source contributions in the measured analytical values. In this way the routine estimation of the soil quality could be improved.     

Electroanalytical and chromatographic determination of pentachlorophenol and related molecules in a contaminated soil: a real case example

Electroanalytical and chromatographic methodologies have been applied for the determination of pentachlorophenol (PCP) and some of its derivatives in real soil samples contaminated by industrial discharge. The analytes were extracted with hexane from soil samples collected at different points of the site and mixed to produce a representative sample. Square wave voltammetry (SWV) experiments were carried out on either a boron-doped diamond (BDD) electrode or a gold ultramicroelectrode (Au-UME) in an analyte composed by the Britton-Robinson (B-R) buffer at pH 5.5 with the direct addition of proper amounts of the extract. The voltammetric responses revealed an irreversible anodic peak at approximately 0.80 V vs. Ag/AgCl with a peak current showing a linear dependence on PCP concentration. This linear relationship yielded a detection limit (DL) of 2×10⁻⁸ mol l⁻¹ (or 5.5 μg l⁻¹) for the BDD electrode and 6.9×10⁻⁸ mol l⁻¹ (18.4 μg l⁻¹) for the Au-UME, while the independently measured HPLC detection limit was 1.1×10⁻⁸ mol l⁻¹ (3.0 μg l⁻¹). The application of electroanalytical and chromatographic methodologies in the analysis of soil extracts revealed, besides the PCP responses, signals for some related molecules such as o-tetrachlorobenzoquinone (o-chloranil), hexachlorobenzene and tetrachlorophenol. Recovering experiments for PCP showed a concentration of 27.5 mg kg⁻¹ for the electroanalytical determinations and 26.8 mg kg⁻¹ for the HPLC analysis, values exceedingly high if considering that the maximum residue limit established for natural waters by the Brazilian Environmental Agency is 10 μg l⁻¹.     

Bioreactor design studies for a hydrogen-producing bacterium

Carbon monoxide (CO) can be metabolized by a number of microorganisms along with water to produce hydrogen (H₂) and carbon dioxide. National Renewable Energy Laboratory researchers have isolated a number of bacteria that perform this so-called water-gas shift reaction at ambient temperatures. We performed experiments to measure the rate of CO conversion and H₂ production in a trickle-bed reactor (TBR). The liquid recirculation rate and the reactor support material both affected the mass transfer coefficient, which controls the overall performance of the reactor. A simple reactor model taken from the literature was used to quantitatively compare the performance of the TBR geometry at two different size scales. Good agreement between the two reactor scales was obtained.     

Development of a systems analysis approach for resolving the structure of biodegrading soil systems

An experimental and mathematical method is developed for the microbial systems analysis of polyaromatic hydrocarbon (PAH)-degrading mixed cultures in PAH-contaminated “town gas” soil systems. Frequency response is the primary experimental and data analysis tool used to probe the structure of these complicated systems. The objective is to provide a fundamental protocol for evaluating the performance of specific mixed microbial cultures on specific soil systems by elucidating the salient system variables and their interactions. Two well-described reactor systems, a constant volume stirred tank reactor (CSTR) and a plug flow differential volume reactor, are used in order to remove performance effects that are related to reactor type as opposed to system structure. These two reactor systems are well-defined systems that can be described mathematically and represent the two extremes of one potentially important system variable, macroscopic mass transfer. The experimental and mathematical structure of the protocol is described, experimental data is presented, and data analysis is demonstrated for the stripping, sorption, and biodegradation of napththalene.     

<Superscript>13</Superscript>C NMR and mass spectrometry of soil organic matter

Liquid state, high resolution ¹³C NMR spectroscopy and mass spectrometry were used to study the composition and structure of soil organic matter (SOM) using soil extracts from two long-term experiments at the Rothamsted Experimental Station. Both one- and two-dimensional NMR techniques were applied. ¹³C NMR sub-spectra of the CH _n (n=0...3) groups, obtained by the Distortionless Enhancement by Polarisation Transfer (DEPT) technique, were used for the elucidation of the qualitative and quantitative composition of humic and fulvic acids in the soils. The chemical structure of SOM was further analysed at the molecular level through Fast Atom Bombardment Mass Spectrometry (FABMS) and Gas Chromatography-Mass Spectrometry (GC/MS). Humic and fulvic extract results were not only compared to each other, but also to the solid state ¹³C NMR results for the complete soil sample.     

Use of a Graphical Method to Predict the Retention Times of Selected Flavonoids in HPLC from Thin-Layer Chromatographic Data

Similarities and differences between the retention characteristics of octadecylsilica wettable with water used in TLC and RP-18 used in HPLC have been elucidated by use of the linear relationships between log k and R_M. The stationary phases compared were investigated with the same mobile phases—binary mixtures of methanol and water, acetonitrile and water, and tetrahydrofuran and water. For these adsorbents of the same type but differing in specific surface area the correlation line was shifted by log (_systemI/_systemII). High values of the correlation coefficients obtained over the whole range of mobile phase organic modifier concentration examined indicated that the TLC systems could be used to predict HPLC conditions for flavonoid separation.     

Bench Scale Studies of the Soil Aeration Process for Bioremediation of Petroleum Hydrocarbons

An alternative to traditional hydrocarbon bioremediation is to pump air through unsaturated soils to create aerobic conditions and induce biodegradation. This study examines the effects of moisture and nutrient augmentation on biodegradation of petroleum hydrocarbons in aerated soils. Findings indicate that forced aeration, coupled with additions of nutrients and moisture, stimulate hydrocarbon-degrading microorganisms and present a feasible approach to bioremediation management.     

Rapid selection system of strains with higher avicel degrading ability in a cellulolytic fungus,Trichoderma

We have attempted to develop an active selection system for strains that have a higher potential for Avicel degradation using haploidized conidia from colchicine-treated Trichoderma reesei Rut C-30 as a model strain. Avicel, absorbent cotton, and wood powder were used as substrates for selection. It appeared that the strains that degrade Avicel actively could be effectively selected when the solid medium containing the selection substrate and the liquid medium containing Avicel were used.     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

硫丹在土壤中的降解特性与污染修复模拟研究

硫丹是具有很高毒性的有机氯农药,历史上我国硫丹产量较大,因此急需硫丹污染场地的修复技术．在实验室条件研究了硫丹在土壤中的降解行为,使用模拟生物堆降解研究了硫丹污染土壤的生物堆修复和化学修复条件．结果表明：在土壤介质中,α-硫丹可部分转化为β-硫丹：硫丹为微生物好氧降解;添加秸秆和绿肥及适当的通气可促进硫丹的降解;添加EM菌或葡萄糖和硝酸铵或复合肥均会抑制硫丹降解．碱性条件有利于硫丹快速降解,因而向污染土壤中添加石灰是一种高效的化学修复方法．     

Continuous production of succinic acid by a fumarate-reducing bacterium immobilized in a hollow-fiber bioreactor

Enterococcus faecalis RKY1, a fumarate-reducing bacterium, was immobilized in an asymmetric hollow-fiber bioreactor (HFBR) for the continuous production of succinic acid. The cells were inoculated into the shell side of the HFBR, which was operated in transverse mode. Since the pH values in the HFBR declined during continuous operation to about 5.7, it was necessary to change the feed pH from 7.0 to 8.0 after 24 h of operation in order to enhance production of succinic acid. During continuous operation with a medium containing fumarate and glycerol, the productivity of succinate was 3.0–10.9 g/(L·h) with an initial concentration of 30 g/L of fumarate, 4.9–14.9 g/(L·h) with 50 g/L of fumarate, and 7.2–17.1 g/(L·h) with 80 g/L of fumarate for dilution rates between 0.1 and 0.4 h⁻¹. The maximum productivity of succinate obtained by the HFBR (17.1 g of succinate /[L·h]) was 1.7 times higher than that of the batch bioconversions (9.9 g of succinate /[L·h]) with 80 g/L of fumarate. Furthermore, the long-term stability of the HFBR was demonstrated with a continuously efficient production of succinate for more than 15 d (360 h).     

Isolation of strains degrading poly(Vinyl alcohol) at high temperatures and their biodegradation ability

Thermophilic strains were isolated for the first time using activated sludge retrieved from waste water treatment plant of a poly(vinyl alcohol) (PVA) producing factory for biodegradation of PVA at relatively high temperatures. The isolated strains were identified to be Geobacillus tepidamans, Brevibacillus brevis and Brevibacillus limnophilus. The former strain degraded PVA for itself, while the latter 2 strains digested PVA symbiotically. PVA degradation activity of the isolated strains was assessed at first by the halo zone size formed around the colonies and finally by the modified Sturm test. The biodegradation rate of PVA was explored also in the presence of different dyes, because most of the waste water from PVA-consuming factories contains waste dyes.     

Culture medium optimization for acetic acid production by a persimmon vinegar-derived bacterium

A new acetic acid-producing microorganism, Acetobacter sp. RKY4, was isolated from Korean traditional persimmon vinegar, and we optimized the culture medium for acetic acid production from ethanol using the newly isolated Acetobacter sp. RKY4. The optimized culture medium for acetic acid production using this microorganism was found to be 40 g/L ethanol, 10 g/L glycerol, 10 g/L corn steep liquor, 0.5 g/L MgSO₄·7H₂O, and 1.0 g/L (NH₄H₂PO₄. Acetobacter sp. RKY4 produced 47.1 g/L of acetic acid after 48 h of fermentation in a 250 mL Erlenmeyer flask containing 50 mL of the optimized medium.     

A comparison of sequential extraction procedures for fractionation of arsenic,cadmium, lead,and zinc in soil

Twelve soil samples differing in physicochemical properties and total element contents were extracted by three sequential extraction procedures to determine As, Cd, Pb, and Zn bound to individual soil fractions and are defined by individual operational procedures. In the case of arsenic, two additional sequential extraction schemes were designed entirely for fractionation of soil containing arsenic were tested. The results confirmed that determination of element proportions bound to individual soil fractions is strongly dependent on the extracting agent and/or procedure applied within individual extracting schemes. As expected, absolute values of the elements released among the individual extracting procedures are weakly comparable. More reliable results were determined for the more mobile soil elements i.e. cadmium and zinc, in the fractions characterizing the most mobile proportions of investigated elements where significant correlations with basic soil characteristics were observed. In contrast, ambiguous results were observed for As and Pb, for both the individual extraction procedures and the effect of the soil characteristics. Regardless of the studied element, the poorest results were determined for reducible and oxidizable soil fractions. The application of at least two independent procedures or modification of the extraction scheme according to element investigated and/or particular soil characteristics can also be helpful in definition of element pattern in soils in further research.     

Production of poly-3-hydroxyalkanoates from CO and H2 by a novel photosynthetic bacterium

A novel process is described to efficiently photoconvert low-grade organic materials such as waste biomass into natural biological plastics. When heterogeneous forms of dry biomass are thermally gasified, relatively homogeneous synthesis gas mixtures composed primarily of carbon monoxide and hydrogen are produced. Unique strains of photosynthetic bacteria were isolated that nearly quantitatively photoassimilate the carbon monoxide and hydrogen components of synthesis gas into new cell mass. Under unbalanced culture conditions when cellular growth is limited by shortages of nitrogen, calcium, magnesium, iron, or essential vitamins, up to 28% of the new cell mass is found as granules of poly-3-hydroxyalkanoate (PHA), a highmolecular-weight thermoplastic that can be solvent-extracted. The dominant monomeric unit of PHAs is 3-hydroxybutyrate (3HB), which is polymerized into the homopolymeric poly-3-hydroxybutyrate (PHB). PHB is marketed as a biodegradable plastic with physical properties similar to polystyrene. When a green alga was cocultured with the photosynthetic bacterium in light-dark (day-night) cycles, the bacteria synthesized a polymer of poly-3-hydroxybutyrate-3-hydroxyvalerate (PHB-V) with a composition of 70% 3HB and 30% 3-hydroxyvalerate (3HV) to an extent of 18% of the new cell mass. PHB-V is commercially marketed as Biopol and has physical properties similar to polypropylene or polyethylene. Our results demonstrate that a strain of photosynthetic bacteria capable of photoassimilating synthesis gas or producer gas is a potential candidate for large-scale production of biological polyesters.