The effects of carbon sources and micronutrients in fermented whey on the biodegradation of n-hexadecane in diesel fuel contaminated soil

Fermented whey has previously been shown to stimulate biodegradation of n-hexadecane in diesel contaminated soils. The proposed explanation for the stimulatory effect is that fermented whey provides easily accessible carbon and micronutrients, which give rise to an increased degrading biomass.The objective of this work has been to investigate the role of the different carbon sources and vitamins in fermented whey on the microbial degradation of n-hexadecane in soil.The effects of lactose, lactate, vitamins and free amino acids were tested in combinations according to a full factorial design experiment, at concentrations corresponding to those present in fermented whey. The target substance was ¹⁴C-labeled n-hexadecane in nutrient amended soil microcosms contaminated with 5000 mg diesel fuel kg⁻¹ dw. Biodegradation was monitored by determination of evolved ¹⁴CO₂.Significant effects on the biodegradation of n-hexadecane were observed for lactate and amino acids additions in a sandy soil. Lactate showed both an inhibitory effect in the early phase of the experiment and a stimulatory effect in the later phase. The effect of amino acids was slightly stimulatory, mainly evident as a shortening of the lag time.The degree of n-hexadecane degradation at the end of the experiment was correlated with the total concentration of organic compounds added to the soil.Scientific relevanceThere are a handful papers describing the potential of using organic amendments (often industrial by-products) with a content of both easily accessible carbon and micronutrients, to enhance the bioremediation of polluted soils. Enhanced biodegradation is often reported and the proposed explanations are that the combination of easily accessible carbon and micronutrients increases the degrading biomass.In this paper, we examine the effect of fermented whey on the degradation of n-hexadecane and correlate the observed effects on the biodegradation with the main components lactate, amino acids, lactose and B-vitamins. This has to our knowledge never been done before.     

Enhanced Degradation of n-Hexadecane in Diesel Fuel Contaminated Soil by the Addition of Fermented Whey

The objective of this work has been to investigate the possibility of using fermented whey as an organic growth supplement in order to enhance the aerobic degradation of n-hexadecane in soil. Fermented whey was added at different dosages to nutrient amended soil microcosms contaminated with 5000 mg diesel fuel kg^?1 dry weight (dw). The target substance was ¹⁴C-labeled n-hexadecane, and the biodegradation was monitored by analysis of evolved ¹⁴CO₂. Biodegradation curves were fitted to a three-half-order kinetics model. Enhanced biodegradation was observed in sand at 7 and 22°C and in loamy sand at 22°C but the effect was most pronounced in the sand soil at 22°C. The addition of 6 or 60 ml fermented whey kg^{? 1} soil dw increased the degree of n-hexadecane biodegradation at the end of the experiment, 167 days, from 49% in the untreated sand to 60 or 67%, respectively. This increase in biodegradation was characterized by an increase in the amount of substrate biodegradation by first-order kinetics despite a decrease in the first order rate constant, k₁. The highest concentration of fermented whey, 60 ml kg^?1, gave rise to substrate competition, diauxie, which resulted in an extended lag phase.     

Optimization of environmental parameters for biodegradation of alpha and beta endosulfan in soil slurry by Pseudomonas aeruginosa

Aim: To determine optimal environmental conditions for achieving biodegradation of α‐ and β‐endosulfan in soil slurries following inoculation with an endosulfan degrading strain of Pseudomonas aeruginosa. Methods and Results: Parameters that were investigated included soil texture, soil slurry: water ratios, initial inoculum size, pH, incubation temperature, aeration, and the use of exogenous sources of organic and amino acids. The results showed that endosulfan degradation was most effectively achieved at an initial inoculum size of 600 μl (OD = 0·86), incubation temperature of 30°C, in aerated slurries at pH 8, in loam soil. Under these conditions, the bacterium removed more than 85% of spiked α‐ and β‐endosulfan (100 mg l^?1) after 16 days. Abiotic degradation in noninoculated control medium within same incubation period was about 16%. Biodegradation of endosulfan varied in different textured soils, being more rapid in course textured soil than in fine textured soil. Increasing the soil contents in the slurry above 15% resulted in less biodegradation of endosulfan. Exogenous application of organic acids (citric acid and acetic acid) and amino acids (l ‐methionine and l ‐cystein) had stimulatory and inhibitory effects, respectively, on biodegradation of endosulfan. Conclusion: The results of this study demonstrated that biodegradation of endosulfan by Ps. aeruginosa in soil sediments enhanced significantly under optimized environmental conditions. Significance and Impact of the Study: Endosulfan is a commonly used pesticide that can contaminate soil, wetlands and groundwater. Our study demonstrates that bioaugmentation of contaminated soils with an endosulfan degrading bacterium under optimized conditions provides an effective bioremediation strategy.     

Effects of Zeolite-Containing Material and Ammonium Nitrate on Biodegradation of n-Tridecane in Heavy Clay-Loam Leached Chernozem with High Organic Matter Content

Biostimulation based on usage of soil amendments is growing due to their efficiency in removing different petroleum hydrocarbons (PHC) from contaminated sand or loam-sand soils. However, the research on clay-rich soils with higher organic carbon content, in which PHC biodegradation may proceed differently and which are more difficult to clean up, has been less extensive. In a pot experiment, we studied and compared the effects of two soil amendments, natural zeolite-containing material (ZCM, 50 g kg^?1) as a bulking agent and ammonium nitrate (0.3 g N kg^?1) as a nitrogen fertilizer, on biodegradation of n-tridecane (1 wt.%) in a weakly acidic heavy clay loam leached chernozem with fairly high organic carbon content (3.71%). After 48 days, the nitrogen-amended contaminated soil showed enhancement of both respiratory activity (basal and substrate-induced respiration rates) and the number of n-tridecane- degraders. As a consequence, the extent of n-tridecane biodegradation (86.5%) was essentially higher in the presence of added nitrogen than that in the non-amended soil (73.7%). In contrast, due to the partial retention of n-tridecane molecules in its pores, ZCM retarded biodegradation to 56.0%, showed no significant effect on the number of n-tridecane-degraders and, moreover, enhanced the decomposition of the soil intrinsic organic matter. The obtained data indicate that more precautions should be considered when using porous sorbents such as ZCM for remedial arrangements in PHC-contaminated soils.     

Formation of Glutaric and Adipic Acids from n-Alkanes with Odd and Even Numbers of Carbons by Candida tropicalis OH23

Many strains of yeast which can utilize n-alkanes as the sole source of carbon were isolated from flowers and fruits. Among them, a strain, OH23, identified as Candida tropicalis, formed acidic substances from n-alkanes. The principal products from n-alkanes with odd and even numbers of carbons were identified as glutaric and adipic acids, respectively. The culture conditions for their formation were investigated. n-Pentadecane and n-hexadecane were the best substrates for the formation of glutaric and adipic acids, respectively. Yields of 170 mg of glutaric and 64 mg of adipic acid were obtained from 100 ml of media containing 4% (v/v) n-pentadecane and n-hexadecane, respectively, and 0.5% casamino acids.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

Studies on whey fermentation using lactic acid bacteria L. acidophilus and L. bulgaricus

The fermentation process of acid curd whey using pure cultures of L. bulgaricus and L. acidophilus was investigated. The influence of the starter culture amount on the acidification rate in the fermentation was specified, the biological value of fermented and fermented-ammoniated curd whey was determined, and the ability of fermented whey to prevent the injurious effect of Bac. mesenthericus on the wheat bread quality was examined. Acid curd whey was fermented up to a titratable acidity of 19.8–21.6 g lactic acid/kg whey using L. acidophylus and L. bulgaricus. Mathematical equations were developed on the basis of experimental data to calculate the titratable acidity (A) as a functionof fermentation time (τ) and temperature (t). Fermentation and fermentation-ammoniation processes increase the biological value of whey (the content of the vitamins B₁, B₂, B₆, PP and the free amino acids increase). A new dry fodder BIOLAKTS was developed from fermented curd whey and was recommended for use in veterinary medicine. The fermentation-ammoniation process of curd whey was carried out by adding calculated amounts of non-protein nitrogen NH₄OH to increase the total protein equivalent and to achieve mutual proportions of protein and lactose 1:1.4, as in skimmed milk. Fermented-ammoniated curd whey was used to obtain a skimmed milk substitute. A dry flour lactic acid concentrate (FLC) was created as a mixture of high quality wheat flour and evaporated fermented whey in established ratios. As our experiments prove, it can be used as an additive in bread-making to prevent the spoiling of wheat bread by Bac. mesenthericus.     

Production of Fluorescent Compounds from Guanine by Microorganisms

One species of hydrocarbon utilizing bacteria was isolated from soil. This strain was named as Achromobacter petrophilum No. 4017. This bacterial species utilizes normal hydrocarbons with carbon chains of nC₁₀ to nC₁₈, but does not utilize glucose or other carbohydrates. Achromobacter petrophilum forms small amounts of green-yellow, green-blue and violet fluorescent compounds in the medium containing n-hexadecane (nC₁₆) as a carbon source. The mutant strain, No. 4510, which requires hypoxanthine and thiamine for growth, was obtained from Achromobacter petrophilum No. 4017 by ultraviolet irradiation and formed considerable amounts of green-yellow fluorescent compound by the addition of guanine to the n-hexadecane medium. This fluorescent compound was crystallized from culture broth.     

Hydrophobic cell surface and bioflocculation behavior of Rhodococcus erythropolis

An alkane-biodegrading bacterium identified as Rhodococcus erythropolis (NTU-1 strain) was isolated from petroleum contaminated soil. The major purpose of the current research was to study the issues regarding biofloccules formation and cell surface hydrophobicity of NTU-1. When long-chain alkanes are supplied as the carbon source, NTU-1 tends to form biofloccules and remove significant amount of alkanes by biodegradation and physical trapping. Approximately, more than 95% of each alkane could be efficiently removed within 40–68 h. The bioremediation process was accompanied by formation of biofloccules with size ranging from 0.1 to 2 cm in diameter. The MATH test and the hydrophobic slide experiment suggested that NTU-1 might possess a hydrophobic cell surface which is one of the important factors in the formation of biofloccules. It provides the interaction of cells with hydrocarbon droplets effectively and further aggregate into larger clumps. Besides, when grown on n-hexadecane, experimental results revealed that there were at least 11 different growth-associated fatty acids produced, with carbon chain length ranging from 12 to 24, and cell surface hydrophobicity was enhanced via accumulation at the cell surface.     

Data consistency,yield, maintenance,and hysteresis in batch cultures of Candida lipolytica cultured on n-hexadecane

Candida lipolytica was cultured batchwise using n-hexadecane as the main carbon source. Biomass production, n-hexadecane consumption, oxygen consumption, and carbon dioxide evolution were measured to follow the fermentation. The consistency of the measured data was examined using integrated and instantaneous available electron and carbon balances. Values of the “true” growth yield, η_max, and maintenance coefficient, m_e were estimated using three different sets of data (biomass and n-hexadecane, oxygen and biomass, and CO₂ and biomass), and the results were compared with estimates obtained from literature data. Hysteresis patterns were observed in plots of specific rates of oxygen consumption and carbon dioxide evolution versus specific growth rate.     

Synthesis of dl-Phyllostine

“Protein-like activator (PA)” for n-alkane oxidation was formed by Pseudomonas aeruginosa S₇B₁ from long-chain n-alkanes, 1-hexadecene and cetyl alcohol but not from glucose, glycerol and palmitic acid. The molecular weight and the total amino acid residues of PA were estimated at about 14,300 and 147, respectively. PA was relatively stable to low pH and high temperature, and completely inactivated upon heating at 98°C for 45 min. The cultural fluid obtained from n-hexadecane medium stimulated the growth of the strain on n-hexadecane. The degree of the growth stimulation by the fluid depended on the amount of PA and rhamnolipid (RL) in the fluid. The heat-treated PA lost the growth-stimulaing effect and the emulsifying power on the n-hexadecane medium in the presence of RL.     

Composition of cell material and biological value of the cellular protein of a Micrococcus strain grown on hydrocarbons

A Micrococcus cerificans strain was grown on simple media with n-hexadecane or gas oil as sole carbon sources. Samples of cellular material recovered from hexadecane or gas oil fermentations do not appear to differ significantly in their composition. The protein content varied from 68 to 75%. With the exception of sulfur amino acids the amino acid distribution compares favorably with the FAO standard reference protein. The biological value of cell protein recoveered from hexadecane fermentations was 67 (cascin, 70). In the case of gas oil grown cells, the cell material recovered had to be completely purified in order to improve its protein quality. After fully extraction of undersirable fraction with petroleum ether in a Soxhlet apparatus the biological value observed was 63.     

Fatty Acid Composition of Hydrocarbon-Assimilating Yeast

As a part of extensive program on microbial utilization of hydrocarbons, lipid components of Candida petrophillum SD-14 grown on n-alkanes and glucose as carbon sources were studied. In any carbon source, cellular fatty acids of the yeast contained palmitic, palmitoleic, stearic, oleic and linoleic acids as major components.

When n-tridecane was fed to the yeast, fatty acids with odd- and even-number of carbon atoms were produced in almost identical quantity. Another yeast, Torulopsis petrophillum SD-77, also gave a very similar fatty acid pattern by n-tridecane substrate. These phenomena indicate the existence of C₂ addition and β-oxidation of the fatty acid formed in the yeasts.

In the cases of n-tridecane, n-hexadecane and glucose as substrate, about a half of SD-14’s lipid was phospholipid, which consisted of phosphatidyl ethanolamine and phosphatidyl choline principally. Free alcohol and wax were not detected in any case.     

Evaluation of Three Electron-Donor Permeable Reactive Barrier Materials for Enhanced Reductive Dechlorination of Trichloroethene

Understanding the fate of complex electron-donor materials is important for developing efficient biostimulation strategies to treat ground water contamination by chlorinated ethenes (CEs). The fermentation product distributions and H₂ production of common permeable reactive barrier (PRB) carbon substrates (dairy whey, sodium lactate syrup, and Hydrogen Release Compound [HRC]) were monitored as measures of substrate efficiency in aquifer microcosms spiked with trichloroethene (TCE). In long-term experiments, the fermentation of PRB substrates to slow-degrading organic acids maintained low H₂ partial pressures (≤ 10^?3.5) that, as previous studies suggest, may give competitive advantage to dechlorinators over hydrogenotrophic methanogens. Whey-amended and lactate-amended microcosms exhibited faster complete dechlorination and, according to organic acid carbon flow, higher rates of fermentation to acetate. In HRC-amended microcosms, propionate appeared to serve as a carbon sink that prolonged dechlorination. Upon complete dechlorination, whey microcosms contained the highest percentage of organic acid carbon. Native Dehalococcoides populations increased by 3 orders of magnitude (per g sediment) in whey-amended microcosms. Whey's efficiency improved in microcosms prepared with aquifer sediment and water from within a downgradient whey PRB. Results suggested whey loading values of 0.2 kg/m³ may be appropriate under sufficiently reducing conditions to efficiently stimulate hydrogenotrophic and potentially actetotrophic dechlorinating populations. Renewal of whey PRBs may, however, be required. Implications for further long-term study of cost-efficiencies are discussed.     

Dibenzothiophene desulfurization in hydrocarbon environment by Staphylococcus sp. resting cells

Staphylococcus sp. strain S3/C desulfurized dibenzothiophene/n-hexadecane (3 mg ml^–1) in a hydrocarbon aqueous biphasic culture. The resting cells decreased the sulfur content of the hydrocarbon phase by 57% at 2.2 mg l^–1 h^–1 in the absence of any additional carbon and sulfur source.     

Comparative lipid content ofCandida lipolytica grown on glucose and onn-hexadecane

Candida lipolytica, grown onn-hexadecane as the sole source of carbon and energy, contained 17.1% lipids in the logarithmic phase of growth, and 7.3% lipids in the stationary phase of growth. When the yeast was grown on glucose, it contained 6.2% lipids in the logarithmic phase of growth, and 3.6% lipids in the stationary phase of growth. Fatty acids, that could be extracted by petroleum ether after saponification, constituted the major part of the fatty acids ofC. lipolytica in its logarithmic phase of growth on glucose. They constituted only a minor amount of the fatty acids in the stationary phase of growth on glucose. The reverse was true when the yeast was grown onn-hexadecane. The broth contained more free, petroleum ether-soluble fatty acids when the cellular lipid content was high than when it was low. Overnight starvation ofC. lipolytica grown onn-hexadecane in a carbon-free nutrient medium, removed the residual cell-bound hydrocarbon, increased the cell population by one half and decreased the cellular lipid content (as % of dry yeast) by one third. Various methods for the determination of lipids, described as appropriate for yeasts were compared. The highest yields were obtained by extraction of the freeze-dried paste, at room temperature, with a 1:1 chloroform-methanol mixture.     

Effect of several environmental parameters on carbon metabolism in histosols

High specific activity¹⁴C-labeled glucose, succinate, acetate, salicylate, and amino acids were used to examine carbon metabolism by the microbial community of Pahokee muck (aLithic medisaprist), a drained, cultivated soil of the Florida Everglades. Variations in carbon oxidation were observed from the end of the wet season through the dry season in a fallow (bare) field. Evolution of¹⁴CO₂ varied with the substrate added and time. Calculation of¹⁴CO₂ evolution for each substrate as a proportion of total respiration of the microbial community which was measured by succinate oxidation (relative oxidation) allowed for determination of the proportion of metabolic activity contributed by the oxidation of each carbon source. Except for the May sample when an approximate 30% decline in relative salicylate oxidation activity was observed, the proportion of total catabolic activity contributed by salicylate oxidation and acetate degradation was constant with time. Relative oxidation of glucose and amino acids ranged from 0.12 to 0.52 and 0.10 to 0.23, respectively. At two times during the dry season, the effect of depth of soil and crop on the carbon oxidation was examined. Relative acetate and amino acid oxidation were constant with depth whereas statistically significant variation was observed in glucose and salicylate oxidation. Generally, with the latter substrates, the activity declined with increased soil depth. Greatest effect of crop on these metabolic activities was noted with oxidation of salicylate in soils from a St. Augustinegrass [Stenatophrum secundatum (Walt.) Kuntz] pasture. In these soils, oxidation of salicylate was nearly double that of the fallow field or of soil planted with sugarcane (Saccharum sp.).     

Enhancement of phenol degradation by soil bioaugmentation with Pseudomonas sp. JS150

Aims: To test whether bioaugmentation with genetically modified Pseudomonas sp. JS150 strain could be used to enhance phenol degradation in contaminated soils. Methods and Results: The efficiency of phenol removal, content of humic carbon, survival of inoculant, number of total culturable autochthonous bacteria and changes in fatty acid methyl esters (FAME) profiling obtained directly from soils were examined. Bioaugmentation significantly accelerated phenol biodegradation rate in tested soils. Phenol applied at the highest concentration (5·0 mg g^?1 soil) was completely degraded in clay soil (FC) within 65 days, whereas in sand soil (FS) within 72 days. In comparison, phenol biodegradation proceeded for 68 and 96 days in nonbioaugmented FC and FS soils, respectively. The content of humic carbon remained at the same level at the beginning and the end of incubation time in all soil treatments. The number of introduced bacteria (2·50 × 10⁹ g^?1 soil) markedly decreased during the first 4 or 8 days depending on contamination level and type of soil; however, inoculant survived over the experimental period of time. Analysis of FAME patterns indicated that changes in the percentages of cyclopropane fatty acids 17:0 cy and 19:0 cyω10c and branched fatty acids might be useful markers for monitoring the progress of phenol removal from soil. Conclusions: It was confirmed that soil bioaugmentation with Pseudomonas sp. JS150 significantly enhanced soil activity towards phenol degradation. Cyclopropane and branched fatty acids were sensitive probes for degree of phenol utilization. Significance and Impact of the Study: In future, genetically modified Pseudomonas sp. JS150 strain could be of use in the bioaugmentation of phenol‐contaminated areas.     

Effects of excitatory neurotransmitter amino acids on swelling of rat brain cortical slices.

Abstract— With the single rat brain cortical slice serving as an in vitro bio-assay system, the effects of neurotransmitter amino acids (1 mm ) on brain swelling, water, sodium and potassium content, inulin space, and lactate production were studied. The putative dicarboxylic amino acid neurotransmitters, l -glutamic acid and l -aspartic acids, greatly increased intracellular brain swelling with increased intracellular Na⁺, water content and lactate production, and decreased inulin space and intracellular K⁺. Equimolar GABA, taurine, glycine, the putative inhibitory neurotransmitter amino acids, and equimolar α-amino-isobutyric acid had no effect. Brain swelling and intracellular Na⁺/K⁺ ratios were greatly increased by l -glutamate and l -aspartate at a concentration of 10 mm . However, l -aspartate at these concentrations greatly depleted the K⁺ content and lactate production as compared to l -glutamate. Further studies indicated that only the structural analogs and isomers of the dicarboxylic amino acids possessing two acidic groups and an α-amino group had a similar effect on the induction of brain swelling. Among the analogs of glutamic acid, dl -homocysteic acid and kainic acid had a greater effect on brain swelling, as observed from the total adenosine 5′-triphosphate (ATP) levels and the time-course and dose-response. A biphasic response in lactate production was induced by dl -homocysteic acid and kainic acid, suggesting that these analogs had a neurotoxic effect on cellular metabolism at higher concentrations.     

Pyrene mineralization capacity increases with compost maturity

Experiments were conducted to determine the effects of composting or simple addition of compost to the mineralization of n-hexadecane, pyrene and benzo(a)pyrene in soil. Soil (contaminated or clean) was composted with maple leaves and alfalfa. Samples from different composting phases were spiked with radiolabeled and cold n-hexadecane, pyrene or benzo(a)pyrene, placed in aerated microcosms at different temperatures, and monitored for mineralization. It was determined that neither composting nor the addition of compost had any effect on n-alkane or benzo(a)pyrene mineralization. In contrast, the pyrene mineralization rate increased dramatically with the amount of time that soil had been composted. Highest pyrene mineralization rates and extents (more than 60% after 20 days) were obtained when pyrene was in contact with composted soil from the curing stage. Neither thermophiles (55 °C) nor fungi were responsible for pyrene mineralization.