Hexadecane mineralization activity in ornithogenic soil from Seabee Hook,Cape Hallett,Antarctica

Ornithogenic soils that form in penguin rookeries contain high levels of organic carbon and nitrogen. On Seabee Hook, Cape Hallett, Antartica, ornithogenic soil was contaminated with hydrocarbons following establishment of a scientific research station. In these soils hydrocarbon biodegradation could be supported by available soil nitrogen. Hexadecane mineralization activity was detected in vitro in ornithogenic soil when incubated at 5 or 15°C. At 5°C the extent of hexadecane mineralization was higher in hydrocarbon-contaminated soil than in uncontaminated soil. Alkane-degrading bacteria isolated from Seabee Hook soil were identified as Rhodococcus or Gordonia spp. or an unclassified Corynebacterineae. The alkane degraders grew on n-alkanes from heptane (C8) to eicosane (C20) and pristane, and utilized uric acid or ammonium nitrate as nitrogen source. All of the isolates possessed urease activity. Results of this study indicate biodegradation of hydrocarbons may contribute to the natural attenuation of oil spills in ornithogenic surface soils in summer.     

Studies on the Utilization of Hydrocarbons by Microorganisms

The production of microbial cell substances from hydrocarbons has become the object of commercial attention. We have tested hydrocarbon-utilizing bacterial strains for cell production, and found out one strain of high cell yield, which is very similar to Pseudomonas aeruginosa. The effect of medium composition on cell yield and the utilization of individual hydrocarbons by this strain were investigated. Sixty percent of the added kerosene was converted into cell materials in the following medium of optimum composition: kerosene 2.5%, urea 0.13%, dipotassium phosphate 0.25%, magnesium sulfate 7 aq. 0.1% and tap water. Aliphatic series lower than C₁₀, aromatic and naphthenic hydrocarbons were not or very slightly assimilated. However, aliphatic members higher than C₁₂ were utilized with increasing case. Especially, n-docosane and octadecene-1 were utilized very effectively, and 70% of them were converted into cell substances.     

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.     

Statistical analysis and optimization of nitrogen,phosphorus, and inoculum concentrations for the biodegradation of petroleum hydrocarbons by response surface methodology

In order to optimize and evaluate the influence of nitrogen, phosphorus, and inoculum concentrations on the biodegradation of hydrocarbon contaminated effluents, experiments based on central composite design (CCD) method were carried out for 3 days, employing C1 mixed culture and intermittent aeration. The independent variables were nitrogen concentration (X ₁), phosphorus concentration (X ₂), and inoculum concentration (X ₃) and the removal of total petroleum hydrocarbons (TPH) was the dependent variable. The optimized nutrients ratio (C:N:P = 100:20:2.7) and inoculum concentration (1.32 g/l) provided TPH removal of 71.8% after processing for three days. Analysis using gas chromatography identified five hydrocarbons classes: paraffins, isoparaffins, olefins, naphthenics, and aromatics. The naphthenic compounds did not degrade as readily as the other hydrocarbons that were identified. The following degradation percentages were obtained: 87.1% for the paraffins, 77.7% for the isoparaffins, 78.6% for the olefins, 38.4% for the naphthenics, and 71.7% for the aromatics.     

Ethane and n-pentane in exhaled breath are biomarkers of exposure not effect

The relationship of exhaled ethane and n-pentane to exhaled NO, carbonylated proteins, and indoor/outdoor atmospheric pollutants were examined in order to evaluate ethane and n-pentane as potential markers of airway inflammation and/or oxidative stress. Exhaled NO and carbonylated proteins were found to have no significant associations with either ethane (p = 0.96 and p = 0.81, respectively) or n-pentane (p = 0.44 and 0.28, respectively) when outliers were included. In the case where outliers were removed n-pentane was found to be inversely associated with carbonylated proteins. Exhaled hydrocarbons adjusted for indoor hydrocarbon concentrations were instead found to be positively associated with air pollutants (NO, NO₂ and CO), suggesting pollutant exposure is driving exhaled hydrocarbon concentrations. Given these findings, ethane and n-pentane do not appear to be markers of airway inflammation or oxidative stress.     

Studies on Production of Biotin by Microorganisms

The utilization of hydrocarbons by microorganisms was studied in many fields, but the production of biotin vitamers by hydrocarbon-utilizing bacteria has never been reported.

We have screened many hydrocarbon-utilizing bacteria which produce biotin vitamers in the culture broth. The effects of cultural conditions on biotin vitamers production by strain 5–2, tentatively assigned to the genus Pseudomonas, were studied.

More than 98% of biotin vitamers produced from hydrocarbons by strain 5–2 was chromatographically determined as desthiobiotin. As nitrogen source, natural nutrients were more effective than inorganic nitrogen sources. The production of biotin vitamers was increased under the condition of good aeration. Exogenous pimelic or azelaic acid enhanced biotin vitamers production by strain 5–2.

The production of biotin vitamers from n-alkanes, n-alkenes or glucose by an isolated bacterium, strain 5-2, tentatively assigned to the genus Pseudomonas, was investigated. Among these carbon sources, n-undecane was the most excellent for biotin vitamers production.

The biosynthetic pathway of biotin vitamers, especially desthiobiotin, from n-undecane was also studied. It was found by thin-layer and gas-liquid chromatographical methods that pimelic and azelaic acids were the main acid components in n-undecane culture.

This result, together with previously reported enhancement of biotin vitamers production by these acids, suggests that pimelic and azelaic acids may be the intermediates of biotin vitamers biosynthesis from n-undecane.     

Metagenomic analysis of microbial communities across a transect from low to highly hydrocarbon-contaminated soils in King George Island,Maritime Antarctica

Soil samples from a transect from low to highly hydrocarbon-contaminated soils were collected around the Brazilian Antarctic Station Comandante Ferraz (EACF), located at King George Island, Antarctica. Quantitative PCR (qPCR) analysis of bacterial 16S rRNA genes, 16S rRNA gene (iTag), and shotgun metagenomic sequencing were used to characterize microbial community structure and the potential for petroleum degradation by indigenous microbes. Hydrocarbon contamination did not affect bacterial abundance in EACF soils (bacterial 16S rRNA gene qPCR). However, analysis of 16S rRNA gene sequences revealed a successive change in the microbial community along the pollution gradient. Microbial richness and diversity decreased with the increase of hydrocarbon concentration in EACF soils. The abundance of Cytophaga, Methyloversatilis, Polaromonas, and Williamsia was positively correlated (p-value = <.05) with the concentration of total petroleum hydrocarbons (TPH) and/or polycyclic aromatic hydrocarbons (PAH). Annotation of metagenomic data revealed that the most abundant hydrocarbon degradation pathway in EACF soils was related to alkyl derivative-PAH degradation (mainly methylnaphthalenes) via the CYP450 enzyme family. The abundance of genes related to nitrogen fixation increased in EACF soils as the concentration of hydrocarbons increased. The results obtained here are valuable for the future of bioremediation of petroleum hydrocarbon-contaminated soils in polar environments.     

n-Alkane uptake and utilisation by Streptomyces strains

Streptomyces strains isolated from the Kuwait Burgan oil field were defined as S. griseoflavus, S. parvus, and S. plicatus utilised n-hexadecane, n-octadecane (purified fractions of mineral oil), kerosene, and crude oil as sole carbon and energy sources. The strains were incubated with n-alkanes and increase of the fatty acid content with chain length equivalent to the employed n-alkanes was observed. Signal transducing GTP-binding proteins (GBPs) play an important role in n-alkane uptake in streptomycetes. Specific activators of GBPs increased the uptake of hydrocarbons. Using the hydrophobic fluorescent dye diphenylhexatrien (DPH) as a probe, it was found that the microviscosity of the hydrophobic inner region of the cellular membrane is significantly lower in hydrocarbon utilisers than in non-utilisers. This difference probably reflects differences in the fatty acid composition of the strains. When cultures were grown in n-alkane containing media, electron microscopy revealed that the hydrocarbon utilisers showed less-electron dense areas as inclusions in the cytoplasm. Soil samples inoculated with Streptomyces strains eliminated hydrocarbons much faster than those not containing these strains, serving as control. When inorganic medium was supplied with n-hexadecane-1-¹⁴C as sole carbon and energy source, radioactive CO₂ was detected. Since streptomycetes have not been used until now for oil elimination, though they are known as abundant soil bacteria tolerating extreme conditions, their possible use for bioremediation of hydrocarbon contaminated soils is discussed.     

Ecological and site historical aspects of N dynamics and current N status in temperate forests

Ecological developments during Holocene age and high atmospheric depositions since industrialization have changed the N dynamics of temperate forest ecosystems. A number of different parameters are used to indicate whether the forests are N‐saturated or not, most common among them is the occurrence of nitrates in the seepage water below the rooting zone. The use of different definitions to describe N saturation implies that the N status of ecosystems is not always appropriately assessed. Data on N dynamics from 53 different German forests were used to classify various development states of forest ecosystems according to the forest ecosystem theory proposed by Ulrich for which N balances of input – (output plus plant N increment) were used. Those systems where N output equals N input minus plant N increment are described as (quasi‐) Steady State Type. Those forests where N output does not equal N input minus plant N increment as in a ‘transient state.’ Forests of the transient state may lose nitrogen from the soil (Degradation Type) or gain nitrogen [e.g., from atmospheric depositions (Accumulation Type)]. Forest ecosystems may occur in four different N states: (a) (quasi‐) Steady State Type with mull type humus, (b) Degradation Type with mull type humus, (c) Accumulation Type with moder type humus, and (d) (quasi‐) Steady State Type with moder type humus. Forests with the (quasi‐) steady state with mull type humus in the forest floor (n= 8) have high‐soil pH values, high N retention by plant increment, high N contents in the mineral soils, and have not undergone large changes in the N status. Forests of the Degradation Type lose nitrogen from the mineral soil (currently degradation is occurring on one site). Most forests that have moder or mor type humus and low‐soil pH values, and low N contents in the mineral soil have gone through the transient state of organic matter loss in the mineral soils. They accumulate organic matter in the forest floor (accumulation phase, currently 21 sites are accumulating 6–21 kg N ha^?1 yr^?1) or have reached a new (quasi‐) steady state with moder/mor type humus (n= 15). N retention in the accumulation phase has significantly increased in soil with N deposition (r²= 0.38), soil acidity (considering thickness of the forest floor as indices of soil acidity, r²= 0.43) and acid deposition (sulfate deposition, r²= 0.39). Retention of N (4–20 kg N ha^?1 yr^?1) by trees decreased and of soils increased with a decrease in the availability of base cations indicating the important role of trees for N retention in less acid soils and those of soils in more acid soils. Ecosystem theory could be successfully applied on the current data to understand the dynamics of N in temperate forest ecosystems.     

Utilization of gas oil by a yeast culture

A mixed yeast culture (Culture 4) was grown on representative gas oil samples as well as paraffin wax. Culture 4 was found to utilize n-paraffinic hydrocarbons almost quantitatively from most gas oil fractions; significant alteration of other hydrocarbon components was not detected. Generation times of 4.0–9.0hr. were typical during the exponential growth phase in fermentations with various gas oil fractions. Cell yields were 70–90% based on n-paraffin utilization. The culture appeared to exhibit maximum efficiency of n-alkane removal in the C₁₉ to C₂₄ range. The cells recovered from the fermentations contained 8.8–9.3% nitrogen. Paraffin wax also served as a suitable carbon source when dissolved in 2,6,10,14-tertramethylpentadecane (pristane). However, substrate utilization appeared to be incomplete.     

Taxonomic value of the property of fungi to assimilate hydrocarbons

A wide range of fungi were tested for their ability to assimilate a series of hydrocarbons, which includedn-paraffins, aromatic hydrocarbons and petroleum fractions.The property is not evenly distributed among the various fungal classes, but is to be found mainly in two orders, the Mucorales and the Moniliales. Within the latter order, the generaAspergillus andPenicillium are rich in hydrocarbon-assimilating strains. In other genera, the property of assimilating hydrocarbons is relatively rare.Hydrocarbon assimilation is not necessarily common to related species, nor proper to one species, but more the property of individual strains. Different strains belonging to the same species differ in metabolic activity when they are tested against a series of hydrocarbons. The property of assimilating hydrocarbons appears to lack taxonomic value. Species of the same genus show only a tendency to behave in a similar way, e.g.Penicillium strains usually assimilaten-decane and light gas oil whereasAspergillus strains seldom do so. Aspergillus species sporulate better on long chainn-paraffins. On some hydrocarbons, they develop particular pigments. n-Paraffins with at least ten carbon atoms support better growth than petroleum fractions. Individual strains are very sensitive to minor changes in hydrocarbon composition or structure. Only sparse delayed growth is observed on aromatic hydrocarbons.n-Heptane, petroleum ether, naphtha and kerosene are often toxic whereas aromatic hydrocarbons are usually non-toxic.     

Variation in tissue element concentrations in Quercus ilex L. over a range of different soils

In order to study the variability in nutrient concentrations in four tissues of Q. ilex in relation to soil properties, we selected fifteen stands in both Quercus ilex forests and Q. ilex-Pinus halepensis mixed forests. These stands had developed on soils derived from eight different parent materials. Three soil groups were differentiated according to their chemical properties: calcareous soils, siliceous soils, and volcanic soils. Across sites, nutrient concentrations were generally less variable in current-year tissues than in older tissues. Nitrogen and potassium showed the lowest variability among sites, their concentrations in current-year leaves ranging from 1.17% to 1.39% for N and from 0.53% to 0.68% for K. There were few statistically significant correlations between tissue element concentrations, the most frequent being the antagonistic relationship between calcium and magnesium. Nitrogen concentration in current-year leaves was negatively correlated with soil chemical fertility (nitrogen, phosphorus and potassium). This may reflect a nutritional imbalance between nitrogen and other nutrients, some of which may be more limiting than nitrogen to Q. ilex growth in Catalonia forests. Negative correlations were also found between plant magnesium and soil calcium, and positive correlations between plant calcium and soil calcium.     

Emulsifying and surface active agents from Corynebacterium hydrocarboclastus

A Corynebacterium hydrocarboclastus culture isolated in our laboratory (see, Zajic and Knettig, Developments in Industrial Microbiology, 1971, p. 87) has been shown to produce an extracellular biopolymer with emulsifying properties when grown on a mixture of linear hydrocarbons. This microorganism was found to grow well on a variety of carbohydrates and hydrocarbons. However, the best substrates were pure linear hydrocarbons and particularly, n-C₁₂, n-C₁₃, and n-C₁₄. The substrates supporting good growth gave good polymer production. Maximum cell mass of 10–11 g/liter and a maximum amount of polymer of 5–6 g/liter were recorded. The polymers recovered from the different substrates were found to be complex molecules or mixtures with a protein, a lipid, and a carbohydrate moiety. All the polymers are surface active and have two critical micelle concentrations.     

Biostimulation of n-Alkane Degradation in Diesel Fuel-Spiked Soils

Nutrient enhancement of bioremediation with nitrogen, namely biostimulation, increases process performance. Selection of a proper nitrogen source is critical for bioremediation applications. In this study, the effects of different nitrogen sources on biodegradation of C₁₀–C₂₅ n-alkane compounds in diesel fuel-spiked soil were revealed, and the most appropriate nitrogen source for biodegradation of semi- and non-volatile n-alkanes was investigated. Bioremediation of diesel fuel contaminated soil was monitored in lab-scale reactors for 15 days. Ammonium sulfate, potassium nitrate and urea were used as nitrogen sources. Carbon dioxide and oxygen levels in the reactors were recorded to monitor microbiological activity. Contaminant removal process was investigated by pH, heterotrophic plate count, total petroleum hydrocarbons (TPH) and C₁₀–C₂₅ n-alkane analyses. First-order kinetic constants were calculated via respirometric and contaminant concentration data. According to total C₁₀–C₂₅ n-alkane removal levels and degradation rate constants, ammonium sulfate addition resulted in the most efficient contaminant removal followed by potassium nitrate and urea. Simultaneous degradation of individual n-alkanes was observed for all of the nitrogen sources. Urea addition changed the distribution of individual n-alkane concentrations relative to the pre-experimental concentrations. Nitrogen source type had no differential effect on degradation rates of semi- (C₁₀–C₁₆) and non-volatile (C₁₇–C₂₅) fractions.     

Headspace solid-phase microextraction and gas chromatographic–mass spectrometric screening for volatile hydrocarbons in blood

Optimization for headspace solid-phase microextraction (SPME) was studied with a view to performing gas chromatographic–mass spectrometric (GC–MS) screening of volatile hydrocarbons (VHCs) in blood. Twenty hydrocarbons comprising aliphatic hydrocarbons ranging from n-hexane to n-tridecane, and aromatic hydrocarbons ranging from benzene to trimethylbenzenes were used in this study. This method can be used for examining a burned body to ascertain whether the victim had been alive or not when the burning incident took place. n-Hexane, n-heptane and benzene, the main indicators of gasoline components, were found as detectable peaks through the use of cryogenic oven trapping upon SPME injection into a GC–MS instrument. The optimal screening procedure was performed as follows. The analytes in the headspace of 0.2 g of blood mixed with 0.8 ml of water plus 0.2 μg of toluene-d₈ at −5°C were adsorbed to a 100-μm polydimethylsiloxane (PDMS) fiber for 30 min, and measured using the full-mass-scanning GC–MS method. The lower detection limits of all the compounds were 0.01 μg per 1 g of blood. Linearities (r²) within the range 0.01 to 4 μg per 1 g of blood were only obtained for the aromatic hydrocarbons at between 0.9638 (pseudocumene) and 0.9994 (toluene), but not for aliphatic hydrocarbons at between 0.9392 (n-tridecane) and 0.9935 (n-hexane). The coefficients of variation at 0.2 μg/g were less than 8.6% (n-undecane). In conclusion, this method is feasible for the screening of volatile hydrocarbons from blood in forensic medicine.     

Studies on Utilization of Hydrocarbons by Yeasts

The production of microbial cell substances from hydrocarbons has been attracting attention of people for many years. Production of bacterial cell from hydrocarbons is disadvantageous because of the difficulty in separating cell from the broth.

We have tested hydrocarbon-utilizing yeasts isolated from garden soil for cell production. The effect of medium composition on yeast growth and the utilization of individual hydrocarbon by yeast, strain Y-3, were investigated.

As a nitrogen source, urea was more effective than ammonium nitrate. When a very smal! amount of corn steep liquor was added, yeast growth was very improved. Aliphatic series of hydrocarbon lower than C₉ were not or very slightly assimilated by this yeast.

Generally speaking, series of even-number hydrocarbons were more effective than those of odd-number hydrocarbons.

We found that the yeast Y-3 strain reported in the previous paper¹⁾ has a diterminal oxidation system of hydrocarbon.

This yeast capable of growing in mineral-salts solution with hydrocarbons as sole source of carbon produced a series of dioic acid from n-undecane. These acids are 1,11-undecane dioic acid, 1,9-nonane dioic acid (azelaic acid), 1,7-heptane dioic acid (pimelic acid) and 1,5-pentane dioic acid (glutaric acid). 1,10-Decane dioic acid (sebacic acid) was also isolated from n-decane cultures.

Azelaic acid was partially transformed into pimelic acid and glutaric acid by treating it with resting cells of this yeast.

1,11-Undecane dioic was also transformed into azelaic acid pimelic acid, and glutaric acid by the same treatment as described above.     

Microbial transformation of hydrocarbons. II. Growth constants and cell composition of microbial cells derived from n-alkanes

Cultivation of Norcardia sp., Mycobacterium phlei, and Candida lipolytica in inorganic salt solution containing n-alkanes C₁₀–C₂₀ as solo carbon and energy source was investigated. Generation times of 0.5–7.0 hr were typical during the exponential growth phase. The final cell concentrations (dry weight) were usually 9–26 g/l with n-alkane mixtures ranging from n-decane through n-eicosane. A linear dependence was found between the production of cell mass and the consumption of n-alkanes. The rest concentration of n-alkanes in the cell mass is in all experiments smaller than 0.5% (w/w). Cell yields were Y_sub 60–142% and for Y_e 50–97% based on n-alkane utilization. In one case, with the Nocardia NBZ 23, the substrate specifity on hydrocarbons and on a n-alkane mixture C₁₀-C₂₀ was studied. The cell mass recovered from the fermentations contained 47.8–57.7% carbon, 5.6–9.95% nitrogen, 7.2–9.4% hydrogen, 35–62% crude protein, and 6–36% lipid. Cellular protein and lipid synthesized by an organism is influenced by the type of nitrogen source. The amino acid, glucosamine, muramic acid, 2,6-diaminopimelinic acid, and fatty acid distribution in organisms grown on n-alkanes compared with a corresponding fermentation on glucose as sole carbon source were also estimated.     

Chemical Studies on Components of Dried Bonito, “Katsuobushi”

Volatile components obtained by the extraction of “Katsuobushi” with 80% ethanol and by the subsequent steam distillation of the extract were fractionated by the usual methods, and the resulting hydrocarbon fraction was investigated. Gas chromatographic study on this fraction originated from “Katsuobushi” of bonito (Katsuwonus pelamis) revealed 9 hydrocarbons, including n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nonadecane, n-eicosane, n-heneicosane and n-docosane, which were tentatively identified by the retention times with the aid of authentic hydrocarbons. n-Pentadecane and n-heptadecane that were main components among these hydrocarbons were identified further by NMR and IR spectrometry. “Katsuobushi” of frigate mackerel (Auxis thazard), mackerel (Scomber Japonicus Houttuyn) or muroaji (Decapterus muroadsi) also contained n-penta-decane and n-heptadecane in large amounts, but did other hydrocarbons in negligible amounts.

Possible mechanisms of the hydrocarbon formation during the processing of “Katsuobushi” were discussed.     

Genomic insights into the Acidobacteria reveal strategies for their success in terrestrial environments

Members of the phylum Acidobacteria are abundant and ubiquitous across soils. We performed a large‐scale comparative genome analysis spanning subdivisions 1, 3, 4, 6, 8 and 23 (n = 24) with the goal to identify features to help explain their prevalence in soils and understand their ecophysiology. Our analysis revealed that bacteriophage integration events along with transposable and mobile elements influenced the structure and plasticity of these genomes. Low‐ and high‐affinity respiratory oxygen reductases were detected in multiple genomes, suggesting the capacity for growing across different oxygen gradients. Among many genomes, the capacity to use a diverse collection of carbohydrates, as well as inorganic and organic nitrogen sources (such as via extracellular peptidases), was detected – both advantageous traits in environments with fluctuating nutrient environments. We also identified multiple soil acidobacteria with the potential to scavenge atmospheric concentrations of H₂, now encompassing mesophilic soil strains within the subdivision 1 and 3, in addition to a previously identified thermophilic strain in subdivision 4. This large‐scale acidobacteria genome analysis reveal traits that provide genomic, physiological and metabolic versatility, presumably allowing flexibility and versatility in the challenging and fluctuating soil environment.     

Utilization of Hydrocarbons by Microorganisms

Corynebacterium hydrocarboclastus S10BI or S489BI can accumulate a good deal of Lglutamate in a thiamine-deficient medium at the expence of hydrocarbon, but can not form L-glutamate in a thiamine-sufficient medium in spite of rapid cell growth, as already reported. In order to establish the optimal culture condition for L-glutamate formation, the influence of the following factors was first studied: hydrocarbon concentration, pH control, nitrogen sources, temperature, aeration and supplement of metal ions or amino acids. Then L-glutamate production from a variety of hydrocarbons or petroleum fractions was examined. Sodium oleate was found to stimulate growth remarkably instead of thiamine. Ferrous ion was found to be obligatory for L-glutamate formation and was suggested to have taken part in the earliest step of n-alkane oxidation.