In submerged fermentation of filamentous fungi, pelleted and dispersed types of growth are the two main morphological forms largely affecting the productivity of industrial processes. Despite the immense importance of fungal biotechnology various aspects of the biology of these two growth forms have been rarely studied. Whether there are any differences in architecture and properties of hyphal cell wall between pelleted and dispersed mycelia of the same non-genetically modified fungus was unknown. This comparative study characterized the cell wall structure and properties of pelleted and dispersed forms of Thielavia terrestris growth. Infrared spectroscopy revealed that the hyphal cell wall of pellets, in contrast to dispersed mycelium, contained considerably more chitin (22 % and 1-2 % of dry weight [dw], respectively), glucans (28 % and 10 % dw), and lipids (8 % and 6 % dw). The dispersed form was characterised by much higher content of phosphate-rich polysaccharide (dispersed vs pelleted: 23 % and 1 % dw) and protein (55 % and 40 % dw, respectively). TEM-microscopy showed the thicker cell wall of the dispersed form, which did not manifest the third well-defined electron-dense smooth outer layer present in pellets. The cell wall of the pelleted form was less susceptible to mechanical and enzymatic destruction and demonstrated in AFM hardness tests 5-10 times greater nano-hardness than the dispersed form. Thus, the cell wall structure and properties are very different in the pelleted than the dispersed growth forms, where the narrower and chitin/glucans-richer cell wall of the pelleted form provides more effective protection from mechanical and enzymatic damages.

Currently, commercial microalgal biomass production is limited mainly due to high production costs. The use of cheap alternative media, such as dairy industry by-products and wastewater, can facilitate the commercialization of prospective microalgal strains. However, the use of dairy by-products is hampered by poor understanding of mechanisms of lactose utilization, i.e. the main carbon (C) source in such substrates, leading to inconsistent performance of microalgae. This study focuses on the ability of an isolated freshwater axenic microalgal strain Graesiella emersonii MSCL 1718 to utilize lactose and its monomers, i.e. glucose and galactose, in modified Bold's Basal Medium with triple nitrogen and vitamins (3 N-BBM-V). It aims to develop a better understanding of microalgal interactions with C sources available in dairy by-products. The strain can successfully utilize lactose under mixotrophic and heterotrophic conditions when pH is maintained at approximately 7 and efficiently grow in lactose concentrations up to 30 g l-1. Under mixotrophic cultivation conditions, G. emersonii was capable of utilizing all three tested C sources. However, under heterotrophic cultivation conditions, G. emersonii could not utilize galactose as the main C added to 3 N-BBM-V, highlighting the importance of selecting appropriate cultivation conditions. Additionally, the enzyme beta-galactosidase, responsible for lactose hydrolysis, was detected in the microalgal biomass, with activity up to 17.03 +/- 1.23 U l-1. FTIR analysis of biomass demonstrated an increase in total lipid content and polyunsaturated fatty acids specifically in the mixotrophic group. Overall, G. emersonii demonstrated features suitable for bioconversion of dairy by-products which should be investigated in further studies.

Plants and algae play a crucial role in the earth's ecosystems. Through photosynthesis they convert light energy into chemical energy, capture CO2 and produce oxygen and energy-rich organic compounds. Photosynthetic organisms are primary producers and synthesize the essential omega 3 and omega 6 fatty acids. They have also unique and highly diverse complex lipids, such as glycolipids, phospholipids, triglycerides, sphingolipids and phytosterols, with nutritional and health benefits. Plant and algal lipids are useful in food, feed, nutraceutical, cosmeceutical and pharmaceutical industries but also for green chemistry and bioenergy. The analysis of plant and algal lipidomes represents a significant challenge due to the intricate and diverse nature of their composition, as well as their plasticity under changing environmental conditions. Optimization of analytical tools is crucial for an in-depth exploration of the lipidome of plants and algae. This review highlights how lipidomics analytical tools can be used to establish a complete mapping of plant and algal lipidomes. Acquiring this knowledge will pave the way for the use of plants and algae as sources of tailored lipids for both industrial and environmental applications. This aligns with the main challenges for society, upholding the natural resources of our planet and respecting their limits.

Crypthecodinium cohnii is a marine microalga that can accumulate high amounts of polyunsaturated fatty acids (PUFAs) and thus replace conventional routes of fish oil production. They are associated with the destruction of marine resources and multiple downstream/purification complications. The major drawbacks of using C. cohnii for industrial-scale production are associated with low PUFA productivity. One of the means of increasing the PUFA synthesis rate is to maintain the medium component concentrations at optimal values throughout cultivation, thus increasing PUFA production efficiency, which can result in the successful transfer of the process to pilot and/or industrial scale. The goal of the present research was to develop techniques for increasing the efficiency of PUFA production via C. cohnii cultivation. Multiple experiments were carried out to test and fine-tune the cultivation medium composition and oxygen transfer factors. The biomass yields from individual components, yeast extract, sea salts, and glucose amounted to 5.5, 0.65, and 0.61 g·g−1, respectively. C. cohnii cell susceptibility to mechanical damage was experimentally evaluated. Power inputs of <276.5 W/m3 did not seem to promote cell destruction when Pitched-blade impellers were used. The obtained cultivation conditions were shown to be efficient in terms of increasing the biomass productivity and the omega-3 fatty acid content in C. cohnii. By using the applied methods, the maximal biomass productivity reached 8.0 g·L−1·day−1, while the highest obtained biomass concentration reached 110 g·L−1. A steady increase in the concentration of PUFAs during cultivation was observed from the FTIR data.

The incidence of autoimmunity is increasing, to ensure timely and comprehensive treatment, there must be a diagnostic method or markers that would be available to the general public. Fourier-transform infrared spectroscopy (FTIR) is a relatively inexpensive and accurate method for determining metabolic fingerprint. The metabolism, molecular composition and function of blood cells vary according to individual physiological and pathological conditions. Thus, by obtaining autoimmune disease-specific metabolic fingerprint markers in peripheral blood mononuclear cells (PBMC) and subsequently using machine learning algorithms, it might be possible to create a tool that will allow the diagnosis of autoimmune diseases. In this preliminary study, it was found that the peak shift at 1545 cm-1 could be considered specific for autoimmune disease type 1 diabetes (T1D), while the shifts at 1070 and 1417 cm-1 could be more attributed to the autoimmune condition per se. The prediction of T1D, despite the small number of participants in the study, showed an inverse AUC = 0.33 & PLUSMN; 0.096, n = 15, indicating a stable trend in the prediction of T1D based on FTIR metabolic fingerprint data in the PBMC. A promising trend has been found to predict the presence of type 1 diabetes using PBMC metabolic fingerprinting and machine learning. This will enable extensive monitoring of public health, timely diagnosis of diseases and initiation of treatment.

Understanding the energy metabolism and its regulation is one of the clues to metabolic engineering of stress-resistant lignocellulose-converting microbial strains, also including the promising ethanologen Zymomonas mobilis. Z. mobilis is an obligately fermentative, facultatively anaerobic bacterium, carrying an active respiratory chain with low energy-coupling efficiency. Its respiration does not supply energy to aerobically growing cultures on sugary media, yet oxidative phosphorylation has been demonstrated in non-growing cells with ethanol. Here, we show, for the first time, that in respiring, non-growing Z. mobilis cells receiving regular small amounts of ethanol, oxidative phosphorylation significantly contributes to the maintenance of their viability. No improvement of viability is seen in the NADH dehydrogenase (ndh)-deficient respiratory mutant, which is unable to oxidize ethanol. The ethanol effect is also hampered by the protonophoric uncoupler CCCP, or the inhibitor of ATP synthase, DCCD. At higher concentrations (6% v/v), ethanol causes stress that slows down culture growth. By monitoring the activity of several respiratory gene promoters under ethanol stress with the green fluorescent protein reporter system, we demonstrate downregulation of these promoters, in particular the ndh promoter. We speculate that the decrease in respiratory chain activity in response to stress conditions mitigates the production of reactive oxygen species.

Crypthecodinium cohnii is a marine heterotrophic dinoflagellate that can accumulate high amounts of omega-3 polyunsaturated fatty acids (PUFAs), and thus has the potential to replace conventional PUFAs production with eco-friendlier technology. So far, C. cohnii cultivation has been mainly carried out with the use of yeast extract (YE) as a nitrogen source. In the present study, alternative carbon and nitrogen sources were studied: the extraction ethanol (EE), remaining after lipid extraction, as a carbon source, and dinoflagellate extract (DE) from recycled algae biomass C. cohnii as a source of carbon, nitrogen, and vitamins. In mediums with glucose and DE, the highest specific biomass growth rate reached a maximum of 1.012 h−1, while the biomass yield from substrate reached 0.601 g·g−1. EE as the carbon source, in comparison to pure ethanol, showed good results in terms of stimulating the biomass growth rate (an 18.5% increase in specific biomass growth rate was observed). DE supplement to the EE-based mediums promoted both the biomass growth (the specific growth rate reached 0.701 h−1) and yield from the substrate (0.234 g·g−1). The FTIR spectroscopy data showed that mediums supplemented with EE or DE promoted the accumulation of PUFAs/docosahexaenoic acid (DHA), when compared to mediums containing glucose and commercial YE.

Docosahexaenoic acid (DHA) is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), with numerous health benefits. Crypthecodinium cohnii, a marine heterotrophic dinoflagellate, is successfully used for the industrial production of DHA because it can accumulate DHA at high concentrations within the cells. Glycerol is an interesting renewable substrate for DHA production since it is a by-product of biodiesel production and other industries, and is globally generated in large quantities. The DHA production potential from glycerol, ethanol and glucose is compared by combining fermentation experiments with the pathway-scale kinetic modeling and constraint-based stoichiometric modeling of C. cohnii metabolism. Glycerol has the slowest biomass growth rate among the tested substrates. This is partially compensated by the highest PUFAs fraction, where DHA is dominant. Mathematical modeling reveals that glycerol has the best experimentally observed carbon transformation rate into biomass, reaching the closest values to the theoretical upper limit. In addition to our observations, the published experimental evidence indicates that crude glycerol is readily consumed by C. cohnii, making glycerol an attractive substrate for DHA production.

It is well known that sheep wool is an agricultural product consumed by the textile industry. However, a large part of this material is frequently wasted due to strict industrial requirements. Considering the cheapness, natural origin, and biodegradability of this product, it was proposed that the wool can be used as an adsorbent for isolation of harmful azo dyes from wastewater. Using genotoxic and mutagenic Congo Red (CR) dye as a model compound, different adsorptive properties of the non-irradiated sheep wool have been studied in an aqueous solution and simulated textile effluent. For the purpose of comparison, the surface of the wool was irradiated with gamma rays up to about 100 kGy absorbed dose and the performance of the obtained samples has been measured. The characterization of the wool surface was based on SEM, BET, FTIR, EPR, and determination of zero-point charge. The adsorptive performance of the samples was compared by constructing and analyzing adsorption isotherms and uptake kinetics curves. It was shown that the CR adsorption on all of the samples followed the Langmuir model and adsorption kinetics was described by the Elovich equation. The non-irradiated sheep wool demonstrated the highest value of saturation adsorption capacity for the CR of 5.7 µmol/g which is comparable to the capacities of a large group of unmodified natural adsorbents. Finally, it was shown that 20 mg of the non-irradiated sheep wool can be used repeatedly without cleaning/regeneration at least three times to isolate the CR from simulated effluent at a concentration of 25 µM.

Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated omega-3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6-ndh, with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6-ndh- sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii, either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6-ndh-sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2-3 % of cell dry weight, corresponding to previously reported results for this strain on glucose.

In this paper, results of feasibility study on microplastics (MPs) assessment in leachates from the Latvian solid municipal landfill Getliņi are discussed. The application of leachates for the treatment of cigarette butts (CGB) was evaluated. Methods of fluorescent microscopy, Fourier transform infrared (FTIR) spectroscopy and FTIR-microscopy were used for the identification and characterization of MPs in the leachates and analysis of CGB. Presence of the secondary MPs (e.g., degraded polyolefin mixtures) was determined in the tested landfill leachates, while cellulose acetate (CA) was not determined in these products. The leachates were tested as potential media for the thermophilic (55°C) fermentation of CGB without air supply. Degradation of CGB was determined after one-week fermentation that was confirmed comparing the changes in FTIR spectra of CA prior and after the treatment. This study provoked a path for further experimental studies of controlled degradation of cigarette butts under natural conditions in landfill environments.

Two isothiocyanates, i.e., sulforaphane (SFA) and sulforaphene (SFE), are suggested to be used as an alternative chemopreventive diet. This study was focused on the effect of SFA and SFE on Lactobacillus plantarum, which has been subjected to the irradiation (2-50 Gy). The cultures grown in De Man, Rogosa and Sharpe (MRS) and Tryptone Soya Broth (TSB) were compared in terms of bacteria physiological activity under tested conditions. Broth composition notably influenced the bacteria growth kinetic parameters, as well as culture response to the oxidative stress. Activity of L. plantarum cells after irradiation was evaluated by their dehydrogenase (DHA) and quinone-reductase (QR) activities. The enzyme activity was quantified in living cells. Bacterial cultures obtained in MRS and TSB broth, demonstrated contrasting characteristics in their enzyme activities. The MRS-grown culture did not show any QR activity, whereas the TSB-grown cells showed a non-linear response towards gamma-irradiation with a maximum inhibition being at 10 Gy. Addition of SFA or SFE in concentration of 1 µg/mL to the cultures before irradiation exposure recovered the QR activity from 23% in a non-amended variant up to 102% and 121%, respectively, taking the non-irradiated non-amended variant as 100%.

Zymomonas mobilis is the most efficient bacterial ethanol producer and its physiology is potentially applicable to industrial-scale bioethanol production. However, compared to other industrially important microorganisms, the Z. mobilis metabolome and adaptation to various nutritional and genetic perturbations have been poorly characterized. For rational metabolic engineering, it is essential to understand how central metabolism and intracellular redox balance are maintained in Z. mobilis under various conditions. In this study, we applied quantitative mass spectrometry-based metabolomics to explore how glucose-fed non-growing Z. mobilis Zm6 cells metabolically adapt to change of oxygen availability. Mutants partially impaired in ethanol synthesis (Zm6 adhB) or oxidative stress response (Zm6 cat) were also examined. Distinct patterns of adaptation of central metabolite pools due to the change in cultivation condition and between the mutants and Zm6 reference strain were observed. Decreased NADH/NAD ratio under aerobic incubation corresponded to higher concentrations of the phosphorylated glycolytic intermediates, in accordance with predictions of the kinetic model of Entner-Doudoroff pathway. The effects on the metabolite pools of aerobic to anaerobic transition were similar in the mutants, yet less pronounced. The present data on metabolic plasticity of non-growing Z. mobilis cells will facilitate the further metabolic engineering of the respective strains and their application as biocatalysts.

1,4-dihydropyridines (1,4-DHP) possess important biochemical and pharmacological properties, including antimutagenic and DNA-binding activity. The latter activity was first described for water-soluble 1,4-DHP with carboxylic group in position 4, the sodium salt of the 1,4-DHP derivative AV-153 among others. Some data show the modification of physicochemical properties and biological activities of organic compounds by metal ions that form the salts. We demonstrated the different affinity to DNA and DNA-protecting capacity of AV-153 salts, depending on the salt-forming ion (Na, K, Li, Rb, Ca, Mg). This study aimed to use different approaches to collate data on the DNA-binding mode of AV-153-Na and five other AV-153 salts. All the AV-153 salts in this study quenched the ethidium bromide and DNA complex fluorescence, which points to an intercalation binding mode. For some of them, the intercalation binding was confirmed using cyclic voltammetry and circular dichroism spectroscopy. It was shown that in vitro all AV-153 salts can interact with four DNA bases. The FTIR spectroscopy data showed the interaction of AV-153 salts with both DNA bases and phosphate groups. A preference for base interaction was observed as the AV-153 salts interacted mostly with G and C bases. However, the highest differences were detected in the spectral region assigned to phosphate groups, which might indicate either conformational changes of DNA molecule (B form to A or H form) or partial denaturation of the molecule. According to the UV/VIS spectroscopy data, the salts also interact with the human telomere repeat, both in guanine quadruplex (G4) and single-stranded form; Na and K salts manifested higher affinity to G4, Li and Rb –to single-stranded DNA.

The resistance of lactobacilli to oxidative stress is of great importance for their applicability as probiotics. This study aimed to evaluate the response of Lactobacillus plantarum strain ATCC (R) 14917 (TM), grown in either de Man, Rogosa, and Sharpe agar (MRS medium) or tryptic soy broth (TSB medium), to 1-2 mM H2O2 after the exposure to different doses of ionising radiation. Two bacterial extracellular enzyme groups, dehydrogenases (DHAs) and quinone reductases (QRs), served as the criteria of viability and antioxidant activity, respectively. The irradiated L. plantarum culture grown in TSB showed increased QR activity at irradiation doses of 2-50 Gy, with the maximum activity at 10 Gy, compared to non-irradiated cells. The QR activity of the MRS-grown culture was increased significantly (p < 0.05) in the presence of H2O2 for the cells subjected to irradiation, compared to the control. The cultivation of L. plantarum in TSB resulted in considerable morphological changes in the 24 h culture. Our results indicate that the cultivation of L. plantarum in TSB activated the antioxidative potential of these bacteria. The most important aspect of this phenomenon is the extracellular enzyme activity that may directly influence the host organism by providing antioxidant activity.

Glyphosate-based herbicides (GBHs) are the most widespread commonly used broad-spectrum herbicides that contaminate soils and waters, are toxic to bacteria, plants and animals, and have been classified as 'probably carcinogenic to humans' by the International Agency for Research on Cancer in 2015. Particular soil bacteria and fungi can degrade GBHs, hence, search for new GBH-degrading strains or microbial consortia, effective under specific growth conditions and local environment, seems to be a promising solution for bio-remediation of glyphosate-contaminated environment. Consequently, there is a need for rapid and informative methods to evaluate the GBH-induced changes of the metabolic pathways in cells, that may serve as indicators of GBH degrading potential. Three new GBH-degrading bacterial strains, Pseudomonas sp., Actinobacteria and Serratia sp. were isolated from sludge of municipal waste water treatment plant (Daugavgriva, Riga, Latvia), agricultural soil and plant tissue, respectively. This study examined the response of these isolates to elevated concentrations of glyphosate (GLP) (100 and 500 mg/L) in GBH Klinik (R) 360 SL. The GBH -induced shift of metabolic activity in cells of Pseudomonas sp. was shown by tests on EcoPlates (TM). Fourier transform infrared (FTIR) spectroscopy analyses were used to evaluate the metabolomic response of bacteria to elevated concentrations of GBH in the growth environment. The spectra of Pseudomonas sp. and Serratia sp., incubated with and without GBH, were similar, thus indicating their GBH -resistance. The absorption at 1736 cm(-1), assigned to ester carbonyl stretch vibrations, was detected in spectra of all three bacteria. The highest ester content was detected in Actinobacteria grown in medium with 1.0% molasses and 100 or 500 mg/L GLP in GBH Klinik (R). An increase of cellular amounts of esters, either those of phospholipids or poly-beta-hydroxybutyrates, indicates degradation of GLP. Therefore, monitoring the ester carbonyl stretch vibration band in FTIR spectra of bacterial biomass may speed up the search GBH degrading strains. Microbiological tests and cell metabolic response studies by FTIR spectroscopy showed that the three new isolates of Pseudomonas sp Actinobacteria and Serratia sp. were resistant to elevated concentrations of GBH Klinik (R) in growth environment and exhibited the potential for GBH degradation.

Glyphosate (GLP) currently is one of the most widely used herbicides worldwide. The persistence of GLP and its major metabolite, aminomethylphosphonic acid (AMPA) in the environment has been described by other authors. This study was aimed at comparing the GLP and AMPA behavior in sandy and loamy sand soils after spiking with enhanced (445 mu g g(-1)) concentrations of GLP in herbicide KLINIK (R) (Nufarm, Austria) and bioaugmentation followed by 40 days weathering and a consistent three-stage leaching in a laboratory column experiment. Soil samples were obtained from mineral topsoil (0-10 cm) within former agricultural lands where soil parent material was formed by glacigenic deposits. The total amount of GLP and AMPA collected during three leaching stages was significantly (p<.05) higher from columns with sandy soil, compared to loamy sand soil. Bioaugmentation resulted in considerably lower concentrations of AMPA in leachates, especially in the sets with sandy soil (p=.01). Leachates were tested using FTIR spectroscopy and Daphnia magna. Statistical analysis of the changes in N-tot, C-tot, K+, Mg2+, Al3+, Ca2+, Mn2+ and Fe3+ concentrations in soils after the leaching experiment revealed that the loamy sand soil was likely to be more sensitive to the addition of GLP and bioaugmentation than sandy soil.

High pressure processing (HPP) allows to extend the shelf life of meat and meat products by pressurization of microorganisms. At the same time, HPP can induce changes of the protein structure. Vacuum-packed pork chops were HPP-treated at 300, 600 MPa for 1 or 15 min. Samples of raw, cooked and HPPtreated meat muscles and juice were analysed to evaluate the structure of macromolecules. HPP caused visible discolouration of pork chops; hence, the colour of pork meat surface was tested. The lightness (colour component L*) was directly proportional to the applied pressure, probably due to the increased protein denaturation by high pressure. Pork meat muscle and juice samples were analysed with FTIR high-throughput screening (FTIR HTS-XT, Bruker, Germany). FTIR spectra of meat muscle showed pressure and time dependent decrease of numbers of a-structures and 13-sheet conformations of proteins. In spectra of cooked meat muscle, the number of a-helix structures and 13-sheets, identified at 1682 cm(-1), was the lowest. The decrease of intensities of absorption bands at 1655 cm(-1) and 1454 cm(-1), assigned to the collagen type I, was directly proportional to the pressure and treatment time, thus, suggesting the release of denatured collagen into meat juice. In spectra of meat released juice, changes of Amide band shapes, frequencies and the minimum between the Amide I and II bands, compared to those of control, indicated changes in the protein secondary structure and the ratio of a-helix to 13-sheet. In spectra of all juice samples an absorption band at 1394 cm(-1) assigned to CH3 groups in proteins Amide I and lipids was detected. The intensities of bands at 1394 and 1124 cm(-1) increased proportionally to the increase of pressure and treatment time. It was shown that the absorption band at 1394 cm(-1) can be used as a marker for assessment of the HPP induced changes in proteins. This study showed that HPP-induced changes in protein structure can be evaluated by FTIR high-throughput screening of pork meat muscles as well as released juice. FTIR analyses of the meat released juice is significantly faster and simpler method than analyses of muscle. This approach is especially effective for quick assessment of large sample sets and search of the most effective meat HPP treatment mode. (C) 2019 Elsevier B.V. All rights reserved.

Bacteria with hydrocarbon (HC)-degrading activity coexist and dominate in oil-polluted areas in consortia. Investigation of cooperation between different microbial species is vital for assessing the potential efficiency of various environmental biotechnologies. In this study, the metabolic activity of eight phenotypically distinct isolates with HC-degrading activity was tested. Pairwise comparison of the 16S rRNA gene sequences of the soil isolates revealed that 5 of these showed high similarity with Stenotrophomonas and 3 with Pseudomonas genus. A hierarchical cluster analysis based on the Fourier Transform Infrared Spectroscopy, has demonstrated the grouping of one Pseudomonas and two Stenotrophomonas isolates within one cluster while the rest (5 isolates) formed another cluster. The response of bacteria to 1% diesel oil addition against background of molasses concentrations ranging from 0.05 % to 3.00 % has revealed that molasses concentrations below 1 % are likely to be more efficient for microbial activity when grown with diesel oil. Comparative testing of bacterial activity in a Bushnell Haas broth supplemented with six nitrogen sources showed ammonium salts as a preferential nitrogen source for HC degradation. That was proved using 2,6-dichlorophenol indophenol (DCPIP) indicator. Serial batch experiments with a stepwise increase of diesel oil concentration from 1% to 3% and up to 5% showed that step 2 with 3 % diesel oil represented the highest biomass concentration and the highest phenotypical diversity of colonies among the tested variants. These results provide additional tools towards optimization of bacterial biomass production for its use in bioaugmentation of the HC-contaminated sites.