Balancing Trade-Offs Imposed by Growth Media and Mass Spectrometry for Bacterial Exometabolomics.

The bacterial exometabolome consists of a vast array of specialized metabolites, many of which are only produced in response to specific environmental stimuli. For this reason, it is desirable to control the extracellular environment with a defined growth medium composed of pure ingredients. However, complex (undefined) media are expected to support the robust growth of a greater variety of microorganisms than defined media. Here, we investigate the trade-offs inherent to a range of complex and defined solid media for the growth of soil microorganisms, production of specialized metabolites, and detection of these compounds using direct infusion mass spectrometry. We find that complex media support growth of more soil microorganisms, as well as allowing for the detection of more previously discovered natural products as a fraction of total m/z features detected in each sample. However, the use of complex media often caused mass spectrometer injection failures and poor-quality mass spectra, which in some cases resulted in over a quarter of samples being removed from analysis. Defined media, while more limiting in growth, generated higher quality spectra and yielded more m/z features after background subtraction. These results inform future exometabolomic experiments requiring a medium that supports the robust growth of many soil microorganisms. IMPORTANCE Bacteria are capable of producing and secreting a rich diversity of specialized metabolites. Yet, much of their exometabolome remains hidden due to challenges associated with eliciting specialized metabolite production, labor-intensive sample preparation, and time-consuming analysis techniques. Using our versatile three-dimensional (3D)-printed culturing platform, SubTap, we demonstrate that rapid exometabolomic data collection from a diverse set of environmental bacteria is feasible. We optimized our platform by surveying Streptomyces isolated from soil on a variety of media types to assess viability, degree of specialized metabolite production, and compatibility with downstream LESA-DIMS analysis. Ultimately, this will enable data-rich experimentation, allowing for a better understanding of bacterial exometabolomes.

Effect of Culture Conditions on Fatty Acid Profiles of Bacteria and Lipopolysaccharides of the Genus Pseudomonas-GC-MS Analysis on Ionic Liquid-Based Column.

The profiling of bacterial fatty acids is a well-established technique in identifying and classifying bacteria. Cultivation conditions may affect the biosynthesis, thereby, changing the fatty acid profile in bacteria. The effect of the culture conditions on the fatty acid components of Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa polyresistant and Pseudomonas putida all are aligned to the genus Pseudomonas. The fatty acids in the lipopolysaccharides of Pseudomonas aeruginosa PAO1 were also examined. The effects of the cultivation conditions were followed by using agar and blood agar media at the characteristic temperatures, 25 °C, 37 °C and 42 °C, respectively, and an analysis was made during the 1st, 3rd and 5th day following inoculation. In addition to quantitative differences, we also experienced qualitative differences in the fatty acid profiles which detect newly appearing fatty acids, due to changes in environmental factors. The application of ionic liquid-based column unveils new possibilities for the analyses of fatty acids in GC-MS experiments for bacterial fatty acid profiling. The validation results (response linearity, limit of detection, limit of quantification, system suitability, intraday and interday repeatability and accuracy) show the high separation efficiency of the ionic liquid-based column in the analyses.

Effect of culture conditions on biomass yield of acclimatized microalgae in ozone pre-treated tannery effluent: A simultaneous exploration of bioremediation and lipid accumulation potential.

Microalgae has huge potential towards biological nutrient removal, but the challenges are remains in maximizing the biomass yield and so nutrient/pollutant removal efficiency. In this study, a response surface methodology-central composite design was applied to investigate the significant process variables (temperature, light intensity, inoculum density and light period) and its interaction effect on biomass yield of effluent acclimatized microalgae Nannochloropsis oculata, Chlorella vulgaris and Chlorella sorokiniana in ozone pre-treated tannery effluent (OPTE). At optimum culture condition N. oculata, C. vulgaris, and C. sorokiniana have yielded 0.67 g/L, 0.85 g/L, and 1.06 g/L biomass. Besides, correlation and regression analysis revealed the strong correlation between microalgal growth and nutrient removal rate. Among the species, C. sorokiniana has shown better remediation potential, at 27.5 °C, 150 µmol m-2 s-1 light intensity, 30% (v/v) inoculum, 16 h light period with the specific growth rate of 0.559 day-1 and nutrient/pollutant removal efficiency of 90% C, 90% N, 100% P, 82% COD, and 100% chromium. But, N. oculata has revealed the better lipid accumulation potential (40%) in OPTE. Thus, the present study established the appropriate strains and conditions required for OPTE treatment along with the value-added biomass production in large scale.

Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM-1 ) and recombinant protein productivity per unit area-time (g m-2 /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m-2 than at a low plant density of 100 plants m-2 . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc.

Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.

Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6.

Glyphosate acetylation as a specific trait of Achromobacter sp. Kg 16 physiology.

The growth parameters of Achromobacter sp. Kg 16 (VKM B-2534 D), such as biomass and maximum specific growth rate, depended only on the source of phosphorus in the medium, but not on the carbon source or the presence of growth factors. With glyphosate as a sole phosphorus source, they were still 40-50 % lower than in media supplemented with orthophosphate or other organophosphonate-methylphosphonic acid. At the first time process of glyphosate acetylation and accumulation of acetylglyphosate in culture medium were revealed in this strain. Acetylglyphosate isolated from cultural liquid was identified by mass spectroscopy; its mass spectrum fully corresponded with that of chemically synthesized acetylglyphosate. Even poorer growth was observed in media with acetylglyphosate: although the strain was able to utilize this compound as a sole source of phosphorus, the maximum biomass was still 58-70 % lower than with glyphosate. The presence of acetylglyphosate in culture medium could also hinder the utilization of glyphosate as a phosphorus source. Therefore, the acetylation of glyphosate may be a specific feature of Achromobacter sp. Kg 16 responsible for its poor growth on this compound.

Advances in Culturomics Research on the Human Gut Microbiome: Optimizing Medium Composition and Culture Techniques for Enhanced Microbial Discovery.

Despite considerable advancements achieved using next-generation sequencing technologies in exploring microbial diversity, several species of the gut microbiome remain unknown. In this transformative era, culturomics has risen to prominence as a pivotal approach in unveiling realms of microbial diversity that were previously deemed inaccessible. Utilizing innovative strategies to optimize growth and culture medium composition, scientists have successfully cultured hard-to-cultivate microbes. This progress has fostered the discovery and understanding of elusive microbial entities, highlighting their essential role in human health and disease paradigms. In this review, we emphasize the importance of culturomics research on the gut microbiome and provide new theories and insights for expanding microbial diversity via the optimization of cultivation conditions.

Comparative uptake, translocation and metabolism of phenamacril in crops under hydroponic and soil cultivation conditions.

Many studies investigate the plant uptake and metabolism of xenobiotics by hydroponic experiments, however, plants grown in different conditions (hydroponic vs. soil) may result in different behaviors. To explore the potential differences, a comparative study on the uptake, translocation and metabolism of the fungicide phenamacril in crops (wheat/rice) under hydroponic and soil cultivation conditions was conducted. During 7-14 days of exposure, the translocation factors (TFs) of phenamacril were greatly overestimated in hydroponic-wheat (3.6-5.2) than those in soil-wheat systems (1.1-2.0), with up to 3.3 times of difference between the two cultivation systems, implying it should be cautious to extrapolate the results obtained from hydroponic to field conditions. M-144 was formed in soil pore water (19.1-29.9 µg/L) in soil-wheat systems but not in the hydroponic solution in hydroponics; M-232 was only formed in wheat shoots (89.7-103.0 µg/kg) under soil cultivation conditions, however, it was detected in hydroponic solution (20.1-21.2 µg/L), wheat roots (146.8-166.0 µg/kg), and shoots (239.2-348.1 µg/kg) under hydroponic conditions. The root concentration factors (RCFs) and TFs of phenamacril in rice were up to 2.4 and 3.6 times higher than that in wheat for 28 days of the hydroponic exposure, respectively. These results highlighted that cultivation conditions and plant species could influence the fate of pesticides in crops, which should be considered to better assess the potential accumulation and transformation of pesticides in crops.

Importance of Intracellular Energy Status on High-Hydrostatic-Pressure Inactivation of sake Yeast Saccharomyces cerevisiae.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

Characterizing isoprene production in cyanobacteria - Insights into the effects of light, temperature, and isoprene on Synechocystis sp. PCC 6803.

Engineering cyanobacteria for the production of isoprene and other terpenoids has gained increasing attention in the field of biotechnology. Several studies have addressed optimization of isoprene synthesis in cyanobacteria via enzyme and pathway engineering. However, only little attention has been paid to the optimization of cultivation conditions. In this study, an isoprene-producing strain of Synechocystis sp. PCC 6803 and two control strains were grown under a variety of cultivation conditions. Isoprene production, as quantified by modified membrane inlet mass spectrometer (MIMS) and interpreted using Flux Balance Analysis (FBA), increased under violet light and at elevated temperature. Increase of thermotolerance in the isoprene producer was attributed to the physical presence of isoprene, similar to plants. The results demonstrate a beneficial effect of isoprene on cell survival at higher temperatures. This increased thermotolerance opens new possibilities for sustainable bio-production of isoprene and other products.

Identification of Floral Volatile Components and Expression Analysis of Controlling Gene in Paeonia ostii 'Fengdan' under Different Cultivation Conditions.

In order to explore the release rule of floral volatile substances and the diurnal variation of different flower development stages of Paeonia ostii 'Fengdan' in potted and ground-planted conditions, dynamic headspace adsorption combined with gas chromatography-mass spectrometry(GC-MS) was used to analyze the dynamic changes in floral volatile components and contents. Quantitative real-time PCR (qRT-PCR) was used to analyze changes in flower fragrance-regulating genes PsPAL, PsTPSs, and PsbHLH at different flower development stages and a daily change process at the full-blooming stage. The results show that there were differences in aroma components and contents of Paeonia ostii 'Fengdan' at different flower development stages and different time quantum of every day. There were 25 and 28 aroma components identified in 7 flower development stages of tree peonies planted in pots and in the field, respectively, and 23 and 22 aroma components identified at different time quantum of the day, of which the largest and highest content was alkanes. The main characteristic aroma substances were (E)-ß-ocimene, 1,3,5-trimethoxybenzene, 2,4-di-tert-butylphenol, methyl jasmonate, nerol, and cinnamyl alcohol; released amounts of the abovementioned substances varied depending on the development stage and the time of the day. The expression of flower fragrance-controlling genes (PsPAL, PsTPSs, and PsbHLH) in tree peonies varied greatly in different conditions. The results of this study provide a valuable resource to investigate floral fragrance formation in tree peonies.

Effects of Temperature and pH on Recombinant Thaumatin II Production by Pichia pastoris.

The sweet protein thaumatin is emerging as a promising sugar replacer in the market today, especially in the food and beverage sector. Rising demand for its production necessitates the large-scale extraction of this protein from its natural plant source, which can be limited in terms of raw material availability and production costs. Using a recombinant production technique via a yeast platform, specifically, Pichia pastoris, is more promising to achieve the product economically while maintaining batch-to-batch consistency. However, the bioproduction of recombinant proteins requires the identification of optimal process variables, constituting the maximal yield of the product of interest. These variables have a direct effect on the growth of the host organism and the secretion levels of the recombinant protein. In this study, two important environmental factors, pH, and temperature were assessed by cultivating P. pastoris in shake flasks to understand their influence on growth and the production levels of thaumatin II protein. The results from the pH study indicate that P. pastoris attained a higher viable cell density and secretion of protein at pH 6.0 compared to 5.0 when grown at 30 °C. Furthermore, within the three levels of temperatures investigated when grown at pH 6.0, the protein levels were the highest at 30 °C compared to 20 and 25 °C, whereas 25 °C exhibited the highest viable cell density. Interestingly, the trend observed from the qualitative effects of temperature and pH occurred in all the media that was investigated. These results broaden our understanding of how pH and temperature adjustment during P. pastoris cultivation aid in enhancing the production yields of thaumatin II prior to optimising the fed batch bioreactor operation.

Bacillusvelezensis Strains for Protecting Cucumber Plants from Root-Knot Nematode Meloidogyne incognita in a Greenhouse.

Meloidogyne incognita Kofoid et White is one of the most dangerous root-knot nematodes in greenhouses. In this study, we evaluated two Bacillus strains (Bacillus velezensis BZR 86 and Bacillus velezensis BZR 277) as promising microbiological agents for protecting cucumber plants from the root-knot nematode M. incognita Kof. The morphological and cultural characteristics and enzymatic activity of the strains have been studied and the optimal conditions for its cultivation have been developed. We have shown the nematicidal activity of these strains against M. incognita. Experiments with the cucumber variety Courage were conducted under greenhouse conditions in 2016-2018. We determined the effect of plant damage with M. incognita to plants on the biometric parameters of underground and aboveground parts of cucumber plants, as well as on the gall formation index and yield. It was found that the treatment of plants with Bacillus strains contributed to an increase in the height of cucumber plants by 7.4-43.1%, an increase in leaf area by 2.7-17.8%, and an increase in root mass by 3.2-16.1% compared with the control plants without treatment. The application of these strains was proved to contribute to an increase in yield by 4.6-45.8% compared to control. Our experiments suggest that the treatment of cucumber plants with two Bacillus strains improved plant health and crop productivity in the greenhouse. B. velezensis BZR 86 and B. velezensis BZR 277 may form the basis for bionematicides to protect cucumber plants from the root-knot nematode M. incognita.

Expression of α-Tubulin Acetyltransferase 1 and Tubulin Acetylation as Selective Forces in Cell Competition.

The wound healing response of fibroblasts critically depends on the primary cilium, a sensory organelle protruding into the environment and comprising a stable axonemal structure. A characteristic marker for primary cilia is acetylation of axonemal tubulin. Although formation of primary cilia is under cell cycle control, the environmental cues affecting ciliation are not fully understood. Our purpose was, therefore, to study the impact of culture conditions on cilia formation in NIH3T3 fibroblasts. We quantified ciliation in different NIH3T3 sub-cell lines and culture conditions by immunodetection of primary cilia and counting. Quantitative Western blotting, qRT-PCR, and proliferation assays completed our investigation. We observed large differences between NIH3T3 sub-cell lines in their ability to generate acetylated primary cilia that correlated with cytoplasmic tubulin acetylation. We found no increased activity of the major tubulin deacetylase, HDAC6, but instead reduced expression of the α-tubulin acetyltransferase 1 (Atat1) as being causative. Our observations demonstrate that cells with reduced expression of Atat1 and tubulin acetylation proliferate faster, eventually displacing all other cells in the population. Expression of Atat1 and tubulin acetylation are therefore selective forces in cell competition.

[Novel angucycline/angucyclinone family of natural products discovered between 2010 and 2020].

Angucyclines/angucyclinones are a large group of polycyclic aromatic polyketides and their producers are widely distributed in nature. This family of natural products attracts great attention because of their diverse biological activities and unique chemical structures. With the development of synthetic biology and the exploitation of the actinomycetes from previously unexplored environments, angucyclines/angucyclinones-like natural products with new skeletons were continuously discovered, thus enriching the structural diversity of this family. In this review we summarize the new angucyclines/angucyclinones analogues discovered in the last decade (2010-2020) by using different strategies, such as changing cultivation conditions, genetic modification, genome mining, bioactivity-guided compound isolation, and fermentation of actinomycetes from underexplored environments. We also discuss the role of synthetic biology in the discovery and development of new compounds of the angucycline/angucyclinone family.

Hydroponic cultivation conditions allowing the reproducible investigation of poplar root suberization and water transport.

BACKGROUND: With increasing joint research cooperation on national and international levels, there is a high need for harmonized and reproducible cultivation conditions and experimental protocols in order to ensure the best comparability and reliability of acquired data. As a result, not only comparisons of findings of different laboratories working with the same species but also of entirely different species would be facilitated. As Populus is becoming an increasingly important genus in modern science and agroforestry, the integration of findings with previously gained knowledge of other crop species is of high significance. RESULTS: To ease and ensure the comparability of investigations of root suberization and water transport, on a high degree of methodological reproducibility, we set up a hydroponics-based experimental pipeline. This includes plant cultivation, root histochemistry, analytical investigation, and root water transport measurement. A 5-week-long hydroponic cultivation period including an optional final week of stress application resulted in a highly consistent poplar root development. The poplar roots were of conical geometry and exhibited a typical Casparian band development with subsequent continuously increasing suberization of the endodermis. Poplar root suberin was composed of the most frequently described suberin substance classes, but also high amounts of benzoic acid derivatives could be identified. Root transport physiology experiments revealed that poplar roots in this developmental stage have a two- to tenfold higher hydrostatic than osmotic hydraulic conductivity. Lastly, the hydroponic cultivation allowed the application of gradually defined osmotic stress conditions illustrating the precise adjustability of hydroponic experiments as well as the previously reported sensitivity of poplar plants to water deficits. CONCLUSIONS: By maintaining a high degree of harmonization, we were able to compare our results to previously published data on root suberization and water transport of barley and other crop species. Regarding hydroponic poplar cultivation, we enabled high reliability, reproducibility, and comparability for future experiments. In contrast to abiotic stress conditions applied during axenic tissue culture cultivation, this experimental pipeline offers great advantages including the growth of roots in the dark, easy access to root systems before, during, and after stress conditions, and the more accurate definition of the developmental stages of the roots.

Data on proteome of Mycoplasma hominis cultivated with arginine or thymidine as a carbon source.

Mycoplasma hominis is an opportunistic bacterium that can cause acute and chronic infections of the urogenital tract. This bacterium, like all other Mycoplasma species, is characterized by the reduced genome size, and, consequently, reduction of the main metabolic pathways. M. hominis cells cannot effectively use glucose as a carbon and energy source. Therefore, the main pathway of energy metabolism is the arginine dihydrolase pathway. However, several bacteria can use nucleosides as the sole energy source. Biochemical studies using Salmonella typhimurium have shown that three enzymes (thymidine phosphorylase, phosphopentose mutase and deoxyribose-phosphate aldolase) are involved in the thymidine catabolic pathway. All these enzymes are present in M. hominis. For understanding changes in the energy metabolism of M. hominis we performed shotgun proteome analysis of M. hominis cells in liquid medium with arginine or thymidine as a carbon source. LC-MS analysis was performed with an Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Data are available via ProteomeXchange with identifier PXD018714 (https://www.ebi.ac.uk/pride/archive/projects/PXD018714).

Recent Advances in the Physiology of Spore Formation for Bacillus Probiotic Production.

Spore-forming probiotic bacteria have received a wide and constantly increasing scientific and commercial interest. Among them, Bacillus species are the most studied and well-characterized Gram-positive bacteria. The use of bacilli as probiotic products is expanding especially rapidly due to their inherent ability to form endospores with unique survivability and tolerance to extreme environments and to produce a large number of valuable metabolites coupled with their bio-therapeutic potential demonstrating immune stimulation, antimicrobial activities and competitive exclusion. Ease of Bacillus spp. production and stability during processing and storage make them a suitable candidate for commercial manufacture of novel foods or dietary supplements for human and animal feeds for livestock, especially in the poultry and aquaculture industries. Therefore, the development of low-cost and competitive technologies for the production of spore-forming probiotic bacteria through understanding physiological peculiarities and mechanisms determining the growth and spore production by Bacillus spp. became necessary. This review summarizes the recent literature and our own data on the physiology of bacilli growth and spore production in the submerged and solid-state fermentation conditions, focusing on the common characteristics and unique properties of individual bacteria as well as on several approaches providing enhanced spore formation.

Performance of nanocellulose-producing bacterial strains in static and agitated cultures with different starting pH.

The impact of strain selection and culture conditions on bacterial nanocellulose (BNC) productivity and quality was investigated by using four strains, static and agitated cultures, and an initial pH in the range 4-6. With agitation, strain DHU-ATCC-1 displayed highest productivity [1.14 g/(L × d)]. In static cultures, DHU-ZGD-1186 exhibited superior BNC yield on consumed glucose (0.79 g/g), and lowest by-product formation with respect to gluconic acids [≤0.07 g/(L × d)]. By-product formation typically decreased in the order gluconic acid >2-keto-gluconic acid >5-keto-gluconic acid, and was lowest in cultures with high initial pH. The BNC from DHU-ZGD-1186 exhibited higher average viscometric degree of polymerization (DPv), higher crystallinity index, and higher tear index. In conclusion, both strain selection and cultivation conditions had an impact on BNC productivity and properties. Productivity, DPv, crystallinity, and mechanical strength of BNC from agitated cultures could be similar to or even higher than the corresponding values for static cultures.

Tuning culturing conditions towards the production of neutral lipids from lubricant-based wastewater in open mixed bacterial communities.

Production of bacterial lipid-based biofuels using inexpensive substrates, as wastes, is an emerging approach. In this work, a selective process using carbon feast-famine cycles was applied to obtain an indigenous microbial community of hydrocarbon-degrading and lipid-accumulating bacteria, using a real lubricant-based wastewater as carbon source. In the conditions applied, the enriched bacterial community, dominated by members of the genus Rhodococcus, Pseudomonas and Acinetobacter, was able to degrade almost all hydrocarbons present in the wastewater within 24 h' incubation and to accumulate, although in low levels, triacylglycerol (TAG) (<5% of cell dry weight (CDW)) and polyhydroxyalkanoates (PHA) (3.8% ±â€¯1.1% of the CDW) as well as an unknown lipid (29% ±â€¯6% of CDW), presumably a wax ester-like compound. The influence of culture conditions, namely carbon and nitrogen concentrations (and C/N ratio) and cultivation time, on the amount and profile of produced storage compounds was further assessed using a statistical approach based on a central composite circumscribed design and surface response methodology. The regression analysis of the experimental design revealed that only nitrogen concentration and C/N ratio are significant for neutral lipid biosynthesis (p < 0.05). Maximum neutral lipid content, i.e. 33% (CDW basis), was achieved for the lowest carbon and nitrogen concentrations evaluated (10 g COD L-1 and 0.02 g N L-1). PHA accounted for less than 5% of CDW. In these conditions, neutral lipid content was mainly composed by TAG, about 70% (w/w). TAG precursors, namely monoacylglycerols (MAG), diacylglycerols (DAG) and fatty acids (FA), accounted for 22% of total neutral lipids and WE for about 7%. Nevertheless, according to the applied response surface model, further improvement of neutral lipids content is still possible if even lower nitrogen concentrations are used. The fatty acids detected in TAG extracts ranged from myristic acid (C14:0) to linoleic acid (C18:2), being the most abundant palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1). This study shows the feasibility of combining treatment of hydrocarbon contaminated wastewater, herein demonstrated for lubricant-based wastewater, with the production of bacterial neutral lipids using open mixed bacterial communities. This approach can decrease the costs associated to both processes and contribute to a more sustainable waste management and production of lipid-based biofuels.