Dry Cell Mass Research Articles

Monitoring reactivation of latent HIV by label-free gradient light interference microscopy

SummaryHuman immunodeficiency virus (HIV) can infect cells and take a quiescent and nonexpressive state called latency. In this study, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that the mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-α, are higher for latent cells that reactivate than those of the cells that did not reactivate. Cells with mean dry mass and diameter less than approximately 10 pg and 8 μm, respectively, remain exclusively in the latent state. Also, cells with mean dry mass greater than approximately 28-30 pg and mean diameter greater than 11–12 μm have a higher probability of reactivating. This study is significant as it presents a new label-free approach to quantify latent reactivation of a virus in single cells.

Quantitative phase microscopy monitors subcellular dynamics in single cells exposed to nanosecond pulsed electric fields.

A substantial body of literature exists to study the dynamics of single cells exposed to short duration (<1μs), high peak power (~1MV/m) transient electric fields. Much of this research is limited to traditional fluorescence-based microscopy techniques, which introduce exogenous agents to the culture and are only sensitive to a single molecular target. Quantitative phase imaging (QPI) is a coherent imaging modality which uses optical path length as a label-free contrast mechanism, and has proven highly effective for the study of single-cell dynamics. In this work, we introduce QPI as a useful imaging tool for the study of cells undergoing cytoskeletal remodeling after nanosecond pulsed electric field (nsPEF) exposure. In particular, we use cell swelling, dry mass and disorder strength measurements derived from QPI phase images to monitor the cellular response to nsPEFs. We hope this demonstration of QPI's utility will lead to a further adoption of the technique for the study of directed energy bioeffects.

Label-Free and Quantitative Dry Mass Monitoring for Single Cells during In Situ Culture.

Quantitative measurement of single cells can provide in-depth information about cell morphology and metabolism. However, current live-cell imaging techniques have a lack of quantitative detection ability. Herein, we proposed a label-free and quantitative multichannel wide-field interferometric imaging (MWII) technique with femtogram dry mass sensitivity to monitor single-cell metabolism long-term in situ culture. We demonstrated that MWII could reveal the intrinsic status of cells despite fluctuating culture conditions with 3.48 nm optical path difference sensitivity, 0.97 fg dry mass sensitivity and 2.4% average maximum relative change (maximum change/average) in dry mass. Utilizing the MWII system, different intrinsic cell growth characteristics of dry mass between HeLa cells and Human Cervical Epithelial Cells (HCerEpiC) were studied. The dry mass of HeLa cells consistently increased before the M phase, whereas that of HCerEpiC increased and then decreased. The maximum growth rate of HeLa cells was 11.7% higher than that of HCerEpiC. Furthermore, HeLa cells were treated with Gemcitabine to reveal the relationship between single-cell heterogeneity and chemotherapeutic efficacy. The results show that cells with higher nuclear dry mass and nuclear density standard deviations were more likely to survive the chemotherapy. In conclusion, MWII was presented as a technique for single-cell dry mass quantitative measurement, which had significant potential applications for cell growth dynamics research, cell subtype analysis, cell health characterization, medication guidance and adjuvant drug development.

Efficient production of polyhydroxybutyrate from slaughterhouse waste using a recombinant strain of Cupriavidus necator DSM 545

Slaughterhouse residues are greatly available and can pose a threat to the environment if not disposed of correctly. Such by-products can be proficiently processed into polyhydroxyalkanoates by accurately selected and developed bacterial strains. Cupriavidus necator DSM 545, one of the most efficient polyhydroxyalkanoates-producing strain, cannot grow well on fatty substrates. In this work, a recombinant lipolytic C. necator microbe was developed for the efficient conversion of slaughtering by-products into polyhydroxyalkanoates. Two lipase sequences, lipC and lipH of Pseudomonas stutzeri BT3, were effectively expressed in C. necator DSM 545. The engineered strain C. necator DSM 545 JR11, selected for the outstanding extracellular lipolytic activity, produced high levels of polyhydroxyalkanoates (nearly 65% of cell dry mass) from udder, jowl and membrane caul fat. This research is crucial to the cost-effective one-step processing of slaughterhouse waste into polyhydroxyalkanoates with useful applications in several industrial and medical sectors.

Study of Growth Kinetics of High Lipid Content Algae in Local Environment

In recent years algae has been receiving a lot of interest as an alternate source of biofuel. Fast growth and rich lipid content make algae a great candidate for producing eco-friendly and non-toxic biodiesel. The strain Chlorella vulgaris, has shown great potential as a source of oil. To optimize the mass culture of microalgae Chlorella vulgaris, it is necessary to develop growth kinetic model of Chlorella vulgaris. This work presents a comparative study on growth kinetics of Chlorella vulgaris in batch cultures in four different media, namely- CH, modified CH, Bold’s Basal (BB), and modified Bold’s Basal (BB). Spectroscopic analysis was supported by visual change of color in the algal biomass solution and the microscopic imaging, and was validated with dry cell mass measurement in this study. The data were analyzed to produce growth curves to find exponential phase for each medium. Exponential phases are the best period to harvest the culture. Hence the duration of exponential phase and the final biomass concentration at the end of exponential phase are two important parameters to determine the most suitable medium for mass culture. In this study the overall specific growth, doubling time and biomass productivity of Chlorella vulgaris for CH, modified CH, BB and modified BB media were measured. This study will help to find a suitable media to culture Chlorella vulgaris, a high lipid content algae in the local environment. The information can be used to design a system for mass culture of the strain for biodiesel production.&#x0D; Chemical Engineering Research Bulletin 21(2020) 133-138

Partially Coherent Optical Diffraction Tomography Toward Practical Cell Study

Optical diffraction tomography (ODT) is a computational imaging technique based on refractive index (RI) contrast. Its application for microscopic imaging of weakly absorbing and scattering samples has been demonstrated by using a specially designed holographic microscope with angular scanning of the coherent sample illumination direction. Recently, an alternative low cost technique based on partially coherent sample illumination (PC-ODT), which is compatible with the conventional wide-field transmission microscope, has been established. In this case, the 3D refractive index distribution of the sample is obtained by deconvolution from a single stack of through-focus intensity images. The performance of PC-ODT has been successfully tested on various fixed specimens (diatom frustule and biological cells) and moving bacteria. Here, we demonstrate that the PC-ODT is an efficient tool for the analysis of living eukaryotic cell dynamics at short- and long-term periods. The COS-7 cells, which hail from the African green monkey kidney, have been chosen for this study. A fast data acquisition setup comprising an optical scanning module can be easily attached to the microscope, and it allows observing cell 3D organelle movements and RI variations, with the required temporal resolution. In particular, a more rapid nucleoli rotation than previously reported has been found. The long-term cell monitoring during necrosis reveals significant changes in cell dry mass concentration obtained from recovered RI contrast.

Engineering the permeability of Halomonas bluephagenesis enhanced its chassis properties

Bacterial outer membrane (OM), an asymmetric lipid bilayer functioning as a self-protective barrier with reduced permeability for Gram-negative bacteria, yet wasting nutrients and energy to synthesize, has not been studied for its effect on bioproduction. Here we construct several OM-defected halophile Halomonas bluephagenesis strains to investigate the effects of OM on bioproduction. We achieve enhanced chassis properties of H. bluephagenesis based on positive cellular properties among several OM-defected strains. The OM-defected H. bluephagenesis WZY09 demonstrates better adaptation to lower salinity, increasing 28%, 30% and 12% on dry cell mass (DCM), poly(3-hydroxybutyrate) (PHB) accumulation and glucose to PHB conversion rate, respectively, including enlarged cell sizes and 21-folds reduced endotoxin. Interestingly, a poly(3-hydroxybutyrate-co-21mol%4-hydroxybutyrate) (P(3HB-co-21mol%4HB)) is produced by H. bluephagenesis WZY09 derivate WZY249, increasing 60% and 260% on polyhydroxyalkanoate (PHA) production and 4HB content, respectively. Furthermore, increased electroporation efficiency, more sensitive isopropyl β-D-1-thio-galactopyranoside (IPTG) induction, better oxygen uptake, enhanced antibiotics sensitivity and ectoine secretion due to better membrane permeability are observed if OM defected, demonstrating significant OM defection impacts for further metabolic engineering, synthetic biology studies and industrial applications.

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli

Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaAA–04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA–04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA–04, 1770 bp), was identified. Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA–04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA–04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCABA–04 and pColdTF-phaCABA–04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a YP/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCABA–04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a YP/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCABA–04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

The First Insight into Polyhydroxyalkanoates Accumulation in Multi-Extremophilic Rubrobacter xylanophilus and Rubrobacter spartanus.

Actinobacteria belonging to the genus Rubrobacter are known for their multi-extremophilic growth conditions—they are highly radiation-resistant, halotolerant, thermotolerant or even thermophilic. This work demonstrates that the members of the genus are capable of accumulating polyhydroxyalkanoates (PHA) since PHA-related genes are widely distributed among Rubrobacter spp. whose complete genome sequences are available in public databases. Interestingly, all Rubrobacter strains possess both class I and class III synthases (PhaC). We have experimentally investigated the PHA accumulation in two thermophilic species, R. xylanophilus and R. spartanus. The PHA content in both strains reached up to 50% of the cell dry mass, both bacteria were able to accumulate PHA consisting of 3-hydroxybutyrate and 3-hydroxyvalerate monomeric units, none other monomers were incorporated into the polymer chain. The capability of PHA accumulation likely contributes to the multi-extremophilic characteristics since it is known that PHA substantially enhances the stress robustness of bacteria. Hence, PHA can be considered as extremolytes enabling adaptation to extreme conditions. Furthermore, due to the high PHA content in biomass, a wide range of utilizable substrates, Gram-stain positivity, and thermophilic features, the Rubrobacter species, in particular Rubrobacter xylanophilus, could be also interesting candidates for industrial production of PHA within the concept of Next-Generation Industrial Biotechnology.

Production of Biodegradable Polymer from Agro-Wastes in Alcaligenes sp. and Pseudomonas sp.

The present study was aimed to evaluate the suitability of agro-wastes and crude vegetable oils for the cost-effective production of poly-β-hydroxybutyrate (PHB), to evaluate growth kinetics and PHB production in Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1 with these carbon substrates and to study the biodegradation of PHB accumulated by these cultures. Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1 accumulates higher amounts of PHB corn (79.90% of dry cell mass) and rice straw (66.22% of dry cell mass) medium respectively. The kinetic model suggests that the Pseudomonas sp. RZS1 follows the Monod model more closely than A. faecalis RZS4. Both the cultures degrade their PHB extract under the influence of PHB depolymerase. Corn waste and rice straw appear as the best and cost-effective substrates for the sustainable production of PHB from Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1. The biopolymer accumulated by these organisms is biodegradable in nature. The agro-wastes and crude vegetable oils are good and low-cost sources of nutrients for the growth and production of PHB and other metabolites. Their use would lower the production cost of PHB and the low-cost production will reduce the sailing price of PHB-based products. This would promote the large-scale commercialization and popularization of PHB as an ecofriendly bioplastic/biopolymer.

Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-\u03b1-glucosylglycerol

BackgroundGlucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development.ResultsExpression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h−1; 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 104 U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 103 U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction.ConclusionsCompared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis.

The Potential of Oleaginous Filamentous Fungi Isolated from Soil of Baturraden Botanical Garden, Central Java, Indonesia

The present paper introduces potential oleaginous filamentous fungi that have been isolated from the soils of Baturraden Botanical Garden, Central Java, Indonesia. Several types of filamentous fungi were isolated randomly from Baturraden Botanical Garden soils and were screened for their lipid production. Quantitative screening was done using semisynthetic mediums that contain glucose. The potential lipid producers were identified molecularly. The fatty acid profile of three isolates with high lipid content were identified using GC-MS. In the present study, four of nineteen screened fungi accumulated lipid more than 20% of dry cell mass. The lipid content found in BR.2.2, BR.2.3, BR.3.3 and BR.4.4 were 28.44%, 21.06%, 21.76% and 28.27%, respectively. Further analysis showed that three isolates with high lipid content were identified as unclassified strain (BR.2.2), Brevistachys sp. (BR.3.3) and Cerrena sp. (BR.4.4) based on ITS sequence. Isolate BR.2.2 contains 60.47% saturated fatty acid (SFA), 21.12% monounsaturated fatty acid (MUFA) and 5.0% polyunsaturated fatty acid (PUFA). Isolate BR.3.3 contains 36.85% SFA, 17.47% MUFA and 1.59% PUFA. Isolate BR.4.4 contains 70.48% SFA, 19.62% MUFA and 0.56% PUFA. It concludes that lipid produced from isolates BR.2.2, BR.3.3 and BR.4.4 are suitable and have potential for biodiesel production. Further studies to optimize the lipid production are suggested.

Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis

3-Hydroxypropionic acid (3HP), an important three carbon (C3) chemical, is designated as one of the top platform chemicals with an urgent need for improved industrial production. Halomonas bluephagenesis shows the potential as a chassis for competitive bioproduction of various chemicals due to its ability to grow under an open, unsterile and continuous process. Here, we report the strategy for producing 3HP and its copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) by the development of H. bluephagenesis. The transcriptome analysis reveals its 3HP degradation and synthesis pathways involving endogenous synthetic enzymes from 1,3-propanediol. Combing the optimized expression of aldehyde dehydrogenase (AldDHb), an engineered H. bluephagenesis strain of whose 3HP degradation pathway is deleted and that overexpresses alcohol dehydrogenases (AdhP) on its genome under a balanced redox state, is constructed with an enhanced 1.3-propanediol-dependent 3HP biosynthetic pathway to produce 154 g L−1 of 3HP with a yield and productivity of 0.93 g g−1 1,3-propanediol and 2.4 g L−1 h−1, respectively. Moreover, the strain could also accumulate 60% poly(3-hydroxybutyrate-co-32–45% 3-hydroxypropionate) in the dry cell mass, demonstrating to be a suitable chassis for hyperproduction of 3HP and P3HB3HP.

Investigation of the Physiology of the Obligate Alkaliphilic Bacillus marmarensis GMBE 72T Considering Its Alkaline Adaptation Mechanism for Poly(3-hydroxybutyrate) Synthesis.

The novel extreme obligate alkaliphilic Bacillus marmarensis DSM 21297 is known to produce polyhydroxybutyrate (PHB). However, the detailed mechanism of PHB synthesis in B. marmarensis is still unknown. Here, we investigated which metabolic pathways and metabolic enzymes are responsible for PHB synthesis in order to understand the regulatory pathway and optimize PHB synthesis in B. marmarensis. In accordance with the fact that beta-galactosidase, 3-hydroxyacyl-CoA dehydrogenase, and Enoyl-CoA hydratase together with acyl-CoA dehydrogenase and lipase were annotated in B. marmarensis according to the RAST server, we used glucose, lactose, and olive oil to understand the preferred metabolic pathway for the PHB synthesis. It was found that B. marmarensis produces PHB from glucose, lactose, and olive oil. However, the highest PHB titer and the highest amount of PHB synthesized per dry cell mass (YP/X) were achieved in the presence of lactose, as compared to glucose and olive oil. Additionally, in the absence of peptone, the amount of PHB synthesized is reduced for each carbon source. Interestingly, none of the carbon sources studied yielded an efficient PHB synthesis, and supplementation of the medium with potassium ions did not enhance PHB synthesis. According to these experimental results and the presence of annotated metabolic enzymes based on the RAST server, PHB accumulation in the cells of B. marmarensis could be improved by the level of the expression of 3-hydroxybutyryl-CoA dehydrogenase (1.1.1.157), which increases the production of NADPH. Additionally, the accumulation of 3-hydroxyacyl-CoA could enhance the production of PHB in B. marmarensis in the presence of fatty acids. To our knowledge, this is the first report investigating the regulatory system involved in the control of PHB metabolism of B. marmarensis.

Enhanced Production of Astaxanthin by Co-culture of Paracoccus haeundaensis and Lactic Acid Bacteria

This study aimed to determine the increase in astaxanthin production from Paracoccus haeundaensis by optimizing the medium conditions and co-culturing with lactic acid bacteria. In order to select a suitable strain to co-culture with P. haeundaensis, several strains were co-cultured with P. haeundaensis. Lactobacillus fermentum was selected as the optimal strain. Further to optimize the astaxanthin production in the co-culture, various medium conditions were tested and the PMF medium developed in this study was selected as the optimum medium. For the co-culture under optimal conditions, the dried cell mass and astaxanthin concentration were 1.84 ± 0.09 g/L and 821.09 ± 30.98 μg/g-dried cell weight, respectively, which was 2.5 times higher than that of the P. haeundaensis strain in the Luria-Bertani broth medium. In order to confirm the transcription level of the carotenoid biosynthesis genes such as crtE, crtB, crtI, crtY, crtZ, and crtW of P. haeundaensis in the co-culture, real-time qPCR was performed. Among the six carotenoid biosynthesis genes, crtI, crtY, and crtZ showed a significant difference in relative RNA levels. The mass culture was performed using a 5 L jar fermenter and the astaxanthin concentration was the same as that at the laboratory scale. These results may be used as reference for co-culture and astaxanthin production in carotenoid biosynthesis microorganisms.

Isolation of poly(3-hydroxybutyrate) from bacterial biomass using soap made of waste cooking oil

The aim of this work was to develop a soap-based method for the isolation of poly(3-hydroxybutyrate) from bacterial biomass. The method consisted of adding soap derived from waste cooking oil to a concentrated (25%) biomass suspension, heating and centrifugal separation. Purity above 95% could be achieved with soap:cell dry mass ratios at least 0.125 g/g, making the method comparable to other surfactant-based protocols. Molecular weights Mw of products from all experiments were between 350 and 450 kDa, being high enough for future material applications. Addition of hydrochloric acid to the wastewater led to the precipitation of soap and part of non-P3HB cell mass. The resulting precipitate was utilized as a carbon source in biomass production and increased substrate-to-P3HB conversion.

A new ultradian rhythm in mammalian cell dry mass observed by holography

We have discovered a new 4 h ultradian rhythm that occurs during the interphase of the cell cycle in a wide range of individual mammalian cells, including both primary and transformed cells. The rhythm was detected by holographic lens-free microscopy that follows the histories of the dry mass of thousands of single live cells simultaneously, each at a resolution of five minutes. It was vital that the rhythm was observed in inherently heterogeneous cell populations, thus eliminating synchronization and labeling bias. The rhythm is independent of circadian rhythm, and is temperature-compensated. We show that the amplitude of the fundamental frequency provides a way to quantify the effects of, chemical reagents on cells, thus shedding light on its mechanism. The rhythm is suppressed by proteostasis disruptors and is detected only in proliferating cells, suggesting that it represents a massive degradation and re-synthesis of protein every 4 h in growing cells.

Practical application of microsphere samples for benchmarking a quantitative phase imaging system.

Quantitative phase imaging (QPI) provides an approach for monitoring the dry mass of individual cells by measuring the optical pathlength of visible light as it passes through cells. A distinct advantage of QPI is that the measurements result in optical path length quantities that are, in principle, instrument independent. Reference materials that induce a well‐defined optical pathlength shift and are compatible with QPI imaging systems will be valuable in assuring the accuracy of such measurements on different instruments. In this study, we evaluate seven combinations of microspheres embedded in index refraction matching media as candidate reference materials for benchmarking the performance of a QPI system and as calibration standards for the optical pathlength measurement. Poly(methyl metharylate) microspheres and mineral oil were used to evaluate the range of illumination apertures, signal‐to‐noise ratios, and focus positions that allow an accurate quantitative optical pathlength measurement. The microsphere‐based reference material can be used to verify settings on an instrument that are suitable for obtaining an accurate pathlength measurement from biological cells. The microsphere/media reference material is applied to QPI‐based dry mass measurements of a population of HEK293 cells to benchmark and provide evidence that the QPI image data are accurate.

L-asparaginase Production by Leucosporidium scottii in a Bench-Scale Bioreactor With Co-production of Lipids.

L-asparaginase (ASNase) is a therapeutical enzyme used for treatment of acute lymphoblastic leukemia. ASNase products available in the market are produced by bacteria and usually present allergic response and important toxicity effects to the patients. Production of ASNase by yeasts could be an alternative to overcome these problems since yeasts have better compatibility with the human system. Recently, it was found that Leucosporidium scottii, a psychrotolerant yeast, produces ASNase. In order to advance the production of ASNase by this yeast, the present study aimed to select suitable process conditions able to maximize the production of this enzyme in a bench-scale bioreactor. Additionally, the accumulation of lipids during the enzyme production process was also determined and quantified. Experiments were carried out with the aim of selecting the most appropriate conditions of initial cell concentration (1.0, 3.5, and 5.6 g L–1), carbon source (sucrose and glycerol, individually or in mixture) and oxygen transfer rate (kLa in the range of 1.42–123 h–1) to be used on the production of ASNase by this yeast. Results revealed that the enzyme production increased when using an initial cell concentration of 5.6 g L–1, mixture of sucrose and glycerol as carbon source, and kLa of 91.72 h–1. Under these conditions, the enzyme productivity was maximized, reaching 35.11 U L–1 h–1, which is already suitable for the development of scale-up studies. Additionally, accumulation of lipids was observed in all the cultivations, corresponding to 2–7 g L–1 (32–40% of the cell dry mass), with oleic acid (C18:1) being the predominant compound (50.15%). Since the L-asparaginase biopharmaceuticals on the market are highly priced, the co-production of lipids as a secondary high-value product during the ASNase production, as observed in the present study, is an interesting finding that opens up perspectives to increase the economic feasibility of the enzyme production process.

Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Apiotrichum porosum DSM27194

Volatile fatty acids (VFA) derived from waste organics provide economical feedstocks for fuels and oleochemicals production. In this study, the valorization of VFA into microbial lipids by the oleaginous yeast Apiotrichum porosum DSM27194 was investigated. A. porosum DSM 27194 was able to assimilate different VFA mixtures containing acetic, propionic and butyric acid at different ratios for lipid production. By using a two-stage automated fed-batch fermentation strategy with glucose as initial carbon source and VFA mixtures as both carbon source and buffering agent, a dry cell mass concentration of 26.5 g/L with lipid content of 36.2% was achieved with the VFA ratio of 6:1:1 (acetic acid:propionic acid:butyric acid). Fatty acid composition profiles indicated that the lipid derived from VFA is better suited for biodiesel production compared to glucose as carbon source. Moreover, corn stover hydrolysate containing VFA was applied as raw feedstock. Excitingly, A. porosum DSM 27194 could consume all kinds of carbon source within the hydrolysate fast and simultaneously resulting in 28.0 g/L of dry cell mass containing 44.3% of lipid. This study provides a promising oleaginous yeast chassis of A. porosum DSM 27194 and an economical fermentation strategy of two-stage automated fed-batch fermentation for microbial lipid production from low-value VFA derived feedstocks.