Gluconobacter Oxydans Research Articles

Multiwalled Carbon Nanotubes and the Electrocatalytic Activity of Gluconobacter oxydans as the Basis of a Biosensor.

This paper considers the effect of multiwalled carbon nanotubes (MWCNTs) on the parameters of Gluconobacter oxydans microbial biosensors. MWCNTs were shown not to affect the structural integrity of microbial cells and their respiratory activity. The positive results from using MWCNTs were due to a decrease in the impedance of the electrode. The total impedance of the system decreased significantly, from 9000 kOhm (G. oxydans/chitosan composite) to 600 kOhm (G. oxydans/MWCNTs/chitosan). Modification of the amperometric biosensor with nanotubes led to an increase in the maximal signal from 65 to 869 nA for glucose and from 181 to 1048 nA for ethanol. The biosensor sensitivity also increased 4- and 5-fold, respectively, for each of the substrates. However, the addition of MWCNTs reduced the affinity of respiratory chain enzymes to their substrates (both sugars and alcohols). Moreover, the minimal detection limits were not reduced despite a sensitivity increase. The use of MWCNTs thus improved only some microbial biosensor parameters.

Synthesis of the alternative sweetener 5-ketofructose from sucrose by fructose dehydrogenase and invertase producing Gluconobacter strains

A promising alternative to high-calorie sugars and artificial sweeteners is the microbially produced fructose derivative 5-ketofructose (5-KF). The key enzyme for biotransformation, fructose dehydrogenase (Fdh), was overproduced in Gluconobacter (G.) oxydans and G. japonicus LMG 26773. Furthermore, the fdh genes were integrated into the chromosome of G. oxydans (G. oxydans Δmgdh::fdh). All mutants showed high fructose oxidation rates forming 5-KF. G. japonicus LMG 26773 fdh was selected for 5-KF production from the cost-efficient and renewable feedstock sucrose because the organism possessed both, a highly active Fdh and an enzyme able to cleave sucrose. However, 5-KF yield was low because the strain formed levan and consumed 5-KF in the second growth phase. Several Gluconobacter strains were screened for sucrose-hydrolyzing enzymes. One of these proteins (Inv1417) was characterized and it was found that the enzyme showed the highest specific activity compared to all mesophilic invertases described so far (Vmax = 2295 ± 243 U mg protein−1). The corresponding gene was expressed in G. oxydans Δmgdh::fdh. The results clearly indicated that both heterologously produced enzymes Fdh and Inv1417 were active in this single-strain system for 5-KF synthesis. Overall 84 ± 2% of the available fructose units of sucrose were converted to 5-KF.

Membraneless microbial biofuel cell for municipal waste water treatment

Microbial biofuel cell is the promising technology that gives possibilities to generate electricity by degradation of organic contaminations. Thus, this technology became very perspective for municipal waste water treatment to minimize energy consumption of waste water treatment plants. The main limitations of microbial biofuel cell application are related with high cost of membranes and electrodes materials. Absence of membrane and application of cheap materials with low conductivity and low surface area usually leads to decreasing of power output. In this paper we present membraneless microbial fuel cell (MFC) with cheap conductive electrodes that can be applied in future for waste water treatment. Gluconobacter Oxydans VKM V-1280 strain from All-Russian Collection of Microorganisms was used as biocatalyzer for anode. Graphite was applied as electrode material. Immobilization was provided by using mixture of poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and poly (ethyleneglycol) diglycidyl ether (PEGDE). Experiments were provided by using synthetic analog of municipal waste water. No additional chemical mediator was applied. Power output was 1.43 mcW/sm2. Electrodes were stable during biofuel cell (BFC) operation. Thus, it was demonstrated that it is possible to apply membraneless MFC for municipal waste water treatment.

Efficient glycerol transformation by resting Gluconobacter cells.

In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a cleaner and more efficient technology from those previously reported. Starting from the industrial by‐product, crude glycerol, resting cells of Gluconobacter frateurii and Gluconobacter oxydans were able to convert glycerol under batch reactor conditions in water with no other additive but for the substrate. The study of strains, biomass:solution ratio, pH, growth stage, and simplification of media composition in crude glycerol bioconversions facilitated productivities of glyceric acid of 0.03 g/L.h and 2.07 g/L.h (71.5 g/g % pure by NMR) of dihydroxyacetone (DHA). Productivities surmounted recent reported fermentative bioconversions of crude glycerol and were unprecedented for the use of cell suspended solely in water. This work proposes a novel approach that allows higher productivities, cleaner production, and reduction in water and energy consumption, and demonstrates the applicability of the proposed approach.

Identification of NAD-Dependent Xylitol Dehydrogenase from Gluconobacter oxydans WSH-003.

Gluconobacter oxydans plays an important role in the conversion of d-sorbitol to l-sorbose, which is an essential intermediate for the industrial-scale production of vitamin C. In the fermentation process, some d-sorbitol could be converted to d-fructose and other byproducts by uncertain dehydrogenases. Genome sequencing has revealed the presence of diverse genes encoding dehydrogenases in G. oxydans. However, the characteristics of most of these dehydrogenases remain unclear. Therefore, the analyses of these unknown dehydrogenases could be useful for identifying those related to the production of d-fructose and other byproducts. Accordingly, dehydrogenases in G. oxydans WSH-003, an industrial strain used for vitamin C production, were examined. A nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase, which was annotated as xylitol dehydrogenase 2, was identified, codon-optimized, and expressed in Escherichia coli BL21 (DE3) cells. The enzyme exhibited a high preference for NAD+ as the cofactor, while no activity with nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, or pyrroloquinoline quinone was noted. Although this enzyme presented high similarity with NAD-dependent xylitol dehydrogenase, it showed high activity to catalyze d-sorbitol to d-fructose. Unlike the optimum temperature and pH for most of the known NAD-dependent xylitol dehydrogenases (30–40 °C and about 6–8, respectively), those for the identified enzyme were 57 °C and 12, respectively. The values of Km and Vmax of the identified dehydrogenase toward l-sorbitol were 4.92 μM and 196.08 μM/min, respectively. Thus, xylitol dehydrogenase 2 can be useful for the cofactor-reduced nicotinamide adenine dinucleotide regeneration under alkaline conditions, or its knockout can improve the conversion ratio of d-sorbitol to l-sorbose.

Kombucha tea beverage: Microbiological characteristic, antioxidant activity, and phytochemical composition

The aim of the present study was to determine the microbial composition, antioxidant activity, and content of phytochemicals in prepared kombucha tea beverage. Microbiota was identified by MALDI-TOF mass spectrometry, antioxidant activity of beverage was tested by ABTS and phosphomolybdenum method, the total content of phytochemicals (polyphenols, flavonoids, and phenolic acids) was measured by colorimetric methods. The major phenolic acids, flavonoids, and methylxanthines were detected by high performance liquid chromatography (HPLC). Candida krusei, Sphingomonas melonis, Sphingomonas aquatilis, Brevibacillus centrosporus, and Gluconobacter oxydans were the most abundant microorganisms. Antioxidant activity of kombucha tested by ABTS and phosphomolybdenum method was 1.16 mg TEAC/ml and 2.04 mg TEAC/ml, respectively, which values were higher than in black tea 0.67 and 0.81 mg TEAC/ml, respectively. Also, content of total polyphenols (0.42 mg GAE/ ml), flavonoids (0.13 mg QE/ml), and phenolic acids (0.19 mg CAE/ml) was higher in kombucha than in black tea (0.18 mg GAE/ml; 0.02 mg QE/ml; 0.05 mg CAE/ml, respectively). Gallic, chlorogenic, syringic, and protocatechuic acids, and rutin and vitexin from flavonoids were dominant in kombucha beverage detected by HPLC. Strong difference in caffeine contents, 217.81 µg ml−1 (black tea) and 100.72 µg ml−1 (kombucha beverage), was observed. The amounts of theobromine were similar in black tea and kombucha, but theophylline was detected only in black tea in trace amount (0.52 µg ml−1).

Sustainable Bioleaching of Rare Earth Elements from Industrial Waste Materials Using Agricultural Wastes

Agricultural waste was used as substrate for fermentation by Gluconobacter oxydans to produce lixiviant for rare earth element (REE) recovery from industrial waste materials, i.e., spent fluid cata...

Biosynthesis of α-lipoic acid in Gluconobacter oxydans increases the production of vitamin C by one-step fermentation

In a one-step fermentation system of vitamin C production with Gluconobacter oxydans and Ketogulonicigenium vulgare, a functional module of α-lipoic acid biosynthesis was constructed in G. oxydans. The engineered G. oxydans was co-cultured with K. vulgare to enhance the growth and 2-keto-L-gulonic acid (2-KGA) production of K. vulgare. This one-step fermentation system alleviated the growth inhibition during the mono-culture of K. vulgare and strengthened the interaction between the two bacteria. Moreover, the yield of vitamin C precursor (2-KGA) increased to 73.34 g/L (the control group was 59.09 g/L), and the conversion of D-sorbitol to 2-KGA increased to 86.0%. This study provides a new idea for further optimizing the one-step fermentation system of vitamin C production.

Synthetic cell-cell communication in a three-species consortium for one-step vitamin C fermentation.

A three-species consortium for one-step fermentation of 2-keto-L-gulonic acid (2-KGA) was constructed to better strengthen the cell-cell communication. And the programmed cell death module based on the LuxI/LuxR quorum-sensing (QS) system was established in Gluconobacter oxydans to reduce the competition that between G. oxydans and Ketogulonicigenium vulgare. By constructing and optimizing the core region of the promoter, which directly regulated the expression of lethal ccdB genes in QS system, IR3C achieved the best lethal effect. The consortium of IR3C- K. vulgare-Bacillus megaterium (abbreviated as 3C) achieved the highest 2-KGA titer (68.80 ± 4.18g/l), and the molar conversion rate was 80.7% within 36h in 5l fermenter. Metabolomic analysis on intracellular small molecules of consortia 3C and 1C showed that most amino acids (such as glycine, leucine, methionine and proline) and TCA cycle intermediates (such as succinic acid, fumaric acid and malic acid) were significantly affected. These results further validated that the programmed cell death module based on the LuxI/LuxR QS system in G. oxydans could also faciliate better growth and higher production of consortium 3C for one-step fermentation. We successfully constructed a novel three-species consortia for one-step vitamin C fermentation by strengthening the cell-cell communication. This will be very useful for probing the rational design principles of more complex multi-microbial consortia.

An eco-friendly biorefinery strategy for xylooligosaccharides production from sugarcane bagasse using cellulosic derived gluconic acid as efficient catalyst

A novel approach was proposed for the production of xylooligosaccharides by direct pre-hydrolysis using gluconic acid as catalyst. Maximum xylooligosaccharides (degree of polymerization 2–6) yield of 53.2% could be obtained in 60 min through 5% gluconic acid hydrolysis of sugarcane bagasse at 150 °C. Furthermore, the yield of glucose from solids following gluconic acid hydrolysis treatment was 86.2% after fed-batch enzymatic hydrolysis with 10% solids loading. Results indicated that gluconic acid pretreatment combined with enzymatic hydrolysis could be successfully applied to sugarcane bagasse substrate. Subsequently, glucose could be efficiently bio-oxidized to gluconic acid by Gluconobacter oxydans ATCC 621H with 93.1% yield, and sugarcane bagasse derived gluconic acid has been proved to be an effective catalyst for xylooligosaccharides production. In this study, xylooligosaccharides production from sugarcane bagasse by gluconic acid hydrolysis demonstrated a great potential with respect to the production of these probiotics around the world.

Development of a novel selective medium for the isolation and enumeration of acetic acid bacteria from various foods

Acetic acid bacteria (AAB) are gram-negative, obligate aerobic bacteria that produce acetic acid from alcohols or sugars. AAB are ubiquitous and found in many food products, including vinegar and wine. Owing to the lack of a suitable selective culture medium and the fastidious nature of AAB in food samples, ecological studies have been fundamentally limited for decades. Here, we developed a novel selective medium (AAB-selective agar; ABS) for AAB from food samples and evaluated its performance in comparison with glucose-yeast extract-calcium carbonate agar (GYC). In inclusivity tests, 16 species of AAB showed good growth on both ABS and GYC. Remarkably, the growth of 21 non-AAB strains was inhibited on ABS but not on GYC, suggesting the high exclusivity of the novel selective medium (p < 0.05). In artificial AAB-positive food samples, including wine, vinegar, yogurt, fruit, and kimchi, ABS provided better visual differentiation of AAB colonies, showing green colonies with a yellow halo surrounded by no competing colonies. Moreover, in ABS, a significantly greater number of AAB colonies were recovered from yogurts, fruits, and kimchi (Acetobacter aceti ATCC 15973 in yogurt, 4.24 ± 0.17 vs. 3.80 ± 0.14; Gluconobacter oxydans ATCC 15163 in fruit, 4.87 ± 0.07 vs. 4.53 ± 0.15; Gluconacetobacter hansenii ATCC 35959 in kimchi, 4.25 ± 0.14 vs. 3.85 ± 0.12; p < 0.05). Finally, a total of five novel AAB strains were isolated from 10 vinegar and 10 kimchi samples using ABS, while unsuccessful using GYC, demonstrating the superior performance of ABS in isolating new AAB strains or detecting AAB contamination.

Research of 1,3-Dihydroxyacetone Production by Overexpressing Glycerol Transporter and Glycerol Dehydrogenase

1,3-Dihydroxyacetone (DHA), a natural ketose, is widely used in the chemical, cosmetic, and pharmaceutical industries. The current method for DHA production is Gluconobacter oxydans (G. oxydans) fermentation, but the high concentration of glycerol in the fermentation broth inhibits cells growth. To overcome this obstacle, in this study, we overexpressed the glycerol transporter (GlpFp) by the use of promoters PtufB, Pgmr, Pglp1, and Pglp2 in G. oxydans 621H. The results show that the glycerol tolerances of strains overexpressing GlpF were all much better than that of the control strain. The glycerol dehydrogenase gene (Gdh) was overexpressed by the promoters PtufB and Pgdh, which increased the DHA titer by 12.7% compared with that of the control group. When GlpF and Gdh genes were co-overexpressed in G. oxydans 621H, the OD600 value of the engineered strains all increased, but the DHA titers decreased in different degrees, as compared with strains that overexpressed only Gdh. This study provides a reference for future research on DHA production.

Glutamate addition improves the activity of membrane-bound sorbitol dehydrogenase in a pyrroloquinoline quinone-dependent manner: A feasible strategy for the cost-effective fermentation of Gluconobacter oxydans

Abstract Existing industrial fermentations for Gluconobacter oxydans require a rich nitrogen source, which results in a relatively high production cost. In present study, amino acids utilization from yeast extract (YE) (20 g·L−1) was investigated, revealing that glutamate is disproportionately utilized, and consequently becomes a rate-limiting factor for the activity of membrane-bound sorbitol dehydrogenase (mSLDH). Additional supplementation of glutamate (0.1%) improved the abundance of coenzyme pyrroloquinoline quinone (PQQ) by 1.51-fold. Meanwhile, enzyme activity of mSLDH was improved by 1.21-fold, which was mainly dependent on the induction of PQQ. Partial substitution of YE (15 g·L−1) with glutamate (2 g·L−1) as nitrogen source was tested in G. oxydans fermentations and the thus-obtained resting-cells exhibited an high mSLDH activity towards the miglitol precursor N-2-hydroxyethyl-glucamine with an increased productivity (59.2 vs. 42.1 mg· (g DCW)-1 h-1) and an equally high yield (81.56% vs. 80.35%) compared with cells fermented with 25 g·L−1 YE. Taken together, the results provide a feasible strategy for economical fermentation of G. oxydans in achieving high mSLDH activity for biotransformation applications in large-scale.

Adaptive evolution of Gluconobacter oxydans accelerates the conversion rate of non-glucose sugars derived from lignocellulose biomass

Gluconobacter oxydans is capable of oxidizing various lignocellulose derived sugars into the corresponding sugar acids including glucose, xylose, arabinose, galactose and mannose, but simultaneous utilization of these sugars is difficult. This study attempted an adaptive evolution of G. oxydans by alternate transfer in inhibitors containing hydrolysate and inhibitors free hydrolysate for intensifying sugars simultaneous utilization. After 420 days’ continuous culture, the conversion rate of all non-glucose sugars significantly improved by several folds and achieved complete conversion of lignocellulose-derived sugars to the corresponding sugar acids. The significant up-regulation of mGDH gene in the adapted G. oxydans strain (more than 40-fold greater than the parental) was considered as the decisive factor for the improvement of strain performance. This evolution adaptation strategy also could be used to accelerate robust sugars utilization for other fermented strains in lignocellulose biorefinery.

Characterization of novel Gluconobacter species from fruits and fermented food products: Gluconobacter cadivus sp. nov., Gluconobacter vitians sp. nov. and Gluconobacter potus sp. nov.

Strains LMG 1744T, LMG 1745, LMG 31484T, LMG 1764T and R-71646 were isolated from rotting fruits and fermented food products. A phylogenomic analysis based on 107 single-copy core genes revealed that they grouped in a Gluconobacter lineage comprising Gluconobacter oxydans, Gluconobacter roseus, Gluconobacter sphaericus, Gluconobacter kanchanaburiensis, Gluconobacter albidus, Gluconobacter cerevisiae, Gluconobacter kondonii and Gluconobacter aidae. OrthoANIu and digital DNA hybridization analyses demonstrated that these five strains represented three novel Gluconobacter species, which could be differentiated from the type strains of closely related Gluconobacter species by multiple phenotypic characteristics. We therefore propose to classify strains LMG 1744T and LMG 1745 in the novel species Gluconobacter cadivus sp. nov., with LMG 1744T (=CECT 30141T) as the type strain; to classify strain LMG 31484T as the novel species Gluconobacter vitians sp. nov., with LMG 31484T (=CECT 30132T) as the type strain; and to classify strains LMG 1764T and R-71646 in the novel species Gluconobacter potus sp. nov., with LMG 1764T (=CECT 30140T) as the type strain.

Unique glucose oxidation catalysis of Gluconobacter oxydans constitutes an efficient cellulosic gluconic acid fermentation free of inhibitory compounds disturbance.

Toxic inhibitory compounds from lignocellulose pretreatment are the major obstacle to achieve high bioconversion efficiency in biorefinery fermentations. This study shows a unique glucose oxidation catalysis of Gluconobacter oxydans with its gluconic acid productivity free of inhibitor disturbance. The microbial experimentations and the transcriptome analysis revealed that both the activity of the membrane-bound glucose dehydrogenase and the transcription level of the genes in periplasmic glucose oxidation respiratory chain of G. oxydans were essentially not affected in the presence of inhibitory compounds. G. oxydans also rapidly converted furan and phenolic aldehyde inhibitors into the less toxic alcohols or acids. The synergy of the robust periplasmic glucose oxidation and the rapid inhibitor conversion of G. oxydans significantly elevated the efficiency of the oxidative fermentation in lignocellulose hydrolysate. The corresponding genes responsible for the conversion of furan and phenolic aldehyde inhibitors were also mined by DNA microarrays. The synergistic mechanism of G. oxydans provided an important option of metabolic modification for enhancing inhibitor tolerance of general fermentation strains.

Synergistic improvement of PQQ-dependent D-sorbitol dehydrogenase activity from Gluconobacter oxydans for the biosynthesis of miglitol precursor 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose

6-(N-hydroxyethyl) amino-6-deoxy-l-sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L−1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB, all of which presenting a great potential of their industrial application in 6NSL biosynthesis.

Specialized naphthoquinones present in Impatiens glandulifera nectaries inhibit the growth of fungal nectar microbes.

The invasion success of Impatiens glandulifera (Himalayan balsam) in certain parts of Europe and North America has been partially attributed to its ability to compete for bee pollinators with its rich nectar and due to its capacity to produce and release allelopathic 1,4‐naphthoquinones (1,4‐NQs) from its roots and leaves. Given that other 1,4‐NQs present in the digestive fluids of certain carnivorous plants are proposed to control microbial colonization, we investigated the potential for the 1,4‐NQs, 2‐methoxy‐1,4‐naphthoquinone (2‐MNQ) and lawsone, to fulfill an analogous role in the nectaries of I. glandulifera. Both 2‐MNQ and lawsone were detected in the floral nectaries of I. glandulifera at levels comparable to leaves and roots, but were discovered to be at significantly higher levels in its extra‐floral nectaries (EFNs) and to be present in EFN nectar itself. Nectar microbe inhibition assays revealed that the common nectar bacteria Gluconobacter oxydans and Asaia prunellae are not inhibited by 2‐MNQ or lawsone, although both compounds were found to inhibit the growth of the common fungal nectar microbes Metschnikowia reukaufii and Aureobasidium pullulans. Taken together, these findings suggest that 2‐MNQ and lawsone could serve to protect the rich nectar of I. glandulifera against fungal growth. The high abundance of 2‐MNQ and lawsone in I. glandulifera EFNs may also point to an unsuspected mechanism for how allelopathic 1,4‐NQs are leached into the soil where they exhibit their known allelopathic effects.

L-Erythrulose production with a multideletion strain of Gluconobacter oxydans.

Many ketoses or organic acids can be produced by membrane-associated oxidation with Gluconobacter oxydans. In this study, the oxidation of meso-erythritol to L-erythrulose was investigated with the strain G. oxydans 621HΔupp BP.8, a multideletion strain lacking the genes for eight membrane-bound dehydrogenases. First batch biotransformations with growing cells showed re-consumption of L-erythrulose by G. oxydans 621HΔupp BP.8 in contrast to resting cells. The batch biotransformation with 2.8gL-1 resting cells of G. oxydans 621HΔupp BP.8 in a DO-controlled stirred-tank bioreactor resulted in 242gL-1 L-erythrulose with a product yield of 99% (w/w) and a space-time yield of 10gL-1h-1. Reaction engineering studies showed substrate excess inhibition as well as product inhibition of G. oxydans 621HΔupp BP.8 in batch biotransformations. In order to overcome substrate inhibition, a continuous membrane bioreactor with full cell retention was applied for meso-erythritol oxidation with resting cells of G. oxydans 621HΔupp BP.8. At a mean hydraulic residence time of 2h, a space-time yield of 27gL-1h-1 L-erythrulose was achieved without changing the product yield of 99% (w/w) resulting in a cell-specific product yield of up to 4.4gPgX-1 in the steady state. The product concentration (54gL-1 L-erythrulose) was reduced in the continuous biotransformation process compared with the batch process to avoid product inhibition.

Dihydroxyacetone production from glycerol using Gluconobacter oxydans: Study of medium composition and operational conditions in shaken flasks.

The production of dihydroxyacetone from glycerol employing aerobic cultures of Gluconobacter oxydans is studied. Dihydroxyacetone is one of the most important value-added products obtained from glycerol, a by-product of biodiesel production. The effect of organic nitrogen source and initial substrate concentrations has been studied together with the possibility of product inhibition. Afterward, the influence of the main operating conditions (temperature, shaking speed, and initial biomass concentration) on in vivo glycerol dehydrogenase activity has also been considered. The results show no evidence of glycerol inhibition, but an important product inhibition was detected, which has been taken into account in a kinetic model for enzymatic activity description. In terms of operating conditions, pH was found to exert a great impact on glycerol conversion, being necessary to keep it above 4 to ensure complete glycerol conversion. The minimum temperature that maximized enzymatic activity was found to be 30°C. In addition, a surprising decoupling between biomass concentration and dihydroxyacetone production rate was observed when adding increasing nitrogen source concentrations at a fixed shaking speed. Glycerol dehydrogenase activity remains constant despite the increase in biomass concentration, contrary to what would be expected. This fact revealed the existence of a rate limiting factor, identified subsequently as oxygen transfer rate depending on the biomass concentration.