Trace Metal Solution Research Articles

Biodiesel from Dunaliella salina Microalgae Using Base Catalyzed Transesterification – An Assessment through GC/MS, FTIR and NMR Studies

Algal biofuels are a promising renewable feedstock to produce energy that can supplement future energy demands greatly. The present study aims to utilize Dunaliella salina, a hypersaline, unicellular greenish-orange micro-algae, to produce bio-oil. F/2 nutrient media and trace metal and vitamin solution under carbon-dioxide-rich conditions were used to cultivate the microalgae. Ultrasonic extraction method at 60 Hz for 90 min isolated 650 mL of bio-oil. A single-stage based-catalyzed transesterification process with methanol and sodium hydroxide yielded 380 mL of Pure Dunaliella salina biodiesel at % an extraction efficiency of 87%. The Phytochemical screening on the cultivated Dunaliella sp. was performed to understand its feasibility to be used as a fuel for IC engines. Furthermore, the obtained biodiesel was characterized using Fourier Transform Infrared Spectrometer (FTIR), Gas Chromatography Mass Spectrometer (GCMS), and Nuclear Magnetic Resonance (NMR) spectral analysis.

Valorization of lignocellulosic waste (coconut coir) for bio-vanillin production having antioxidant and anticancer activity against human breast cancer cells (MCF-7)

The usage of agro-waste for producing natural flavor has gained research interest. Vanillin is a vital flavoring compound widely used in food industries. In the present study, Bacillus aryabhattai NCIM 5503 efficiently utilizes the temple waste coconut coir as the sole carbon source for bio-vanillin production. The most influential process variables, i.e., temperature, pH, and trace metal solution, were identified based on central composite rotary design. Response surface methodology was employed to optimize these process variables, and an enhanced bio-vanillin yield (14.78 %) was obtained. The result demonstrated that a yield of 0.528 ± 0.04 g/100 g bio-vanillin was obtained under optimum conditions (temperature 30 ℃; pH 9; trace metal solution 1.27 mL) after 72 h of fermentation. The predicted response was experimentally validated, resulting in a maximum bio-vanillin of 0.533 ± 0.03 g/100 g. High-performance thin-layer chromatography (HPTLC) of extracted bio-flavor showed a distinct fingerprinting profile similar to bio-vanillin. The bio-flavor produced by B. aryabhattai was characterized as bio-vanillin by FTIR and GC-MS. The antioxidant and anticancer potential of bio-vanillin were also studied. The result revealed that the extracted bio-vanillin has anticancer activity against human breast cancer cells (MCF-7). The experimental results showed the feasibility of SSF as an alternative for producing bio-vanillin through agro-waste valorization.

Valorization of lignin for the production of vanillin by Bacillus aryabhattai NCIM 5503

Coconut coir waste is a rich lignocellulosic biomass. The coconut coir waste generated from temples is resistant to natural degradation, and its accumulation causes environmental pollution. Ferulic acid, a vanillin precursor, was extracted from the coconut coir waste by hydro-distillation extraction. The extracted ferulic acid was used for vanillin synthesis by Bacillus aryabhattai NCIM 5503 under submerged fermentation. In the present study, the Taguchi DOE (design of experiment) software was used to optimize the fermentation process, which resulted in a 1.3 fold increase in vanillin yield (640.96 ± 0.02 mg/L), as compared to the unoptimized yield of 495.96 ± 0.01 mg/L. The optimized media for enhanced vanillin production comprised; fructose 0.75 % (w/v), beef extract 1 % (w/v), pH 9, temperature 30℃, agitation speed 100 rpm, trace metal solution 1 % (v/v), and ferulic acid 2 % (v/v). The results show that the commercial production of vanillin can be envisioned using coconut coir waste.

Screening and identification of azo dye decolorizers from mangrove rhizospheric soil.

Removal of synthetic textile dyes poses a challenge to the textile industry and a threat to the environment's flora and fauna. These dyes are recalcitrant and not very amenable to physical and chemical techniques of degradation. Hence, several studies on alternative bioremediation methods involving plants, plant roots, single microbes, or a consortium of microbes for the decolorization of dyes have been carried out. In the present study, potent bacteria for dye decolorization were isolated from rhizospheric soil of mangrove plants collected from Kamothe, Navi Mumbai, India. Of the 20 isolates obtained after enrichment, seven isolates were used for further screening of efficient decolorization ability in minimal basal media containing 10% glucose, 2.5% trace metal solution, and 0.1% of Methyl Orange (MO) dye concentration. Physiological parameters to optimize the decolorization of dye at optimum pH, temperature, and incubation time were studied for all the seven isolates. UV-vis and Fourier transform infrared spectroscopy were used to investigate dye decolorization. The seven isolates were characterized morphologically, biochemically, and molecular identification of these bacterial isolates was performed by 16S rRNA sequence analysis. The isolates were identified as Bacillus paramycoides, Pseudomonas taiwanensis, Citrobacter murliniae, Acinetobacter pitti, Exiguobacterium acetylicum, Psychrobacter celer, and Aeromonas taiwanensis. Out of these, Aeromonas taiwanensis has shown exceptional capacity by ~ 100% decolorization of azo dye in minimum time.

Growth enhancement of bioethanol-producing microbe Clostridium autoethanogenum by changing culture medium composition

The aim of this study was to optimize the culture medium to enhance the growth of Clostridium autoethanogenum, an acetogenic bacterium that converts synthesis gas to ethanol and acetic acid. In order to optimize the culture medium, four components, yeast extract, trace metal solution, vitamin solution, and Na2S·9H2O, were investigated. Yeast extract was found to have the biggest influence on growth, and a concentration of 5 g/L, 10 times higher than that in basal medium, leading to 2.3 times greater growth. At 10 times the concentration in basal medium, trace metal solution inhibited the growth of the acetogen. A change in the concentration of vitamin solution did not yield any significant difference in cell growth. Lastly, growth with Na2S·9H2O showed a tendency to decrease after an initial cell mass increase for concentrations of 0.5 g/L (basal medium condition) and 0.05 g/L. However, at 5 g/L, C. autoethanogenum grew steadily.

Impact of Phosphate, Potassium, Yeast Extract, and Trace Metals on Chitosan and Metabolite Production by Mucor indicus

In this study the effects of phosphate, potassium, yeast extract, and trace metals on the growth of Mucor indicus and chitosan, chitin, and metabolite production by the fungus were investigated. Maximum yield of chitosan (0.32 g/g cell wall) was obtained in a phosphate-free medium. Reversely, cell growth and ethanol formation by the fungus were positively affected in the presence of phosphate. In a phosphate-free medium, the highest chitosan content (0.42 g/g cell wall) and cell growth (0.66 g/g sugar) were obtained at 2.5 g/L of KOH. Potassium concentration had no significant effect on ethanol and glycerol yields. The presence of trace metals significantly increased the chitosan yield at an optimal phosphate and potassium concentration (0.50 g/g cell wall). By contrast, production of ethanol by the fungus was negatively affected (0.33 g/g sugars). A remarkable increase in chitin and decrease in chitosan were observed in the absence of yeast extract and concentrations lower than 2 g/L. The maximum chitosan yield of 51% cell wall was obtained at 5 g/L of yeast extract when the medium contained no phosphate, 2.5 g/L KOH, and 1 mL/L trace metal solution.

Enhanced Expression and Purification of a SUMO‐Metallothionein Chimeric Protein Using an Improved Fermentation Method

We have been developing a method for improving the expression and yield of Artemia metallothionein (MT). The protein has the unique ability to selectively bind heavy metals, e.g., lead, mercury, gold, and uranium. Therefore, MT could be used as a “heavy metal sponge” for the removal and/or recovery of toxic and precious metal from aqueous solutions. Preliminary evidence indicates the protein could also be used to remove metals from organic solvents. Our goal was to develop a large‐scale fermentation process to enhance the yield and simplify the purification of MT. This involved the use of an MT chimeric protein. An N‐his‐tagged SUMO (small ubiquitin‐like modifier) MT fusion construct was cloned and the recombinant protein expressed in an inducible T7 expression system in E. coli. The SUMO tag was incorporated to enhance the expression and solubility of MT. The media used for fermentation was supplemented with a trace metal solution and ammonium chloride, and fermentation was maintained at pH 7.0. There was an 8‐fold increase in collected cell mass. Purification was by affinity chromatography using Ni2+‐NTA resin. The chimeric protein retained its metal binding activity. The concentration of eluted protein was four times more than previously purified by other methods. Analysis of the eluted protein by SDS‐PAGE showed a 22 kDa protein consistent with the size of the MT fusion protein. Supported in part by a grant from CSUPERB and the CSULB Mini‐Grant Program.

Changing growth behavior of heavy‐metal tolerant bacteria: Media optimization using droplet‐based microfluidics

Overproduction of secondary metabolites requires know‐how for media optimization and design of effective screening procedures. Micro‐segmented flow was applied to test the growth characteristics of heavy‐metal tolerant soil bacteria (Bacillus sporothermodurans and Streptomyces tendae) toward changing media composition. The applied microfluidic technique was shown to be suitable for media optimization, with effects of media solutes like phosphorus and nitrogen, and a trace metal solution. For inorganic phosphate and trace metals, the synthesis of secondary metabolites had a much narrower tolerance for concentration ranges than vegetative cells. As we worked with metal‐tolerant strains, some tests were done in the presence of copper‐II ions in the bacteria‐specific critical concentration of 0.25 mM. In particular, for the Bacillus strain, the macronutrient phosphorus resulted in a threefold increase in growth with rising concentration. Copper led to a prolonged lag phase. For S. tendae, no similarly enhancing effect was observed. The trace metal solution promoted bacterial growth only in the case of S. tendae, for higher concentration ranges. For the nitrogen source, especially S. tendae responded with changes between the light scattering and the fluorescence signal. This could be a hint for the production of fluorescent secondary metabolites.

Application of response surface methodology for rapid chrysene biodegradation by newly isolated marine-derived fungus Cochliobolus lunatus strain CHR4D.

For the first time, Cochliobolus lunatus strain CHR4D, a marine-derived ascomycete fungus isolated from historically contaminated crude oil polluted shoreline of Alang-Sosiya ship-breaking yard, at Bhavnagar coast, Gujarat has been reported showing the rapid and enhanced biodegradation of chrysene, a four ringed high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH). Mineral Salt Broth (MSB) components such as ammonium tartrate and glucose along with chrysene, pH and trace metal solution have been successfully optimized by Response Surface Methodology (RSM) using central composite design (CCD). A validated, two-step optimization protocol has yielded a substantial 93.10% chrysene degradation on the 4(th) day, against unoptimized 56.37% degradation on the 14(th) day. The results depict 1.65 fold increase in chrysene degradation and 1.40 fold increase in biomass with a considerable decrement in time. Based on the successful laboratory experiments, C. lunatus strain CHR4D can thus be predicted as a potential candidate for mycoremediation of HMW PAHs impacted environments.

Optimization of expression of untagged and histidine-tagged human recombinant thrombin precursors in Escherichia coli.

The present study is focused on preparation of proper Escherichia coli expression system to ensure high yields of various modified precursors of human recombinant thrombin, a potential biopharmaceutical reagent. Two thrombin precursors, the smallest single-chain α-thrombin precursor prethrombin-2 and its shortened form prethrombin-2∆13, and their His-tagged forms were used. In order to determine the effect of the different lengths and amino acid compositions of affinity His-tag on the target protein expression level, a variety of the His-tag sequences were used. We found out that the protein expression efficiency was closely related to the codons used for encoding of amino acids of fusion histidine tag. Optimization of culture medium composition is another way to increase yield of the target protein. Suitable medium composition can ensure cell growth to high densities which is related to total yield of expressed protein. In this study, a new optimized complex medium for batch fermentation was developed. Addition of nutrients like a yeast extract and enzymatic casein hydrolysate to the defined medium components had a positive impact on protein expression, where relatively high expression level of the target protein from total amount of cellular proteins was achieved. Further, we have focused on trace element solution composition, and the optimized nickel and selenium concentrations were determined. Our results show that the composition of essential trace metal solution has a major impact not only on expression level, but it can also affect cell growth rate.

Optimization of culture media for large‐scale lutein production by heterotrophic Chlorella vulgaris

Lutein is a carotenoid with a purported role in protecting eyes from oxidative stress, particularly the high-energy photons of blue light. Statistical optimization was performed to growth media that supports a higher production of lutein by heterotrophically cultivated Chlorella vulgaris. The effect of media composition of C. vulgaris on lutein was examined using fractional factorial design (FFD) and central composite design (CCD). The results indicated that the presence of magnesium sulfate, EDTA-2Na, and trace metal solution significantly affected lutein production. The optimum concentrations for lutein production were found to be 0.34 g/L, 0.06 g/L, and 0.4 mL/L for MgSO4 ·7H2 O, EDTA-2Na, and trace metal solution, respectively. These values were validated using a 5-L jar fermenter. Lutein concentration was increased by almost 80% (139.64 ± 12.88 mg/L to 252.75 ± 12.92 mg/L) after 4 days. Moreover, the lutein concentration was not reduced as the cultivation was scaled up to 25,000 L (260.55 ± 3.23 mg/L) and 240,000 L (263.13 ± 2.72 mg/L). These observations suggest C. vulgaris as a potential lutein source.

Biosorption of trace metals from aqueous multimetal solutions by green microalgae

Two strains of green microalgae C. reinhardti and C. pyrenoidosa were examined for their biosorption of Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+ from aqueous multi-metal solutions. A wide range of biosorption capacities can be observed due to different strains of microalgae and different species of trace metals. This characteristic was ascribed to the distinct components and structures of algal cell walls and the different physicochemical properties of trace metals, such as atomic weight and ion density. C. pyrenoidosa showed higher uptake capacities than C. reinhardti and both of them had a preference for the uptake of cadmium over others in the trace metal solution, suggesting they can be a good biomaterial for biosorption of cadmium. Live microalgal cells displayed a more complex sorption process than dead microalgal cells because of cell assimilation.

Integrating syngas fermentation with the carboxylate platform and yeast fermentation to reduce medium cost and improve biofuel productivity

To ensure economic implementation of syngas fermentation as a fuel-producing platform, engineers and scientists must both lower operating costs and increase product value. A considerable part of the operating costs is spent to procure chemicals for fermentation medium that can sustain sufficient growth of carboxydotrophic bacteria to convert synthesis gas (syngas: carbon monoxide, hydrogen, and carbon dioxide) into products such as ethanol. Recently, we have observed that wild-type carboxydotrophic bacteria (including Clostridium ljungdahlii) can produce alcohols with a longer carbon chain than ethanol via syngas fermentation when supplied with the corresponding carboxylic acid precursors, resulting in possibilities of increasing product value. Here, we evaluated a proof-of-concept system to couple syngas fermentation with the carboxylate platform to both lower medium costs and increase product value. Our carboxylate platform concept consists of an open culture, anaerobic fermentor that is fed with corn beer from conventional yeast fermentation in the corn kernel-to-ethanol industry. The mixed-culture anaerobic fermentor produces a mixture of carboxylic acids at dilute concentrations within the carboxylate platform effluent (CPE). Besides providing carboxylic acid precursors, this effluent may represent an inexpensive growth medium. An elemental analysis demonstrated that the CPE lacked certain essential trace metals, but contained ammonium, phosphate, sodium, chloride, potassium, magnesium, calcium, and sulphate at required concentrations. CPE medium with the addition of a trace metal solution supported growth and alcohol production of C. ljungdahlii at similar or better levels compared with an optimized synthetic medium (modified ATCC 1754 medium). Other expensive supplements, such as yeast extract or macro minerals (ammonium, phosphate), were not required. Finally, n-butyric acid and n-caproic acid within the CPE were converted into their corresponding medium-chain alcohols n-butanol and n-hexanol.

Optimization of Endoglucanase Production from Fomitopsis pinicola Mycelia

The culture conditions to maximize the production of endoglucanase (EC 3.2.1.4) from the brown rot fungus Fomitopsis pinicola MKACC 54347 mycelia were investigated. Among the tested media for endoglucanase production, Mandel's mineral salts medium (MSM; 1% cellulose, 0.1% peptone, 0.14% , 0.03% urea, 0.2% , 0.03% , 0.03% , and 0.1% trace metal solution (19.8 mM , 13.0 mM , 12.2 mM , and 15.4 mM )) produced the highest activity of the enzyme. To optimize the medium composition for enzyme activity, the effects of various carbon, nitrogen, phosphorus, and inorganic sources were investigated in MSM. Maximal enzyme production was accomplished using a medium containing 2% carboxymethyl cellulose (CMC), 2% yeast extract, 0.2% , 0.03% , and 0.3% trace metal solution. Different physiological conditions, like incubation period and temperature, were also examined to assess their influence on enzyme production. Enzyme production from F. pinicola reached its highest level after cultivation for 8 days at . Nondenaturing polyacrylamide gel electrophoresis (PAGE), followed by the endoglucanase activity staining using CMC as the substrate, was performed to identify the endoglucanase under the culture conditions studied. Zymogram analysis of the culture supernatant revealed an endoglucanase band with a molecular mass of 52 kDa. The optimum pH and temperature for enzyme activity were and pH 5.0, respectively.

Response surface optimization of poly (3-hydroxyalkanoic acid) production using oleic acid as an alternative carbon source by Pseudomonas aeruginosa

This study aims at optimizing medium-chain-length Polyhydroxyalkanoate (MCL-PHA) production from Pseudomonas aeruginosa 13003 culture using the statistical design techniques of 2-level factorial design and RSM. Two-level factorial design with eight variables viz. oleic acid, (NH4)2PO4, incubation time, trace metal solution, MgCl2, K2HPO4, KH2PO4 and inoculum was performed to screen for nutrients that were significantly affecting MCL-PHA production. After the values for the less-influential variables were fixed, oleic acid, (NH4)2PO4, K2HPO4 and KH2PO4 having high t-values were selected to study their optimal relationship for maximal PHA production using 24 Central Composite Design. The optimal composition of the medium was found to be at 7.5 g L−1 oleic acid, 0.4 g L−1 (NH4)2PO4, 3.0 g L−1 K2HPO4 and 1.5 g L−1 KH2PO4 which gave 60% of CDW as MCL-PHA. This optimization resulted in a 1.6 times increase in the PHA content (60% mass fraction in contrast to 36%). On analysis carbon to nitrogen ratio of 19 was found to be optimum for PHA production. This is the first report on the use of CCD to improve MCL-PHA production from P. aeruginosa.

Exopolysaccharide production by Bacillus cereus GU812900, a fouling marine bacterium

The aim of the study was to isolate the exopolysaccharides (EPS) from the active biofilm bacterium Bacillus cereus GU 812900 which was isolated from the stainless steel test panel and deployed in the Vellar estuary for a period of about four weeks. The deployed stainless steel test panels when analyzed revealed a direct relationship between the rate of corrosion and the concentration of EPS that is, the rate of corrosion increased with an increase in the concentration of EPS and the period of immersion. Growth and EPS production by B. cereus strain GU812900 was studied in a basalt salt solution (BSS) containing (g/1); NaCl, 30.0; KCl 0.75; MgSO4.7H2O, 7.0; NH4Cl, 1.0; K2HPO4, 0.7; KH2PO4. 0.3; glucose, 10.0 and 1 ml of trace metal solution. The rate of EPS production in batch cultures was highest during the late log phase of the growth then stationary phase. The EPS was recovered from the culture supernatant by using ethanol precipitation – dialysis procedure. Chemical composition of EPS revealed that it is primarily composed of natural sugars, proteins, phosphates and sulphates. The EPS production was influenced by carbon, nitrogen and phosphate concentrations in the growth medium. EPS produced by B. cereus GU812900 is a heteropolysaccharide which influence the corrosion. The FTIR analysis revealed the functional groups of the EPS. The results of the study was expressed the relationship between biocorrosion and biofilm formation of the stainless steel test panels. Key words: Biofilm, exopolysaccharides (EPS), sequencing, secondary metabolite, characterization, FTIR analysis.

Impact of nickel and cobalt on biogas production and process stability during semi-continuous anaerobic fermentation of a model substrate for maize silage

The importance of nickel and cobalt on anaerobic degradation of a defined model substrate for maize was demonstrated. Five semi-continuous reactors were operated for 250 days at 35 °C and a well-defined trace metal solution was added to all reactors. Two reactors each were limited regarding the concentration of Ni2+ and Co2+, respectively, for certain time intervals. The required nickel concentration was depending on the organic loading rates (OLR) while, for example, above 2.6 g ODM L−1 d−1 nickel concentrations below 0.06 mg kg−1 FM in the process significantly decreased biogas production by up to 25% compared to a control reactor containing 0.8 mg Ni2+ kg−1 FM. Similarly, limitation of cobalt to 0.02 mg kg−1 FM decreased biogas production by about 10%. Limitations of nickel as well as cobalt lead to process instability. However, after gradual addition of nickel till 0.6 mg and cobalt till 0.05 mg kg−1 FM the OLR was again increased to 4.3 g ODM L−1 d−1 while process stability was recovered and a fast metabolisation of acetic and propionic acid was detected. An increase of nickel to 0.88 mg kg−1 FM did not enhance biogas performance. Furthermore, the increase of cobalt from 0.05 mg kg−1 FM up to 0.07 mg kg−1 FM did not exhibit a change in anaerobic fermentation and biogas production.

Characterization of an anaerobic population digesting a model substrate for maize in the presence of trace metals

The influence of a defined trace metal solution and additionally Ni2+ on anaerobic digestion of biomass was investigated. A novel synthetic model substrate was designed consisting of cellulose, starch and urea as carbon and nitrogen source in a ratio mimicking the basic composition of maize silage. Two independent batch fermentations were carried out over 21d with the synthetic model substrate in the presence of the trace metal solution. Particularly an increase in nickel concentrations (17 and 34μM) enhanced methane formation by up to 20%. This increased activity was also corroborated by fluorescence microscopy measurements based on cofactor F420. The eubacterial and methanogenic population was characterized with the single strand conformational polymorphism analysis and the amplified 16S rDNA restriction analysis of 16S rRNA genes amplified by different primer systems. Nearly the half of the analyzed bacteria were identified as Firmicutes while 70% in this phylum belonged to the class of Clostridiales and 30% to the class of Bacilli. Bacteroides and uncultured bacteria represented each a quarter of the analyzed community. Methanogenic archaea were investigated with ARDRA, too. The hydrogenotrophic Methanoculleus sp. was the dominant genus which is commonly described for maize digestion thus confirming the value of the model substrate.

Leachate chemical composition effects on OIT depletion in an HDPE geomembrane

Results of series of tests examining the effect of different chemical constituents found in municipal solid waste leachate on the degradation of a high-density polyethylene (HDPE) geomembrane are reported. Geomembrane samples were incubated in four synthetic leachates consisting of different combinations of volatile fatty acids, inorganic nutrients, trace metal solution, and surfactant at temperatures of 85°C, 70°C, 55°C, 40°C and 22°C. It is shown that the leachate should be replaced every two weeks to maximise the depletion of antioxidants from the geomembrane. Arrhenius modelling gave activation energies for antioxidant depletion of between 62.5 and 64.0 kJ/mol. The small difference in activation energies implies that the four leachates examined are similar in terms of antioxidant depletion rate. There was no evident effect of the difference in these leachates on crystallinity, MFI, or tensile properties during the testing period. However, the fastest antioxidant depletion was observed for the simplest leachate, comprising trace metals and surfactant in water. Results are also reported for a second series of tests involving 18 different immersion media with different concentrations of trace metals and surfactant. Based on an examination of solutions with pH between 4 and 10, it is found that antioxidant depletion is the fastest for relatively acidic or basic solutions and the slowest for neutral solutions. Antioxidant depletion is the most sensitive to the presence of surfactant. As the surfactant concentration increases to about 1 ml/l the majority of the effect is evident. There was no further increase in effect for any increase in concentration beyond 5 ml/l.

Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production

A chemically defined medium was optimized for the maximum biomass production of recombinant Pichia pastoris in the fermentor cultures using glycerol as the sole carbon source. Optimization was done using the statistical methods for getting the optimal level of salts, trace metals and vitamins for the growth of recombinant P. pastoris. The response surface methodology was effective in optimizing nutritional requirements using the limited number of experiments. The optimum medium composition was found to be 20g/L glycerol, 7.5g/L (NH4)2SO4, 1g/L MgSO4·7H2O, 8.5g/L KH2PO4, 1.5mL/L vitamin solution and 20mL/L trace metal solution. Using the optimized medium 11.25g DCW/L biomass was produced giving a yield coefficient of 0.55g biomass/g of glycerol in a batch culture. Chemostat cultivation of recombinant P. pastoris was done in the optimized medium at different dilution rates to determine the kinetic parameters for growth on glycerol. Maximum specific growth rate of 0.23h−1 and Monod saturation constant of 0.178g/L were determined by applying Monod model on the steady state data. Products of fermentation pathway, ethanol and acetate, were not detected by HPLC even at higher dilution rates. This supports the notion that P. pastoris cells grow on glycerol by a respiratory route and are therefore an efficient biomass and protein producers.