Bioconversion Process Research Articles

Inorganic characterization of switchgrass biomass using laser-induced breakdown spectroscopy

The inorganic characterization of 74 samples of switchgrass using laser-induced breakdown spectroscopy (LIBS) was undertaken. Determination of ash and inorganic elements content in biomass materials is vital for feedstock screening for bioconversion processes. Hierarchical models using principal component analysis (PCA) and partial least square analysis (PLS) were used to determine the presence of specific elemental micronutrients that are important in determining plant health for robust biomass production. LIBS uses a 532 nm laser with 45 mJ of laser power to excite the samples of switchgrass plant material and the emission of all the elements present in the plant samples were recorded in single spectra with a wide wavelength range of 200–800 nm. The results were compared to the laboratory standard technique, e.g., ICP-OES technique, to determine the true values for major micronutrients such as, silicon (Si), potassium (K), calcium (Ca), magnesium (Mg), phosphorus (P), and sulfur (S). Our objectives were: 1) To determine the spectral features of switchgrass containing different amounts of these elements and 2) To examine the viability of this technique for determining the quality of the feedstock in terms of its inorganic composition. Cross-validation results showed that the broad-based model developed is promising for inorganics prediction in switchgrass. The LIBS validation prediction for the micronutrient elements mentioned here have been obtained. The regression coefficients for Si, were obtained to be 0.995, 0.994 for calibration and validation respectively, in case of Ca the regression coefficients were, 0.994 and 0.992 for calibration and validation. Similarly, in the case of Mg and K these were calculated to be 0.992 and 0.985, and 0.994 and 0.993 respectively. The regression coefficients are not as good as those for the elements mentioned, in case of the two elements S and P. They are 0.957, and 0.878, and 0.952 and 0.894 respectively for calibration, validation for the two elements. This demonstrates that LIBS-based techniques are inherently well suited for diverse environmental applications. Furthermore, LIBS along with PLS model can show capability in determining the viability of switchgrass as a biomass in the production of biofuels and survivability of switchgrass in processes associated with climate change. LIBS can help determining which switchgrass would be appropriate for a specific conversion process that favors low ash content overall or low value of specific inorganics.

Bioconversion of lignin and methane production from Corn cobs (Zea mays) treated by lignin-degrading bacteria

Corn cobs are one of the potential feedstocks consisting of cellulose, hemicellulose and lignin, which provide potential lignocellulose biomass to be converted into renewable energy such as biogas through anaerobic digestion (AD). However, the recalcitrant structure of corn cobs lignocellulose makes it resistant to microbial access to the cell wall, and therefore the effective pre-treatment needs to be conducted. The biological pre-treatment using lignin-degrading bacteria is one of the promising bioconversion processes which will help to break down the lignocellulose structure. This study aims to analyse the ability of bacteria, Agrobacterium sp., Lysinibacillus sphaericus and Paenibacillus sp. in degrading lignin of corn cobs and therefore will enhance the methane released from AD. The ability of bacteria to degrade lignin was observed by analysis of total reducing sugar, total soluble phenols, lignin content, and weight loss, while the methane production was determined by the biochemical methane potential (BMP). The percentage of lignin content of untreated and pre-treated corn cobs with bacteria Agrobacterium sp., L. sphaericus and Paenibacillus sp. is 18.34%; 9.66%; 11.48% and 9.06%, respectively. The methane concentration (specific methane production) produced by using inoculum of Agrobacterium sp., L. sphaericus and Paenibacillus sp. with the addition of pre-treated corn cobs are 1.79%; 1.16% and 2.51%, respectively. These results were higher than the inoculum with the addition of untreated corn cobs.

Study of the Bioconversion Process of Black Soldier Fly (Hermetia Illucens) Larvae in Decomposition of Various Variations of Organic Waste

Poor waste management will affect various aspects such as environmental pollution due to piles of garbage that disturb the community. It needs a waste treatment process and one of them is the bioconversion process. This process uses the biological agent Black Soldier Fly (Hermetia illucens). This research was conducted with three organic matter variations, namely tofu pulp, eggplant pulp, and pumpkin pulp as bioconversion media. The ability of BSF larvae in bioconversion organic waste can be seen from several parameters, namely temperature, media pH, larvae weight growth, WRI (Waste Reduction Index), survival rate, consumption substrate and feed residue. The results were analyzed by one-way ANOVA test then the Least Significant Difference (LSD) test with a significance level of 95%. The research results with different bioconversion media are significantly different results on the ability of BSF larvae in the decomposition process. Tofu pulp media show the best results compared to other media with suitable conditions for the decomposition process with a final temperature of 28.8 oC, pH 7, weight growth 28.35%, WRI 5.38%, and 80.71% substrate consumption.

A shortcut to carbon-neutral bioplastic production: Recent advances in microbial production of polyhydroxyalkanoates from C1 resources

Since the 20th century, plastics that are widely being used in general life and industries are causing enormous plastic waste problems since improperly discarded plastics barely degrade and decompose. Thus, the demand for polyhydroxyalkanoates (PHAs), biodegradable polymers with material properties similar to conventional petroleum-based plastics, has been increased so far. The microbial production of PHAs is an environment-friendly solution for the current plastic crisis, however, the carbon sources for the microbial PHA production is a crucial factor to be considered in terms of carbon-neutrality. One‑carbon (C1) resources, such as methane, carbon monoxide, and carbon dioxide, are greenhouse gases and are abundantly found in nature and industry. C1 resources as the carbon sources for PHA production have a completely closed carbon loop with much advances; i) fast carbon circulation with direct bioconversion process and ii) simple fermentation procedure without sterilization as non-preferable nutrients. This review discusses the biosynthesis of PHAs based on C1 resource utilization by wild-type and metabolically engineered microbial host strains via biorefinery processes.

Method for C/N ratio estimation using Mask R-CNN and a depth camera for organic fraction of municipal solid wastes

Fast assessment of the initial carbon to nitrogen ratio (C/N) of organic fraction of municipal solid waste (OFMSW) is an important prerequisite for automatic composting control to improve efficiency and stability of the bioconversion process. In this study, a novel approach was proposed to estimate the C/N of OFMSW, where an instance segmentation model was applied to predict the masks for the waste images. Then, by combining the instance segmentation model with the depth-camera-based volume calculation algorithm, the volumes occupied by each type of waste were obtained, therefore the C/N could be estimated based on the properties of each type of waste. First, an instance segmentation dataset including three common classes of OFMSW was built to train mask region-based convolutional neural networks (Mask R-CNN) model. Second, a volume measurement algorithm was proposed, where the measurement result of the object was derived by accumulating the volumes of small rectangular cuboids whose bottom area was calculated with the projection property. Then the calculated volume was corrected with linear regression models. The results showed that the trained instance segmentation model performed well with average precision scores AP50 = 82.9, AP75 = 72.5, and mask intersection over unit (Mask IoU) = 45.1. A high correlation was found between the estimated C/N and the ground truth with a coefficient of determination R2=0.97 and root mean square error RMSE = 0.10. The relative average error was 0.42% and the maximum error was only 1.71%, which indicated this approach has potential for practical applications. Keywords: carbon to nitrogen ratio, estimation, volume measurement, organic fraction of municipal solid waste, depth camera, instance segmentation DOI: 10.25165/j.ijabe.20211405.6382 Citation: Huang J J, Zhang H D, Xiao X, Huang J Q, Xie J X, Zhang L, et al. Method for C/N ratio estimation using Mask R-CNN and a depth camera for organic fraction of municipal solid wastes. Int J Agric & Biol Eng, 2021; 14(5): 222–229.

Fatty Acid Photodecarboxylase Is an Interfacial Enzyme That Binds to Lipid-Water Interfaces to Access Its Insoluble Substrate.

Fatty acid photodecarboxylase (FAP), one of the few natural photoenzymes characterized so far, is a promising biocatalyst for lipid-to-hydrocarbon conversion using light. However, the optimum supramolecular organization under which the fatty acid (FA) substrate should be presented to FAP has not been addressed. Using palmitic acid embedded in phospholipid liposomes, phospholipid-stabilized microemulsions, and mixed micelles, we show that FAP displays a preference for FAs present in liposomes and at the surface of microemulsions. The kinetics of adsorption onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with a higher affinity in the presence of FAs. The FAP structure reveals a potential interfacial recognition site with clusters of hydrophobic and basic residues surrounding the active site entrance. The resulting dipolar moment suggests the orientation of FAP at negatively charged interfaces. These findings provide important clues about the mode of action of FAP and the development of FAP-based bioconversion processes.

Application of bioelectrochemical systems to regulate and accelerate the anaerobic digestion processes

Anaerobic digestion (AD) serves as a potential bioconversion process to treat various organic wastes/wastewaters, including sewage sludge, and generate renewable green energy. Despite its efficiency, AD has several limitations that need to be overcome to achieve maximum energy recovery from organic materials while regulating inhibitory substances. Hence, bioelectrochemical systems (BESs) have been widely investigated to treat inhibitory compounds including ammonia in AD processes and improve the AD operational efficiency, stability, and economic viability with various integrations. The BES operations as a pretreatment process, inside AD or after the AD process aids in the upgradation of biogas (CO2 to methane) and residual volatile fatty acids (VFAs) to valuable chemicals and fuels (alcohols) and even directly to electricity generation. This review presents a comprehensive summary of BES technologies and operations for overcoming the limitations of AD in lab-scale applications and suggests upscaling and future opportunities for BES-AD systems.

Multi‐Enzymatic Cascade Reactions for the Synthesis of cis,cis‐Muconic Acid

AbstractLignin valorization allows the generation of a number of value‐added products such as cis,cis‐muconic acid (ccMA), which is widely used for the synthesis of chemicals for the production of biodegradable plastic materials. In the present work, we reported the first multi‐enzymatic, one‐pot bioconversion process of vanillin into ccMA. In details, we used four sequential reactions catalyzed by xanthine oxidase, O‐demethylase LigM (and the tetrahydrofolate‐regeneration enzyme methyl transferase MetE), decarboxylase AroY (based on the use of E. coli transformed cells) and catechol 1,2‐dioxygenase CatA. The optimized lab‐scale procedure allowed to reach, for the first time, the conversion of 5 mM vanillin into ccMA in ∼30 h with a 90% yield: this achievement represents an improvement in terms of yields and time when compared to the use of a whole‐cell system. This multi‐enzymatic system represents a sustainable alternative for the production of a high value added product from a renewable resource.magnified image

The Use of Processing Aids to Control the Water-Binding Properties of Flour Mixtures from Buckwheat Flour

The article discusses the effectiveness of adding various processing aids to determine the effectiveness of replacing a batch of wheat flour with buckwheat and its influence on such parameters as water absorption capacity of flour, formation time and dough stability during kneading. Buckwheat is a traditional agricultural crop, its processed products have a significant potential in the field of food bioconversion of raw materials. The increased interest in this crop is due to its chemical composition and a complex of useful qualities that have benifits on the human body, due to the presence of a high content of vitamins P, PP, B1, B2 and E. All this makes it possible to increase the nutritional and biological values of bakery and flour confectionery products. The specificity of the use of non-traditional types of raw materials and ingredients in this area, the peculiarities of production and the lack of balance in formulations encourage scientific and technological research. The creation of a complex food additive based on buckwheat flour as a carrier, together with enzyme preparations, makes it possible to produce functional mixtures for the production of bakery and flour confectionery products of stable quality. Also, the actual development is the process of bioconversion of buckwheat flour and the creation of dry buckwheat sourdough, which will facilitate the mass production of bakery products with a dietary preventive focus

Selective butanol production from carbon monoxide by an enriched anaerobic culture

An anaerobic mixed culture able to grow on pure carbon monoxide (CO) as well as syngas (CO, CO2 and H2), that produced unusual high concentrations of butanol, was enriched in a bioreactor with intermittent CO gas feeding. At pH 6.2, it mainly produced acids, generally acetic and butyric acid. After adaptation, under stress conditions of CO exposure at a partial pressure of 1.8 bar and low pH (e.g., 5.7), the enrichment accumulated ethanol, but also high amounts of butanol, up to 6.8 g/L, never reported before, with a high butanol/butyric acid molar ratio of 12.6, highlighting the high level of acid to alcohol conversion. At the end of the assay, both the acetic acid and ethanol concentrations decreased, with concomitant butyric acid production, suggesting C2 to C4 acid bioconversion, though this was not a dominant bioconversion process. The reverse reaction of ethanol oxidation to acetic acid was observed in the presence of CO2 produced during CO fermentation. Interestingly, butanol oxidation with simultaneous butyric acid production occurred upon production of CO2 from CO, which has to the best of our knowledge never been reported. Although the sludge inoculum contained a few known solventogenic Clostridia, the relative taxonomic abundance of the enriched sludge was diverse in Clostridia and Bacilli classes, containing known solventogens, e.g., Clostridium ljungdhalii, Clostridium ragsdalei and Clostridium coskatii, confirming their efficient enrichment. The relative abundance of unassigned Clostridium species amounted to 27% with presumably novel ethanol/butanol producers.

Lipids from Hermetia illucens, an Innovative and Sustainable Source

The exponential increase of global demand for proteins and lipids can no longer be satisfied by classical sources. High amounts of CO2 produced by intensive livestock breeding and its effects on the environment are the main factors that prevent the use of animals as primary sources for proteins and lipids, calling for the use of new sustainable sources, such as insects. The massive breeding of bioconverter insects as a feed source has been a major topic in recent years, with both economic and scientific aspects related to rearing and subsequent processing optimization. The larvae of Hermetia illucens (Diptera: Stratiomyidae) (also known as Black Soldier Fly) can be used for the eco-sustainable production of proteins and lipids with high biological and economic value. Lipids can be obtained from BSF bioconversion processes and are present in high quantities in the last instar larvae and prepupae. Fats obtained from BSF are used as animal feed ingredients, in the formulation of several products for personal care, and in biodiesel production. To enable the use of insect-derived lipids, it is important to understand how to optimize their extraction. Here, we summarize the published information on the composition, the extraction methods, and the possible applications of the BSF lipid component.

Ecological approach to the use of secondary products of pea flour and rice grain processing into protein concentrates and phytin

The process of bioconversion of the secondary product of pea flour processing into protein concentrate (serum) into fodder microbial-plant concentrate has been optimized. For this, a composition was selected from the culture of the fungus Geotrichium candidum 977 and the yeast Saccharamyces cerevisiae 121, a mathematical model of concentrate synthesis was developed in the form of an equation that adequately describes the dependence of the biomass yield on technological parameters: pH of the medium, temperature and amount of inoculum. The concentrate from the biomass had a protein mass fraction of 61.68 % of DS, from the biomass with the culture liquid - 57.90 %. Concentrates - biologically complete, the rate of essential amino acids was 107-226 %, out of 30 fatty acids, 97 % were acids that are part of animal fats, vegetable oils or marine organisms. The ratio of saturated and unsaturated acids is 1:3, the content of trans isomers is 5.1 %, omega-6 fatty acids (linoleic) is 19.73 %. The ability of symbiosis between the yeast S. cerevisiae 84/5 and the fungus Trich. cutaneum 656 has been proven. transform the components of whey remaining after the extraction of phytin from rice bran into protein biomass. The ratio of monocultures by mass fraction is 1:1, pH - 5.0…6.0, duration of growth - 72 hours, digestibility – 90 %. The protein is enriched with methionine, isoleucine, leucine, lysine. The amount of essential acids is 18-21 % higher than in concentrates obtained from individual monocultures. The use of concentrates is advisable to use in the diet of animals.

A review on bioconversion processes for hydrogen production from agro-industrial residues

Hydrogen is a renewable gas, efficient to produce energy that makes it a suitable alternative and effective solution for a carbon-free environment. Unlike other fossil fuels, combustion of hydrogen does not produce toxic compounds, such as greenhouse gases, carbon monoxide, hydrocarbons, etc., resulting in less environmental pollution. Agro-industrial residues contain several lignocelluloses that favor the growth of microorganisms to produce valuable products such as hydrogen. Of the diverse techniques in hydrogen production, bioconversion proves to be an efficient method in permuting agro-industrial residues into hydrogen. This review provides detailed information on the bioconversion processes and factors involved in hydrogen production from agro-industrial residues including different fermentation processes such as dark fermentation and photo-fermentation, and fuel cell systems such as microbial electrolysis cell and microbial fuel cell. Different pretreatment techniques to enhance the availability of lignocellulose for hydrogen production have been elaborated in this review. Various factors including pH, temperature and nutrient composition of feed, affecting the production efficiency and purity of the products during fermentation have been discussed.

Organosolv fractionation of spruce bark using ethanol–water mixtures: Towards a novel bio-refinery concept

The aim of this study was to assess the effect of hot water extraction followed by organosolv pretreatment on the enzymatic hydrolysability of spruce bark biomass. To this end, samples were pretreated at five different temperatures in the presence or not of acid catalyst. The cellulose content of pretreated biomass reached 49.6% w/w. During the enzymatic hydrolysis trials with 3% w/w dry solids, the final hydrolysis yield reached up to 70.1%, which corresponded to the release of 7.8 g/L of glucose. Whereas, the final hydrolysis yield obtained during the high-gravity enzymatic hydrolysis reached up to 43.5%. The concentration of released glucose was in range of 33.3 – 40.0 g/L with a hemicellulose sugars in a range of 5.5 – 6.6 g/L. These values are suitable for downstream bioconversion processes and represent a significant improvement over existing steam pretreatment methods.

Microbial Phosphorylation Product of Hesperetin by Bacillus subtilis BCRC 80517 Improves Oral Bioavailability in Rats.

The flavanoid hesperidin (Hsd) is one of the major polyphenols in citrus fruits. Hsd and its aglycone hesperetin (Hst) have a broad array of bioactivities; however, their low aqueous solubility and low intestinal permeability lead to their limited oral bioavailability. In the present study, we generated two water-soluble derivatives of Hst, namely, Hst 7-O-phosphate and Hst3'-O-phosphate, by a unique bioconversion process of Bacillus subtilis var. natto BCRC80517. The phosphorylated products showed superior aqueous solubility and distinct physicochemical properties compared with the original Hst. The Hst phosphate derivatives (HstPs) remained stable in simulated gastric and intestinal fluids for 240 min and could revert to the original Hst form by alkaline phosphatase treatment in Caco-2 cells, showing enhanced intestinal permeability in vitro. After oral administration in rats, HstPs greatly elevated plasma exposure to Hst and showed better bioavailability than did Hsd. HstPs may be a potential and efficient alternative to Hst.

Analysis of microalgal growth kinetic model and carbohydrate biosynthesis cultivated using agro-industrial waste residuals as carbon source

Microalgal carbohydrate is considered one of the potential feedstock for biofuel produced via the bioconversion process. However, the current cultivation process using commercial medium exhibited low biomass production and its carbohydrate productivity which become a bottleneck for sustainable microalgal-carbohydrate-based biofuel production. Thus, the objective of this study is to assess the utilization of industrial waste including molasses and glycerol on the Halochlorella rubescens and Tetraselmis suecica growth as well as its carbohydrate content under different cultivation modes such as autotrophic, heterotrophic and photoheterotrophic conditions. From this study, the highest maximum biomass of H. rubenscens and T. suecica of 0.653 ± 0.009 and 0.669 ± 0.01gL−1 were obtained when the cultivation was performed under photoheterotrophic using molasses. High carbohydrate content of H. rubescens and T. seucica of 56.81 ± 0.39% and 71.52 ± 0.03% with glucose represent the dominant sugar was observed under this condition. The growth kinetic model of the analysis indicated that Huang and Gompertz Models described well the growth of H. rubescens and T. suecica under photoheteroptroph condition with a high significant R 2 of 0.99. The information generated could be beneficial for the future development of low-cost microalgal cultivation media formulation for future microalgal carbohydrate-based products such as bioethanol.

Metabolite characterisation and profiling of Hermetia illucens L. larvae at various growth stages using Sesamum indicum residues as nutrient source

The larvae of black soldier fly (Hermetia illucens L.) (BSFL) is an excellent decomposer for industrial and agricultural applications capable of converting low-value waste into nutritional biomass. Sesame residues is industrial food waste possess high protein and lipid content. To investigate the bioconversion process of BSFL, the larvae were raised on a low-bioburden diet consisting of sesame residue, and liquid chromatography-mass spectrometry (LC-MS) analysis was performed on the BSFL and faeces at various instars. The obtained metabolomes were subsequently evaluated through principal component analysis and orthogonal projection to latent structure-discriminant analysis to explore the differential components. Diacylglycerol was the main component of the sesame residues utilised by BSFL during metabolism. The phenylalanine, arginine, oleic acid and 10-hydroxy-8E-octadecenoic acid were identified as the major components in various instars of the BSFL metabolites. We thus concluded that BSFL have different nutritional compositions at different instar stages. Another compound with a molecular weight of 278.224 prevalent in both the larvae and faeces might play vital roles in BSFL development. The present study revealed a part of metabolic changes important in the BSFL lifecycle. Through this pioneering metabolomics profiles, the discovery of highly-valuable biomolecules on BSFL for food industrial application can be anticipated.

Definitive Screening Design and Artificial Neural Network for Modeling a Rapid Biodegradation of Date Palm Fronds by a New Trichoderma sp. PWN6 into Citric Acid.

Generally, the bioconversion of lignocellulolytics into a new biomolecule is carried out through two or more steps. The current study used one-step bioprocessing of date palm fronds (DPF) into citric acid as a natural product, using a pioneer strain of Trichoderma harzianum (PWN6) that has been selected from six tested isolates based on the highest organic acid (OA) productivity (195.41 µmol/g), with the lowest amount of the released glucose. Trichoderma sp. PWN6 was morphologically and molecularly identified, and the GenBank accession number was MW78912.1. Both definitive screening design (DSD) and artificial neural network (ANN) were applied, for the first time, for modeling the bioconversion process of DPF. Although both models are capable of making accurate predictions, the ANN model outperforms the DSD model in terms of OA production, as ANN is characterized by a higher value of R2 (0.963) and validation R2 (0.967), and lower values of the RMSE (13.44), MDA (11.06), and SSE (9749.5). Citric acid was the only identified OA as was confirmed by GC-MS and UPLC, with a total of 1.5%. In conclusion, DPF together with T. harzianum PWN6 is considered an excellent new combination for citric acid biosynthesis, after modeling with artificial intelligence procedure.

Bi-phasic hydrolysis of corncobs for the extraction of total sugars and ethanol production using inhibitor resistant and thermotolerant yeast, Pichia kudriavzevii

In order to have sustainable energy and to reduce the reliance on fossil fuels, transition to renewable bioenergy is highly desired. Adequate biomass conversion to ethanol requires total carbohydrate extraction from the feedstock with minimum inhibitors. The present study aimed at revealing the fermentation potential of toxin resistant and thermotolerant Pichia kudriavzevii for ethanol production. Corncobs being one of the most abundant renewable biomass, was chosen for sequential bioconversion process to ethanol. Firstly, the biomass was pretreated using 2% NaOH which resulted in 83.12 ± 1.07% delignification. Subsequently biomass was used for biphasic hydrolysis (ultrasound assisted dilute acid followed by enzyme) which resulted in 27.03 ± 0.91 gL-1 of xylose, 0.33 ± 0.03 gL-1 furans in the first phase and 27.33 ± 0.9 gL-1 glucose in the later phase. Fermentation efficiency of P. kudriavzevii was evaluated using enzyme and acid hydrolysates (detoxified and undetoxified) which resulted in 11.98 gL-1 ethanol with glucose, 3.83 gL-1 and 3.64 gL-1 ethanol with detoxified and undetoxified acid hydrolysates with theoretical yield of 85.95, 31.89% and 28% respectively at 42 °C. Though the efficiency with xylose was low, the strain was found to be one among very few Pichia strains that have ability to ferment xylose. Furthermore, developing a potential biological process that utilizes the renewable biomass for energy development will not only save a lot of foreign exchange, but also helps in waste management in a sensible way.

Production of 7,10-dihydroxy-8(E)-octadecenoic acid using cell-free supernatant of Pseudomonas aeruginosa

Cell-free synthesis has been adopted in the bioconversion process due to its known advantages, such as fast production rate, high product content, and no substrate/product inhibition effect. In this study, the cell-free supernatant of Pseudomonas aeruginosa was used to improve the production of 7,10‐dihydroxy‐8(E)‐octadecenoic acid (DOD) from oleic acid. DOD production using cell-free supernatant demonstrated reduction in bioconversion duration and higher product concentration than conventional method using whole cell culture. The maximum DOD concentration (6.41 g/L) was obtained after 36 h of biotransformation using 1 % v/v oleic acid as a substrate with a productivity of 0.178 g/L/h and a yield of 74.8 %. DOD concentration, productivity, and yield using cell-free supernatant were 2.12, 7.12, and 2.22 times higher, respectively, than using the conventional whole cell culture method. Of the carbon and nitrogen sources used in pre-culture, galactose and sodium glutamate along with diammonium phosphate were found to be the most effective for DOD production. An incubation temperature of 27 °C and pH 8.0 were found to be most favorable for DOD production. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis demonstrated the presence of enzymes related to DOD production in the cell-free supernatant, which was substantiated by performing DOD production experiment using the supernatant enzymes extracted from protein gel bands with oleic acid as a substrate. To the best of our knowledge, this is the first report on DOD production using a cell-free supernatant and verifying the existence of the relevant enzymes in the cell-free supernatant. Compared to whole cell process, cell-free DOD production holds several advantages, including higher DOD productivity which could be beneficial for large-scale production.