Advances in sophorolipid-producing strain performance improvement and fermentation optimization technology.

We discovered that Candida floricola ZM1502 is capable of selectively producing the promising hydrophilic biosurfactants, acid-form sophorolipids (SLs), from glycerol. However, productivity was very low (approximately 3.5 g L-1) under the initial culture conditions. Here, we describe the design of culture medium for abundant production of acid-form SLs by C. floricola ZM1502 using waste glycerol and hydrophobic substrates in order to develop a method for SL production and disposal of waste glycerol produced by oleo-chemical industries. Urea provided the best nitrogen source for acid-form SL production from glycerol among four nitrogen sources tested [urea, NaNO3, NH4NO3, and (NH4)2SO4]. Among carbon sources we compared, hydrophobic substrates (soybean oil and oleic acid) led to productivities of approximately 20 g L-1, indicating that hydrophobic substrates provided fatty acid moieties for SL production. Addition of olive oil and oleic acid to waste glycerol enhanced acid-form SL production to 42.1 ± 0.9 and 37.5 ± 3.4 g L-1, respectively. To develop a potential industrial process, we explored other suitable hydrophobic substrates for SL production, which were obtained on site from oleo-chemical industries. Alkyl C18 esters (Pastell M-182), along with waste glycerol, increased acid-form SL production to 48.0 ± 3.4 g L-1 over a 7-d period. Furthermore, we demonstrated abundant production of acidic SLs at the mini-jar fermenter scale, obtaining 169 g L-1 over 180 h using a fed-batch cultivation technique. Efficient acid-form SL production by C. floricola could have a great impact on the development of bio-industrial processes using waste glycerol as a substrate.

To facilitate the development of bio-based chemicals from renewable and inexpensive natural resources, we sought to produce biosurfactants using non-edible jatropha oil. Twenty yeasts known to produce biosurfactants were tested in this study, and Stamerella bombicola NBRC 10243 was found to use jatropha oil efficiently to produce sophorolipids (SLs) as a mixture of lactone-form SL (L-SL) and acid-form SL (A-SL). Under culture conditions using rice bran as the source of organic nutrients, the yield of SLs reached 122.6 g/L in 5-L jar fermentors after 9 d in culture. HPLC analysis of the culture medium showed that the levels of phorbol esters (PEs), major toxic components of the oil, decreased markedly with an increase in culture time, suggesting that the yeast degrades PEs. Although the SLs obtained by solvent extraction of the culture medium contained a small amount of PEs, the sodium salt of A-SL (A-SL-Na) obtained by alkaline treatment (5N NaOH, 80°C) showed no PE peaks upon HPLC analysis. A-SL-Na had excellent surface activity with low CMC (9.0×10⁻⁴ M) and γ(CMC) (29.6 mN/m), which are lower than that of sodium dodecyl sulfate (SDS). The solubilizing ability of A-SL-Na toward for octanoic acid ([octanoic acid]/[A-SL-Na]) was found to be 2.0, which is half that of SDS. Our findings should help improve SL production from non-edible feedstock and broaden the use of promising bio-based surfactants.

Techno-economic evaluation of a biorefinery applying food waste for sophorolipid production – A case study for Hong Kong

Production of sophorolipids by the yeast Candida bombicola using simple and low cost fermentative media

Sophorolipids (SLs) are surface-active molecules produced by the non-pathogenic yeast Starmerella bombicola CGMCC 1576. Several genes involved in the synthesis of SLs have been identified. However, the regulation mechanism of the synthesis pathway for SLs has not been investigated. We recently discovered a protein in S. bombicola, which is structurally related to Yarrowia lipolytica YlBro1. To identify the function of the protein SbBro1 in S. bombicola, the deletion, overexpression, and complementary mutant strains were constructed. We found that the deletion mutant no longer produced SLs. Transcriptome analysis indicated that the expression levels of the key enzyme genes of SLs biosynthetic pathway were significantly down-regulated in the Δbro1, especially the expression level of cyp52m1 encoding the first rate-limiting enzyme in SL synthesis pathway was down-regulated 13-folds and the expression of fatty acid β-oxidation-related enzymes was also down-regulated. This study can give insight into the regulation of SL synthesis.

The production of sophorolipids by Candida bombicola NRRL Y- 17069 grown in a mixture of sunflower oil cake and crude soybean oil as economic substrates with different fermentation techniques was studied. The highest yield (49.5 g·100 g−1 substrates) was obtained from solid state fermentation after employing a new concept for extraction by methanol (E I) followed by ethyl acetate (E II), then partially purified with hexane (E III). The course of time of fermentation was also studied, and E I extracted of the 12th day showed the minimum surface tension (45 mN·m−1) at a critical micelle dilution (CMD) of 10% concentration. The produced sophorolipids were characterized and confirmed by FTIR and 1H NMR spectroscopy. The anticancer activity of the produced compounds was assessed against MCF-7, HepG2, A549, HCT116 cancer cell lines and the results revealed that E III and E IV (a mixture of E I & E III) act as promising anticancer agents in HepG2 and A549 by inhibiting urokinase and histone deacetylase activities.

Computational fluid dynamics simulation of a novel bioreactor for sophorolipid production

Solid-state fermentation (SSF) is a promising technology for bioproduct generation within a circular bioeconomy framework, but its commercial application remains limited by heat and mass transfer constraints. In this study, we present a comprehensive experimental methodology for estimating key parameters required for modelling SSF processes. The objective of this study is to establish an experimental methodology for determining model parameters and, through a sensitivity analysis, evaluate their influence on the validation of a mathematical model for SSF processes. Using sophorolipid (SL) production by Starmerella bombicola as a case study, we determine thermal properties, kinetic parameters, and transfer coefficients, and validate them through temperature profile simulations in a 22-L bioreactor. The experimentally determined values were: specific heat capacity of 1.55 J g -1 °C -1 , thermal conductivity of 0.127 W m -1 ºC -1 , heat transfer coefficient of 9790 ± 2.25 W m -3 ºC -1 , mass transfer coefficient 0.00189 ± 0.00078 s -1 , metabolic heat yield of 1.34×10 7 J kg -1 biomass and a specific growth rate of 0.389 h -1 . A sensitivity analysis identifies microbial growth kinetics and heat yield as the most critical parameters influencing model accuracy, while thermal and transport parameters show low sensitivity. This integrated approach provides both a robust modelling framework and specific values applicable to SSF-based SL production, supporting scale-up efforts and adaptation to other solid matrices or microbial systems. • Key parameters for the solid-state fermentation model were experimentally determined. • Simulated solid temperature at reactor centre agreed with experimental data. • Sensitivity analysis found growth kinetics and heat yield as most important parameters.

A rational high-throughput screening can significantly improve the efficiency of strain screening with high performance. In this study, based on the addition reaction of unsaturated fatty acids in the sophorolipids (SLs) and I2 molecules, a simple and rapid high-throughput detection method for SLs was established which demonstrated a correlation coefficient (R2) of 0.9106 with high-performance liquid chromatography (HPLC) method. Moreover, chlorpromazine, as a rational selecting pressure for enrichment of mutants with high cytochrome P450 enzyme activity, which was a key enzyme in the synthesis of SLs, was introduced into the high-throughput screening model. Consequently, with the aid of this effective screening system, a high-yielding mutant designated as Candida bombicola F6.5 was successfully screened out from 1500 single colonies, which presented improvements of 40.3% and 11.4% on SLs titer and yield, respectively, compared to the parent strain in a 1L bioreactor.

Silk fibroin (SF) protein, produced by silkworm Bombyx mori, is a promising biomaterial, while sophorolipid (SL) is an amphiphilic functional biosurfactant synthesized by nonpathogenic yeast Candida bombicola. SL is a mixture of two forms, acidic (ASL) and lactonic (LSL), which when added to SF results in accelerated gelation of silk fibroin. LSL is known to have multiple biological functionalities and hence hydrogels of these green molecules have promising applications in the biomedical sector. In this work, SANS, NMR, and rheology are employed to examine the assembling properties of individual and mixed SLs and their interactions with SF to understand the mechanism that leads to rapid gelation. SANS and NMR studies show that ASL assembles to form charged micelles, while LSL forms micellar assemblies and aggregates of a mass fractal nature. ASL and LSL together form larger mixed micelles, all of which interact differently with SF. It is shown that preferential binding of LSL to SF causes rapid unfolding of the SF chain leading to the formation of intermolecular beta sheets, which trigger fast gelation. Based on the observations, a mechanism for gelation of SF in the presence of different sophorolipids is proposed.

Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine. First, the 22.5kb staurosporine BGC was captured by CRISPR/Cas9-mediated TAR (transformation-associated recombination) from the native producer (145mg/L), and then introduced into three heterologous hosts Streptomyces avermitilis (ATCC 31267), Streptomyces lividans TK24 and Streptomyces albus J1074 to evaluate the staurosporine production capacity. The highest yield was achieved in S. albus J1074 (750mg/L), which was used for further production improvement. Next, we integrated two additional staurosporine BGCs into the chromosome of strain S-STA via two different attB sites (vwb and TG1), leading to a double increase in the production of staurosporine. And finally, optimization of fermentation process by controlling the pH and glucose feeding could improve the yield of staurosporine to 4568mg/L, which was approximately 30-fold higher than that of the native producer. This is the highest yield ever reported, paving the way for the industrial production of staurosporine. KEYPOINTS: • Streptomyces albus J1074 was the most suitable heterologous host to express the biosynthetic gene cluster of staurosporine. • Amplification of the biosynthetic gene cluster had obvious effect on improving the production of staurosporine. • The highest yield of staurosporine was achieved to 4568mg/L by stepwise increase strategy.

Protective effects of microbial biosurfactants produced by Bacillus halotolerans and Candida parapsilosis on bleomycin-induced pulmonary fibrosis in mice: Impact of antioxidant, anti-inflammatory and anti-fibrotic properties via TGF-β1/Smad-3 pathway and miRNA-326

Fermentation monitor is widely used by industries to control the process effectively. Various online method are used such as flow injection method (FIR), liquid chromatography (HPLC), infrared spectroscopy (IR), gas chromatography (GC), mass spectrometry (MS) and others developed techniques. Those instruments are quite expensive to be used by small and cottage industries e.g. “tapai”, “tempe”, “pekasam” and yoghurt industries. This paper will propose to use simple sensors/probes to gather information using certain variables such as pH value and temperature. From the collected information a pattern or a classification for fermentation bioprocess will be extracted using data mining technologies. The knowledge will be used to monitor fermentation using cheap sensor/probe and also can be used in intelligence system to predict the fermentation process for example fermentation duration and optimal fermentation condition. Data mining is a combination of statistical analysis, machine learning, and database management to extract information from large database systems. Different techniques used in data mining will produce different outcomes based on user specification. The outcomes are classification, association, clustering, prediction, estimation and deviation analysis. With this advantages, suitable data mining techniques will be used in this paper to analyse and classify the fermentation database obtained through the experiments. Certain pattern can be isolated and categorised. A prototype intelligent knowledge based system will then be developed to optimise the fermentation process. The system would be able to facilitate certain components of the fermentation for example estimation of complete fermentation cycle, fermentation status, temperature regulation, etc.

The Wickerhamiella/Starmerella (W/S) yeast clade has recently gained attention as a “treasure trove” of metabolic diversity, characterized by unusual pathways shaped through complex evolutionary events. One of their most distinctive traits is their specialized sugar metabolism, which allows them to thrive in sugar-rich environments. While their role in sugar utilization is well established, emerging evidence suggests that some W/S species can also metabolize hydrophobic compounds, indicating a broader capacity for lipid-related processes. For instance, several W/S species produce sophorolipids (SLs)—a class of glycolipids with commercial applications as sustainable biosurfactants. This ability has sparked growing interest in their potential to synthesize a diverse range of lipids, including glycolipids and dicarboxylic acids. This mini-review explores the evolution and distinctive metabolic features of the W/S clade, focusing not only on its well-characterized sugar metabolism but also on its lesser-known lipid metabolism. Particular attention is given to SL production and the expansion and diversity of cytochrome P450 family 52 enzymes within this underexplored group, emphasizing their biotechnological potential in lipid biosynthesis and their promising applications in sustainable bioprocesses.

Requirement of clean energy sources urges us to find substitutes for fossil fuels. Microorganisms provide an option to produce feedstock for biofuel production by utilizing inexpensive, renewable biomass. Rhodotorula toruloides (Rhodosporidium toruloides), a non-conventional oleaginous yeast, can accumulate intracellular lipids (single cell oil, SCO) more than 70% of its cell dry weight. At present, the SCO-based biodiesel is not a price-competitive fuel to the petroleum diesel. Many efforts are made to cut the cost of SCO by strengthening the performance of genetically modified R. toruloides strains and by valorization of low-cost biomass, including crude glycerol, lignocellulosic hydrolysates, food and agro waste, wastewater, and volatile fatty acids. Besides, optimization of fermentation and SCO recovery processes are carefully studied as well. Recently, new R. toruloides strains are developed via metabolic engineering and synthetic biology methods to produce value-added chemicals, such as sesquiterpenes, fatty acid esters, fatty alcohols, carotenoids, and building block chemicals. This review summarizes recent advances in the main aspects of R. toruloides studies, namely, construction of strains with new traits, valorization of low-cost biomass, process detection and optimization, and product recovery. In general, R. toruloides is a promising microbial cell factory for production of biochemicals.