MICROBIAL SYNTHESIS OF RHAMNOLIPIDS BY Pseudomonas aeruginosa (ATCC 10145) ON WASTE FRYING OIL AS LOW COST CARBON SOURCE

Vegetable edible oils and fats are mainly used for frying purposes in households and the food industry. The oil undergoes degradation during frying and hence has to be replaced from time to time. Rhamnolipids are produced by microbial cultivation using refined vegetable oils as a carbon source and Pseudomonas aeruginosa (ATCC 10145). The raw material cost accounts for 10–30% of the overall cost of biosurfactant production and can be reduced by using low-cost substrates. In this research, attention was focused on the preparation of rhamnolipids, which are biosurfactants, using potential frying edible oils as a carbon source via a microbial fermentation technique. The use of low-cost substrates as a carbon source was emphasized to tilt the cost of production for rhamnolipids. The yield was 2.8 g/L and 7.5 g/L from waste frying oil before and after activated earth treatment, respectively. The crude product contained mainly dirhamnolipids, confirmed by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography–mass spectroscopy (LC-MS), and ¹H-nuclear magnetic resonance (NMR). Hence, the treatment can be used to convert waste frying oil as a low-cost substrate into a cost-effective carbon source.     

Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography–mass spectrophotometry (MS) and by high-performance liquid chromatography–MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l⁻¹. Under these conditions, the surface tension of the culture decreased to 30 mN m⁻¹, and the critical micelle concentration was 26.5 mg l⁻¹. It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.     

废弃食用油脂生物合成鼠李糖脂研究进展

碳源的成本过高限制了鼠李糖脂的工业化生产及应用,废弃食用油脂作为一种廉价易得的碳源,越来越多的研究者开始关注用它发酵生产鼠李糖脂.废弃食用油脂的种类、投加量对鼠李糖脂的产量、结构、性质均会产生影响,目前研究中用废弃食用油脂作碳源,鼠李糖脂产量最高可达24.61g/L、表面张力最低达到24mN/m、产物CMC最低可达40.19mg/L.此外,本文还总结了菌株、氮源、微量元素、pH、溶氧及培养方式等因素对废弃食用油脂生产鼠李糖脂的影响,并展望了利用废弃食用油脂生产鼠李糖脂实现产业化的重点研究方向.     

Sunflower seed oil and oleic acid utilization for the production of rhamnolipids by Thermus thermophilus HB8

The potential production of rhamnolipids was demonstrated using the thermophilic eubacterium Thermus thermophilus HB8 and sunflower seed oil or oleic acid as carbon sources. Sunflower seed oil was directly hydrolyzed by secretion of lipase and became a favorable carbon source for rhamnolipids production. Rhamnolipids levels were attainted high values, comparable to those produced by Pseudomonas strains from similar sources. Rhamnolipids synthesis in oleic acid exhibited a long period of induction, while in sunflower seed oil, the synthesis is more rapid. Glucose resulted in a more protracted period of rhamnolipids production after exhaustion of each or both carbon sources. Both mono- and di-rhamnolipids were identified by thin-layer chromatography (TLC) in the total rhamnolipids extract. The molecular composition of the produced biosurfactant was evaluated by Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and LC-MS analysis. Furthermore, secretion of rhamnolipids was confirmed on agar plates. The antimicrobial activity of rhamnolipids was detected against the bacterium Micrococcus lysodeikticus using a lysoplate assay. These results demonstrate that rhamnolipids produced in these substrates can be useful in both environmental and food industry applications by using cheap oil wastes. The alternative use of this thermophilic microorganism opens a new perspective concerning the valorization of wastes containing plant oils or frying oils to reduce the cost of rhamnolipids production.     

Oil wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI

Oil wastes were evaluated as alternative low-cost substrates for the production of rhamnolipids by Pseudomonas aeruginosa LBI strain. Wastes obtained from soybean, cottonseed, babassu, palm, and corn oil refinery were tested. The soybean soapstock waste was the best substrate, generating 11.7 g/L of rhamnolipids with a surface tension of 26.9 mN/m, a critical micelle concentration of 51.5 mg/L, and a production yield of 75%. The monorhamnolipid RhaC(10)C(10) predominates when P. aeruginosa LBI was cultivated on hydrophobic substrates, whereas hydrophilic carbon sources form the dirhamnolipid Rha(2)C(10)C(10) predominantly.     

Biotechnological processes for biodiesel production using alternative oils

As biodiesel (fatty acid methyl ester (FAME)) is mainly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. The use of waste lipids such as waste frying oils, waste fats, and soapstock has been proposed as low-cost alternative feedstocks. Non-edible oils such as jatropha, pongamia, and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast, and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstocks for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole-cell catalysts, avoiding soap production as the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered, and the purification of FAME is simplified using biological catalysts.     

Isolation and characterization of biosurfactant production under extreme environmental conditions by alkali-halo-thermophilic bacteria from Saudi Arabia

Twenty three morphologically distinct microbial colonies were isolated from soil and sea water samples, which were collected from Jeddah region, Saudi Arabia for screening of the most potent biosurfactant strains. The isolated bacteria were selected by using different methods as drop collapse test, oil displacement test, blue agar test, blood hemolysis test, emulsification activity and surface tension. The results showed that the ability of Virgibacillus salarius to grow and reduce surface tension under a wide range of pH, salinities and temperatures gives bacteria isolate an advantage in many applications such as pharmaceutical, cosmetics, food industries and bioremediation in marine environment. The biosurfactant production by V. salarius decreased surface tension and emulsifying activity (30 mN/m and 80%, respectively). In addition to reducing the production cost of biosurfactants by tested several plant-derived oils such as jatropha oil, castor oils, jojoba oil, canola oil and cottonseed oil. In this respect the feasibility to reusing old frying oil of sunflower for production rhamnolipids and sophorolipids, their use that lead to solve many ecological and industrial problems.     

Synthesis of polyhydroxyalkanoate from palm oil and some new applications

Polyhydroxyalkanoate (PHA) is a potential substitute for some petrochemical-based plastics. This biodegradable plastic is derived from microbial fermentation using various carbon substrates. Since carbon source has been identified as one of the major cost-absorbing factors in PHA production, cheap and renewable substrates are currently being investigated as substitutes for existing sugar-based feedstock. Plant oils have been found to result in high-yield PHA production. Malaysia, being the world’s second largest producer of palm oil, is able to ensure continuous supply of palm oil products for sustainable PHA production. The biosynthesis and characterization of various types of PHA using palm oil products have been described in detail in this review. Besides, by-products and waste stream from palm oil industry have also demonstrated promising results as carbon sources for PHA biosynthesis. Some new applications in cosmetic and wastewater treatment show the diversity of PHA usage. With proper management practices and efficient milling processes, it may be possible to supply enough palm oil-based raw materials for human consumption and other biotechnological applications such as production of PHA in a sustainable manner.     

From agro-industrial wastes to single cell oils: a step towards prospective biorefinery

The reserves of fossil-based fuels, which currently seem sufficient to meet the global demands, is inevitably on the verge of exhaustion. Contemporary raw material for alternate fuel like biodiesel is usually edible plant commodity oils, whose increasing public consumption rate raises the need of finding a non-edible and fungible alternate oil source. In this quest, single cell oils (SCO) from oleaginous yeasts and fungi can provide a sustainable alternate of not only functional but also valuable (polyunsaturated fatty acids (PUFA)-rich) lipids. Researches are been increasingly driven towards increasing the SCO yield in order to realize its commercial importance. However, bulk requirement of expensive synthetic carbon substrate, which inflates the overall SCO production cost, is the major limitation towards complete acceptance of this technology. Even though substrate cost minimization could make the SCO production profitable is uncertain, it is still essential to identify suitable cheap and abundant substrates in an attempt to potentially reduce the overall process economy. One of the most sought-after in-expensive carbon reservoirs, agro-industrial wastes, can be an attractive replacement to expensive synthetic carbon substrates in this regard. The present review assess these possibilities referring to the current experimental investigations on oleaginous yeasts, and fungi reported for conversion of agro-industrial feedstocks into triacylglycerols (TAGs) and PUFA-rich lipids. Multiple associated factors regulating lipid accumulation utilizing such substrates and impeding challenges has been analyzed. The review infers that production of bulk oil in combination to high-value fatty acids, co-production strategies for SCO and different microbial metabolites, and reutilization and value addition to spent wastes could possibly leverage the high operating costs and help in commencing a successful biorefinery. Rigorous research is nevertheless required whether it is PUFA-rich oil production (for competing with algal omega oils) or neutral bulk oil production (for overcoming yield limitations and managing process economy) to establish this potential source as future resource.     

Comparison between waste frying oil and paraffin as carbon source in the production of biodemulsifier by Dietzia sp. S-JS-1

In order to lower the production cost, waste frying oils were used in the biosynthesis of demulsifier by Dietzia sp. S-JS-1, which was isolated from petroleum contaminated soil. After 7 days of cultivation, the biomass concentration of the most suitable waste frying oil (WFO II) culture reached 3.78 g/L, which was 2.4 times the concentration of paraffin culture. The biodemulsifier produced with WFO II culture broke the emulsions more efficiently than that produced with paraffin culture, given the same volume ratio of carbon source in the culture medium and the same cultivation conditions. It achieved 88.3% of oil separation ratio in W/O emulsion and 76.4% of water separation ratio in O/W emulsion within 5 h. With the aid of thin layer chromatography (TLC) and Fourier transform infrared (FTIR) spectrometry, biodemulsifiers produced from both paraffin and WFO II were identified as a mixture of lipopeptide homologues. The subtle variation in the distribution of these homologues and high biomass concentration of WFO II cultures may account for the afore-mentioned good demulsification performance.     

Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils

World production of oils and fats is about 2.5 million tonnes, 75% of which are derived from plants. Most of them are used in the food industry for the manufacture of different products, or directly as salad oil. Great quantities of waste are generated by the oil and fat industries: residual oils, tallow, marine oils, soap stock, frying oils. It is well known that the disposal of wastes is a growing problem and new alternatives for the use of fatty wastes should be studied. Used frying oils, due to their composition, have great potential for microbial growth and transformation. The use of economic substrates such as hydrophobic wastes meets one of the requirements for a competitive process for biosurfactant production. In the Mediterranean countries, the most used vegetable oils are sunflower and olive oil. Here we present a screening process is described for the selection of micro-organism strains with the capacity to grow on these frying oils and accumulate surface-active compounds in the culture media. From the 36 strains screened, nine Pseudomonas strains decreased the surface tension of the medium to 34-36 mN/M; the emulsions with kerosene remained stable for three months. Two Bacillus strains accumulated lipopeptide and decreased the surface tension to 32-34 mN/m. Strain Ps. aeruginosa 47T2 was selected for further studies. The effect of nitrogen and a C/N of 8. 0 gave a final production of rhamnolipid of 2.7 g l-1 as rhamnose, and a production yield of 0.34 g g-1.     

Rhamnolipid surfactants: an update on the general aspects of these remarkable biomolecules

Pseudomonas strains are able to biosynthesize rhamnose-containing surfactants also known as rhamnolipids. These surface-active compounds are reviewed with respect to chemical structure, properties, biosynthesis, and physiological role, focusing on their production and the use of low-cost substrates such as wastes from food industries as alternative carbon sources. The use of inexpensive raw materials such as agroindustrial wastes is an attractive strategy to reduce the production costs associated with biosurfactant production and, at same time, contribute to the reduction of environmental impact generated by the discard of residues, and the treatment costs. Carbohydrate-rich substrates generated low rhamnolipid levels, whereas oils and lipid-rich wastes have shown excellent potential as alternative carbon sources.     

Polyunsaturated fatty acids production by solid-state fermentation on polyurethane foam by Mortierella alpina

Polyunsaturated fatty acids (PUFAs) are essential in healthy diets and their production is extremely important. Natural sources of PUFAs includes animal and aquatic products such as marine fish oil, however there are several limitations such as the decrease of fish stocks throughout the world. Thus, microbial oils are a preferable source of PUFAs. Herein, it was studied the production of PUFAs by Mortierella alpina under solid-state fermentation (SSF) using polyurethane foam as inert substrate and synthetic medium or lignocellulosic hydrolysate as source of C, N, and other nutrients. Several parameters of fermentation conditions were evaluated as carbon source, inductors addition, ratio C/N and temperature. The highest amount of total PUFAs per mass of solid (535.41 ± 24.12 mg/g), linoleic acid (129.66 ± 5.84 mg/g), and α-linoleic acid (401.93 ± 18.10 mg/g) were produced when the culture medium contained 20 g/L glucose, 10% (w/v) linseed oil, the C/N ratio was adjusted to 25 and the incubation temperature was 25°C for 3 days decreasing to 16°C on the remaining 4 days of fermentation. In addition, a hemicellulosic hydrolysate can be used as low-cost substrate to produce PUFAs, although the production was lower than the achieved with synthetic medium. SSF showed an interesting technology for microbial PUFAs production.     

Oleaginous yeasts for biodiesel: Current and future trends in biology and production

Production of biodiesel from edible plant oils is quickly expanding worldwide to fill a need for renewable, environmentally-friendly liquid transportation fuels. Due to concerns over use of edible commodities for fuels, production of biodiesel from non-edible oils including microbial oils is being developed. Microalgae biodiesel is approaching commercial viability, but has some inherent limitations such as requirements for sunlight. While yeast oils have been studied for decades, recent years have seen significant developments including discovery of new oleaginous yeast species and strains, greater understanding of the metabolic pathways that determine oleaginicity, optimization of cultivation processes for conversion of various types of waste plant biomass to oil using oleaginous yeasts, and development of strains with enhanced oil production. This review examines aspects of oleaginous yeasts not covered in depth in other recent reviews. Topics include the history of oleaginous yeast research, especially advances in the early 20th century; the phylogenetic diversity of oleaginous species, beyond the few species commonly studied; and physiological characteristics that should be considered when choosing yeast species and strains to be utilized for conversion of a given type of plant biomass to oleochemicals. Standardized terms are proposed for units that describe yeast cell mass and lipid production.     

Optimization and characterization of rhamnolipid production by Pseudomonas aeruginosa NY3 using waste frying oil as the sole carbon

Yield and cost are two major factors limiting the widespread use of rhamnolipids (RLs). In the present study, waste frying oil (WFO) was used as the sole carbon source to produce environmentally friendly RLs by Pseudomonas aeruginosa NY3. The Plackett–Burman design (PBD) and Box–Behnken design (BBD) methods were used to maximize the production yield of RL. The PBD results showed that the concentrations of NaNO₃, Na₂HPO₄, and trace elements were the key factors affecting the yield of RL. Furthermore, the BBD results showed that at NaNO₃, Na₂HPO₄, and trace elements concentrations were 4.95, 0.66, and 0.64 mL/L, respectively, the average RL yield reached 9.15 ± 0.52 g/L, 1.58-fold higher than that observed before optimization. Fourier transform infrared spectroscopy (FTIR) and liquid chromatography-ion trap-time of flight mass spectrometry (LCMS-IT-TOF) were used to elucidate the diversity of RL congeners. The results showed that, after optimization, the RL congener diversity increased, and the major RL constituent was converted from di-RLs (64.04%) to mono-RLs (60.44%). These results suggested that the concentrations of the components contained in the culture medium of P. aeruginosa NY3 influenced not only the yield of RL, but also its congener distribution.     

原生质体紫外诱变选育可利用廉价碳源的高产油新菌株

【目的】研究并建立利用原生质体紫外诱变技术选育可利用廉价碳源发酵的高产油新菌株的方法。【方法】采用1.5%蜗牛酶和1.0%纤维素酶混合液水解去除细胞壁得到2A00015(近平滑假丝酵母,Candida parapsilosis)的原生质体,将其放于紫外灯下诱变及再生壁培养,筛选获得可利用廉价碳源发酵的高产油酵母,并采用气相色谱质谱联用法(GC-MS)测定其脂肪酸组成。【结果】突变效果最好的突变菌株2A00015/25用葡萄糖发酵培养7 d后,其生物量、油脂产率和产油量分别为17.77 g/L、58.12%和10.32 g/L,较原始菌株分别提高了12.45%、23.32%和38.68%;利用废糖蜜发酵培养,其生物量、油脂产率和产油量分别为18.54 g/L、49.44%和9.17 g/L,较原始菌株分别提高了9.09%、21.16%和32.18%。利用废糖蜜培养其产油效率虽低于利用葡萄糖培养,但从环境保护及原材料成本的角度考虑,用废糖蜜作为碳源发酵培养产生油脂更具优势。诱变菌株利用废糖蜜发酵后产生油脂经检测含有8种脂肪酸,其脂肪酸组成与植物油近似,其中不饱和脂肪酸含量占脂肪酸总量的82.4%。【结论】通过利用原生质体紫外诱变技术,成功选育出一株新的可利用廉价碳源的高产油海洋菌株,产油率达到49.4%,提高了21.2%。     

Single cell oil production from low-cost substrates: The possibility and potential of its industrialization

Currently, single cell oils (SCO) attract much attention because of their bi-function as a supplier of functional oils and feedstock for biodiesel production. However, high fermentation costs prevent their further application, and the possibility and potential of their industrialization is suspected. Therefore, various low-cost, hydrophilic and hydrophobic substrates were utilized for SCO production. Of these substrates, lignocellulosic biomass, which is the most available and renewable source in nature, might be an ideal raw material for SCO production. Although many reviews on SCO have been published, few have focused on SCO production from low-cost substrates or evaluated the possibility and potential of its industrialization. Therefore, this review mainly presents information on SCO and its production using low-cost substrates and mostly focuses on lignocellulosic biomass. Finally, the possibility and potential of SCO industrialization is evaluated.     

Physicochemical characterization and antimicrobial properties of rhamnolipids produced by Pseudomonas aeruginosa 47T2 NCBIM 40044

Pseudomonas aeruginosa 47T2, grown in submerged culture with waste frying oil as a carbon source, produced a mixture of rhamnolipids with surface activity. Up to 11 rhamnolipid homologs (Rha-Rha-C(8)-C(10); Rha-C(10)-C(8)/Rha-C(8)-C(10);Rha-Rha-C(8)-C(12:1); Rha-Rha-C(10)-C(10); Rha-Rha-C(10)-C(12:1); Rha-C(10)-C(10); Rha-Rha-C(10)-C(12)/Rha-Rha-C(12)-C(10); Rha-C(10)-C(12:1)/Rha-C(12:1)-C(10); Rha-Rha-C(12:1)-C(12); Rha-Rha-C(10)-C(14:1); Rha-C(10)-C(12)/Rha-C(12)-C(10)) were isolated from cultures of P. aeruginosa 47T2 from waste frying oil and identified by HPLC-MS analysis. This article deals with the production, isolation, and chemical characterization of the rhamnolipid mixture RL(47T2). The physicochemical and biological properties of RL(47T2) as a new product were also studied. Its surface tension decreased to 32.8 mN/m; and the interfacial tension against kerosene to 1 mN/m. The critical micellar concentration for RL(47T2) was 108.8 mg/mL. The product showed excellent antimicrobial properties. Antimicrobial activity was evaluated according to the minimum inhibitory concentration (MIC), the lowest concentration of an antimicrobial agent that inhibits development of visible microbial growth. Low MIC values were found for bacteria Serratia marcescens (4 microg/mL), Enterobacter aerogenes (8 microg/mL), Klebsiella pneumoniae (0.5 microg/mL), Staphylococcus aureus and Staphylococcus epidermidis (32 microg/mL), Bacillus subtilis (16 microg/mL), and phytopathogenic fungal species: Chaetonium globosum (64 microg/mL), Penicillium funiculosum (16 microg/mL), Gliocadium virens (32 microg/mL) and Fusarium solani (75 microg/mL).     

产油真菌在甘薯淀粉废水中发酵的初步研究

目的:利用甘薯淀粉废水发酵低成本获取微生物油脂。方法:以甘薯淀粉废水为发酵基质,进行菌株筛选、发酵工艺优化及油脂成分的气相色谱分析。结果:筛选出一株刺孢小克银汉霉F7,生物量为19.375g/L,含油量为45.1%。菌株F7发酵第11天生物量达到18.140g/L,含油量达到51.2%,COD去除率87%。研究发现与对照相比,NaAc和KH2PO4对生长、产油以及出水COD去除有显著促进作用,NaAc2g/L时生物量提高了25.4%,含油量提高了4.4%,COD下降了52.0%,KH2PO4的作用稍次之。结论:资源化利用甘薯淀粉废水发酵生产微生物油脂同时降低废水COD是一条可行的途径,可以为生物柴油提供廉价油源。     

Production of rhamnolipids and diesel oil degradation by bacteria isolated from soil contaminated by petroleum

Biosurfactants are microbial secondary metabolites. The most studied are rhamnolipids, which decrease the surface tension and have emulsifying capacity. In this study, the production of biosurfactants, with emphasis on rhamnolipids, and diesel oil degradation by 18 strains of bacteria isolated from waste landfill soil contaminated by petroleum was analyzed. Among the studied bacteria, gram‐positive endospore forming rods (39%), gram positive rods without endospores (17%), and gram‐negative rods (44%) were found. The following methods were used to test for biosurfactant production: oil spreading, emulsification, and hemolytic activity. All strains showed the ability to disperse the diesel oil, while 77% and 44% of the strains showed hemolysis and emulsification of diesel oil, respectively. Rhamnolipids production was observed in four strains that were classified on the basis of the 16S rRNA sequences as Pseudomonas aeruginosa. Only those strains showed the rhlAB gene involved in rhamnolipids synthesis, and antibacterial activity against Escherichia coli, P. aeruginosa, Staphylococcus aureus, Bacillus cereus, Erwinia carotovora, and Ralstonia solanacearum. The highest production of rhamnolipids was 565.7 mg/L observed in mineral medium containing olive oil (pH 8). With regard to the capacity to degrade diesel oil, it was observed that 7 strains were positive in reduction of the dye 2,6‐dichlorophenolindophenol (2,6‐DCPIP) while 16 had the gene alkane mono‐oxygenase (alkB), and the producers of rhamnolipids were positive in both tests. Several bacterial strains have shown high potential to be explored further for bioremediation purposes due to their simultaneous ability to emulsify, disperse, and degrade diesel oil. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:262–270, 2016