Improved rhamnolipid biosurfactant production by Burkholderia thailandensis E264 using agro-industrial waste

Biosurfactants are amphiphilic surface-active compounds, produced by various microorganisms, that reduce surface and interfacial tension. These compounds are attracting increasing interest over their chemical counterparts due to their advantages, such as biodegradability, high stability in extreme environments, low toxicity, low critical micelle concentrations (CMC) and the fact that they can be effectively produced from agro-industrial wastes and renewable resources. Furthermore, their diversity allows for a significant number of uses, including microbial enhanced oil recovery (MEOR), bioremediation and biomedical applications. However, the high operational costs, mainly regarding the use of expensive raw materials in the fermentation and the complex downstream processing, to yield low amounts of product restricts their industrial-scale applications. Several attempts to solve these limitations by reducing the production costs have been conducted and include the use of low-cost agro-industrial wastes and by-products as substrates. One of these low-cost substrates, that has been successfully used to produce biosurfactants by Bacillus subtilis and Pseudomonas aeruginosa strains, is Corn Steep Liquor (CSL). In this research, rhamnolipid biosurfactant production by Burkholderia thailandensis E264 was optimized using this agro-industrial waste as sole substrate. When grown in a culture medium containing CSL (7.5% v/v), this strain produced 1.77 g biosurfactant/L, which is about 2.6 times the amount of biosurfactant produced in the standard synthetic medium. The purified biosurfactant produced in the low-cost medium exhibited similar surface-active properties when compared with that produced in the synthetic medium, reducing the surface tension of water to 29.7 mN/m, with a CMC of 385 mg/L. HPLC analysis showed that the culture medium used contains about 2.6 g/L of fructose and 2.5 g/L of glucose, that are fully consumed within the first 48 h of fermentation. Since the synthetic medium contains 40 g/L of glycerol, results suggest that biosurfactant production is more efficient in the low-cost medium. Furthermore, to the best of the authors knowledge, this is the first experimental research that combines the utilization of B. thailandensis with CSL to produce biosurfactants with very optimistic results in terms of cost and production levels. The rhamnolipid-containing cell-free supernatant could be used directly in bioremediation or MEOR processes.This study was supported by PARTEX Oil and Gas, and the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte 2020 - Programa Operacional Regional do Norte. The authors also acknowledge to the Biomass and Bioenergy Research Infrastructure (BBRI)- LISBOA-01-0145-FEDER-022059, supported by Operational Program for Competitiveness and Internationalization (PORTUGAL2020), by Lisbon Portugal Regional Operational Program (Lisboa 2020) and by North Portugal Regional Operational Program (Norte 2020) under the Portugal 2020 Partnership Agreement, through the ERDF.info:eu-repo/semantics/publishedVersio

Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?

Astaxanthin is a xanthophyll of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from β-carotene by the sequential action of two enzymes: a β-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3') positions of each of the two β-ionone rings of β-carotene, and a β-carotene ketolase that introduces keto groups at carbons 4 and 4' of the β-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of β-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementation studies of X. dendrorhous mutants and expression analysis in Mucor circinelloides reveals that the CrtS enzyme is a β-carotene hydroxylase of the P-450 monooxygenase family that converts β-carotene to the hydroxylated derivatives β-cryptoxanthin and zeaxanthin, but it does not form astaxanthin or the ketolated intermediates in this fungus. A bifunctional β-carotene hydroxylase-ketolase activity has been proposed for the CrtS protein. The evidence for and against this hypothesis is analyzed in detail in this review

Isolation of microorganisms from oil samples for application in microbial enhanced oil recovery

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/1096

Biosurfactant-producing Bacillus subtilis strains isolated from crude oil samples enhance oil recovery at lab scale

Biosurfactant-producing Bacillus subtilis strains isolated from crude oil samples enhance oil recovery at lab scale Eduardo J Gudiña, Lígia R. Rodrigues, José A. Teixeira IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal Microbial Enhanced Oil Recovery (MEOR) is potentially useful to increment oil recovery from reservoirs beyond primary and secondary recovery operations using microorganisms and their metabolites. Stimulation of bacterial growth and biosurfactant production by indigenous microorganisms can reduce the capillary forces that retain the oil into the reservoir. MEOR offers major advantages over conventional EOR, namely low amounts of energy consumption and independence of the price of crude oil [1]. In this work, a sand pack column model was designed to simulate the oil recovery operations in oil reservoirs and evaluate the mobilization of residual oil. Three Bacillus subtilis strains, previously isolated from crude oil samples [2], were used. Those strains grow and produce extracellular biosurfactants at 40ºC under anaerobic conditions in medium supplemented with hydrocarbons. Biosurfactants produced reduce the surface tension of water from 72 to 30 mN/m, exhibit emulsifying activity and are not affected by exposure to high temperatures (121ºC) which makes them good candidates for application in biosurfactant mediated MEOR. Sand pack column assays were performed using paraffin and crude oil. Additional oil recovery using paraffin ranged from 19 to 35% with the different isolates. When crude oil was used as hydrocarbon, the isolates recovered between 19 and 21% of the entrapped oil. The results obtained suggest that stimulation of biosurfactant production by these strains in situ can contribute to mobilize entrapped oil and improve the oil fluidity. [1] Sen R, “Biotechnology in petroleum recovery: The microbial EOR”, Progress in Energy and Combustion Science (2008) 34: 714-724. [2] Gudiña EJ, Pereira JFB, Rodrigues LR, Coutinho JAP, Teixeira JA, “Isolation and study of microorganisms from oil samples for application in Microbial Enhanced Oil Recovery”, International Biodeterioration and Biodegradation (2012) 68: 56-64

Biosurfactant-producing lactobacilli : screening, production profiles and effect of medium composition

Biosurfactant production was screened in four lactobacilli strains. The highest biosurfactant production (excreted and cellbound biosurfactants) was achieved with Lactobacillus paracasei ssp. paracasei A20, a strain isolated from a Portuguese dairy plant, with a decrease in the surface tension of 6.4mNm−1 and 22.0mNm−1, respectively. Biosurfactant production by this strain was evaluated under different culture broth compositions. The use of different nitrogen sources revealed that yeast extract is essential for bacterial growth, while peptone is crucial for biosurfactant synthesis. For biosurfactant production, the use of peptone and meat extract yielded a higher production when compared to the standard medium, with a surface tension reduction of 24.5mNm−1. Furthermore, experiments were also conducted in a reactor with pH and temperature control. Biomass and biosurfactant production in bioreactor was higher comparing with the experiments conducted in shake flaks. The optimization procedure adopted in the current work was found to improve the biosurfactant production and opened new perspectives for the use of L. paracasei ssp. paracasei A20 as a promising biosurfactant-producer

Novel bioemulsifier produced by a Paenibacilus sp. strain and its applicability in microbial enhanced oil recovery

Microbial Enhanced Oil Recovery (MEOR) is potentially useful to increment oil recovery from reservoirs beyond primary and secondary recovery operations using microorganisms and their metabolites. In situ stimulation of microorganisms that produce surface active compounds reduces the capillary forces that retain the oil inside the reservoir, thus promoting its flow and increasing oil production. Paenibacillus sp. #510, isolated from crude oil samples obtained from a Brazilian oil field, produced a bioemulsifier in a mineral medium containing sucrose as the carbon source under aerobic and anaerobic conditions, and its production was induced (up to 7.9 g/l) by the addition of paraffin or crude oil to the culture medium. It formed stable emulsions with several hydrocarbons and its emulsifying ability was not affected by exposure to high salinities (up to 300 g/l), high temperatures (100ºC-121ºC) or a wide range of pH values (2-13). This is the first description of bioemulsifier production by a Paenibacillus strain. A preliminary chemical characterization by Fourier Transform Infrared Spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance (1H NMR and 13C CP-MAS NMR) and size exclusion chromatography indicated that this new bioemulsifier is a low-molecular weight oligosaccharide-lipid complex. Mobilization of heavy crude oil by this isolate was evaluated using a core-flooding equipment working at the oil reservoir pressure (32.4 bar) and temperature (40ºC). Growing in situ Paenibacillus sp. #510 for 14 days at the oil reservoir conditions using a mineral medium resulted in the mobilization of 6% of the entrapped heavy oil, confirming that this isolate can contribute to enhance oil recovery from mature reservoirs. These results will be further validated in a pilot field assay

Potential therapeutic applications of biosurfactants

Biosurfactants have recently emerged as promising molecules for their structural novelty, versatility, and diverse properties that are potentially useful for many therapeutic applications. Mainly due to their surface activity, these molecules interact with cell membranes of several organisms and/or with the surrounding environments, and thus can be viewed as potential cancer therapeutics or as constituents of drug delivery systems. Some types of microbial surfactants, such as lipopeptides and glycolipids, have been shown to selectively inhibit the proliferation of cancer cells and to disrupt cell membranes causing their lysis through apoptosis pathways. Moreover, biosurfactants as drug delivery vehicles offer commercially attractive and scientifically novel applications. This review covers the current state-ofthe- art in biosurfactant research for therapeutic purposes, providing new directions towards the discovery and development of molecules with novel structures and diverse functions for advanced applications.The authors acknowledge Fundacao para a Ciencia e a Tecnologia (FCT) (Portugal) and the Department of Science and Technology (DST) (India) for financial support of the project 'MEDSURF - The potential use of biosurfactants for medical applications' developed under the scope of a bilateral agreement between Portugal and India

Development of a low-cost culture medium for biopolymer production by Rhizobium viscosum CECT 908 and its potential application in Microbial Enhanced Oil Recovery

Polymers are a versatile class of compounds that play an essential role in our society, being their production estimated in more than 180 million tons per year. Nowadays, the world market is dominated by synthetic and plant-derived polymers. Biopolymers of microbial origin are characterized by their better environmental compatibility and biodegradability when compared with the synthetic ones, and their production is faster than those obtained from plants. Microbial biopolymers usually exhibit excellent rheological properties, stability at a wide range of temperatures, salinities and pH values, as well as a broad variety of chemical structures, which results in different physicochemical and rheological properties. However, despite their outstanding properties, their application is still limited by their high production costs. In this work, an alternative low-cost culture medium was developed for biopolymer production by Rhizobium viscosum CECT 908, containing sugarcane molasses (60 g/L) and corn steep liquor (1%, v/v) as carbon and nitrogen sources, respectively. Using this low-cost medium, higher biopolymer production and apparent viscosity values (5.2 g/L and 6700 mPa s, respectively) were obtained comparing with the synthetic medium (2.3 g/L and 1100 mPa s), which contained glucose and yeast extract. As a result, the cost of the culture medium necessary to produce 1 Kg of biopolymer was reduced more than 20 times. The biopolymer produced in the alternative low-cost medium exhibited better rheological properties as compared to xanthan gum, including higher viscosity at the same concentration. Furthermore, it was found to be stable at temperatures up to 80ºC, NaCl concentrations as high as 200 g/L, and high shear rates (300 s-1). Polymers are widely used by the oil industry to increase the oil reservoirs productivity during the tertiary oil recovery processes. In sand-pack column assays performed using a heavy crude oil (40ºC= 170 mPa s), this biopolymer produced using the low-cost medium demonstrated a better performance than xanthan gum, recovering almost 50% of the entrapped oil. Results herein obtained highlight that the R. viscosum biopolymer is a promising candidate for application in MEOR as an alternative to the conventional microbial and synthetic polymers currently used.info:eu-repo/semantics/publishedVersio

Improved rhamnolipid biosurfactant production by Burkholderia thailandensis E264 using agro-industrial wastes

Biosurfactants are amphiphilic surface-active compounds, produced by various microorganisms, that reduce surface and interfacial tension. These compounds are attracting increasing interest over their chemical counterparts due to their advantages, such as biodegradability, high stability in extreme environments, low toxicity, low critical micelle concentrations (CMC) and the fact that they can be effectively produced from agro-industrial wastes and renewable resources. Furthermore, their diversity allows for a significant number of uses, including microbial enhanced oil recovery (MEOR), bioremediation and biomedical applications. However, the high operational costs, mainly regarding the use of expensive raw materials in the fermentation and the complex downstream processing due to the low production yields restricts their industrial-scale applications. Several attempts to solve these limitations by reducing the production costs have been conducted and include the use of low-cost agro-industrial wastes and by-products as substrates. One of these low-cost substrates, that has been successfully used to produce biosurfactants by Bacillus subtilis and Pseudomonas aeruginosa, is corn steep liquor (CSL). In this work, CSL is evaluated for rhamnolipid biosurfactant production by Burkholderia thailandensis E264. When grown in a culture medium containing CSL (7.5% v/v) as sole substrate, this strain produced 1.8 g rhamnolipid/L, which is about 2.6 times the amount of rhamnolipid produced in the standard synthetic medium. By supplementing the culture medium with olive oil mill wastewater (OMW, 10% v/v), a residue originating from the olive oil extraction industry, rhamnolipid production was increased up to 2.6 g/L, most likely due to the inductive effect of long-chain fatty acids present in OMW (mainly oleic, palmitic, linoleic and stearic acids) on rhamnolipid production. Rhamnolipids purification was also done and the biosurfactant produced in the low-cost medium (CSL + OMW) exhibited better surface-active properties when compared with those produced in the synthetic medium, reducing the surface tension of water to 26.8 ± 0.1 mN/m, with a CMC of 280 mg/L (28.8 ± 0.2 mN/m and 460 mg/L, respectively, in the synthetic medium). These results demonstrate that rhamnolipid production is more efficient in the low-cost medium. Furthermore, to the best of the authors knowledge, this is the first experimental research that describes the utilization of CSL and CSL + OMW as substrates for the production of rhamnolipids by B. thailandensis, with very optimistic results in terms of cost and production levels.This work was sponsored by PARTEX Oil and Gas and it was supported by the Portuguese Foundation for Science and Technology(FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio