Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Improving Downstream Processes <s> Great emphasis has recently been given to the environmental impacts caused by chemical surfactants due to their toxicity and difficulty in being degraded in the environment [1]. Increasing environmental concerns, the advance in biotechnology and the emergence of more stringent laws have led to biosurfactants being a potential alternative to the chemical surfac‐ tants available on the market [2, 3]. Although biosurfactants have promising use in bioreme‐ diation processes, their industrial scale production is currently difficult due to high rawmaterial costs, high processing costs and low manufacturing output [3]. As a result, the current research challenges are to increase the yield and to reduce the cost of raw materials [4]. <s> BIB001 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Improving Downstream Processes <s> BACKGROUND ::: ::: There is currently much interest in the use of natural biosurfactants and bioemulsifiers, mainly in the cosmetic, pharmaceutical and food industries. However, there are no studies on the optimization of the extraction conditions of cell-bound biosurfactants. In this work, a biosurfactant with emulsifier properties was extracted from Lactobacillus pentosus cells, under different extraction conditions, and characterized. ::: ::: ::: ::: RESULTS ::: ::: During extraction, the most influential independent variable, concerning the emulsifying capacity of biosurfactant, was the operation time, followed by temperature and salt concentration. Biosurfactant from L. pentosus was evaluated by Fourier transform infrared spectroscopy and the composition of fatty acids was analyzed by gas chromatography–mass spectrometry. The hydrophobic chain of the biosurfactant from L. pentosus comprises 548 g kg−1 linoelaidic acid (C18:2), 221 g kg−1 oleic or elaidic acid (C18:1), 136 g kg−1 palmitic acid (C16) and 95 g kg−1 stearic acid (C18). In addition, emulsions of water and rosemary oil were stabilized with a biosurfactant produced by L. pentosus and compared with emulsions stabilized with polysorbate 20. ::: ::: ::: ::: CONCLUSION ::: ::: The optimum extraction conditions of biosurfactant were achieved at 45 °C at 120 min and using 9 g kg−1 of salt. In all the assays biosurfactant from L. pentosus yielded more stable emulsions and higher emulsion volumes than polysorbate 20. © 2014 Society of Chemical Industry <s> BIB002 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Improving Downstream Processes <s> Biosurfactants are surface-active agents that are produced by a variety of microorganisms including yeasts, filamentous fungi and bacteria. In this work, we report on the ability of Pseudomonas aeruginosa ATCC 9027 and Burkholderia thailandensis E264 to produce rhamnolipids via a 10-L bioreactor and their recovery through foam fractionation studies in a continuous stripping mode. The recovery of Rha-C10-C10 (mono-rhamnolipids) produced by P. aeruginosa ATCC 9027 increased (from 6% to 96%), whilst the enrichment decreased (from 2.9 to 1.2) with the increasing airflow rate. These results are consistent with foam fractionation of a single surfactant system with stable foam. The recovery and enrichment of Rha-Rha-C14-C14 (di-rhamnolipids) produced by B. thailandensis E264 (and an unknown molecule) in a single-component system were found to display different characteristics. Both recovery and enrichment were found to decrease with the airflow rate. It is postulated that a competitive adsorption process could occur between the smaller molecule identified by electrospray ionisation–mass spectrometry (ESI–MS) and Rha-Rha-C14-C14. <s> BIB003 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Improving Downstream Processes <s> BACKGROUND: Multiple aqueous two‐phase systems (ATPS) were evaluated for the partial purification of laccase from a crude aqueous extract. The laccase extract was obtained from residual compost, generated from the production of the edible mushroom Pleurotus ostreatus. A total of 59 ATPS were evaluated; 16 systems using poly (ethylene glycol) (PEG) and phosphates, 20 systems using UCON (ethylene oxide/propylene oxide copolymer) and different salts, and 23 polymer–polymer systems (Ficoll, Dextran and PEG). RESULTS: Protein partitioning was not achieved with the PEG–phosphate systems. ATPS composed of UCON and salts presented great potential for laccase recovery, with up to 95.12 ± 5.27, 96.05 ± 4.99 and 98.31 ± 3.58% recovery, and purification factors of 5.02, 4.14 and 9.97 for the UCON‐NaH₂PO₄ , UCON‐NaSO₄, and UCON‐(NH₄)₂SO₄ systems, respectively. The PEG 10 000/Dextran 10 000 and the PEG 10 000/Dextran 100 000 polymer–polymer systems proved to have potential, presenting purification factors as high as 5.78 and 6.56, respectively. CONCLUSION: The laccase analyzed in this study had a molecular weight of approximately 30 kDa, and thus, is the smallest laccase yet to be reported for P. ostreatus. ATPS represent an attractive alternative for the efficient recovery of laccase from residual compost generated after P. ostreatus production. © 2016 Society of Chemical Industry <s> BIB004 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Improving Downstream Processes <s> Surfactants are one of the most versatile group of chemicals used in various industrial processes. Their market is competitive, and manufacturers will have to expand surfactant production in ecofriendly and cost effective manner. Increasing interest in biosurfactants led to an intense research for environment friendly and cost-efficient production of biosurfactant. Structural diversity and functional properties of biosurfactants make them an attractive group of compounds for potential use in wide variety of industrial, environmental and biotechnological applications. Screening methods make task easier to obtain potential biosurfactant producing microorganisms. Variety of purification and analytical methods are available for biosurfactant structural characterization. This review aims to compile information on types and properties of biosurfactant, microbial screening methods as well as biosynthesis, extraction, purification and structural characterization of biosurfactant using rhamnolipid as a model biosurfactant. It also describes factors affecting rhamnolipid production. It gives an overview of oil recovery using biosurfactant from Pseudomonas aeruginosa. <s> BIB005 </s> Statistical Design, a Powerful Tool for Optimizing Biosurfactant Production: A Review <s> Improving Downstream Processes <s> Since 60–80% of total costs of production are usually associated with downstream collection, separation and purification processes, it has become advantageous to investigate how to replace traditional methods with efficient and cost-effective alternative techniques for recovery and purification of biosurfactants. In the traditional techniques, large volumes of organic solvents are usually used increasing production cost and the overall environmental burden. In addition, traditional production and separation methods typically carried out in batch cultures reduce biosurfactant yields due to product inhibition and lower biosurfactants activity as a result of interaction with the organic solvents used. However, some in situ recovery methods that allow continuous separation of bioproducts from culture broth leading to an improvement in yield production and fermentation efficiency. For biosurfactants commercialization, enhancement of product capacity of the separation methods and the rate of product removal is critical. Recently, interest in the integration of separation methods with production step as rapid and efficient techniques has been increasing. This review focuses on the technology gains and potentials for the most common methods used in in situ product removal (ISPR): Foam fractionation and Ultrafiltration, especially used to recover and purify two well-known biosurfactants: glycolipids (rhamnolipids) and lipopeptides (surfactins). ::: ::: This article is protected by copyright. All rights reserved <s> BIB006
Although cost effective Bs production is the major priority for feasible industrial application, downstream processing should be taken into account. Once the secondary metabolite has been produced, the need for high purity of the tenso-active agent will depend on the application intended BIB006 . Recovery and purification has been shown to greatly increase production costs, especially in the case of proteins, and Bs are no exception BIB005 . Traditional strategies for Bs recovery and purification include precipitation with acids, salts, or organic solvents which are toxic and cause air pollution. Other more sophisticated methods include foam fractionation, ultrafiltration, adsorption-desorption on polystyrene resins, ion exchange chromatography, and High Pressure Liquid Chromatography (HPLC) BIB001 BIB003 . However, the latter present very low yields, as only small amounts of the crude extract can be treated. A new approach which has recently gained popularity for downstream processing in other biomolecules is Aqueous Two-Phase Systems (ATPS). This strategy has been successful for the recovery and partial purification of enzymes and other micro and macro molecules, but has been hardly used for the treatment of crude Bs extracts BIB004 . Up to date, the application of ATPS for Bs downstream processing remains basically unexplored in the biotechnological field. In fact, we found only one study dedicated to the use of statistical design for the improvement of Bs recovery BIB002 . They optimized the extraction conditions of a Lactobacillus pentosus cell bound biosurfactant/emulsifier using a Box-Behnken response surface methodology. Their results showed that operation time was the most influential independent variable, followed by temperature and salt concentration. The optimum extraction conditions of the biosurfactant were achieved at 45 • C at 120 min and using 9 gkg −1 of salt. Bs yield improved from 9.49-13.76 mgL −1 . Their study serves as an excellent example for others in this field. Researchers can and should exploit the possibilities for enhancing Bs yields after production optimization. Here we propose the use of designs such as the PBD for the screening of important factors affecting Bs recovery, and subsequently, the implementation of other designs for determining the most adequate levels of the relevant factors such as solvent concentration, temperature, and exposition time.