Biochemical Engineering Journal 95 (2015) 48–55 Contents lists available at ScienceDirect Biochemical Engineering Journal jo ur nal home p age: www.elsevier.com/locate/ bej Regular Article Production of fatty-acyl-glutamate biosurfactant by Bacillus subtilis on soybean co-products Mustafa E. Marti a,∗ , William J. Colonna a,b , Gabriel Reznik c , Michelle Pynn c , Kevin Jarrell c , Buddhi Lamsal b,d , Charles E. Glatz a a Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA b Center for Crops Utilization Research, Iowa State University, Ames, IA 50011, USA c Modular Genetics, Inc., Woburn, MA 01801, USA d Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA a r t i c l e i n f o Article history: Received 16 April 2014 Received in revised form 20 October 2014 Accepted 17 November 2014 Available online 25 November 2014 Keywords: FA-Glu Biosurfactant Soybean hull Enzyme Technology Fermentation Substrate Inhibition a b s t r a c t Fatty-acyl-glutamate (FA-Glu), a surfactin variant has been successfully produced using a genetically modified strain of Bacillus subtilis grown on glucose. However, yields with soybean hulls (SBH) replacing glucose were lower. This work was undertaken to reduce the yield loss when using SBH as the carbon source and to evaluate two other soy by-products, namely fiber and skim from aqueous oil extraction as alternative carbon and nitrogen sources. Fermentation of soybean hulls, fibers and skim at various concentrations produced lower FA-Glu titers compared to S-7 medium. Neither increasing their amount nor supplementing with glucose increased the FA-Glu titer, suggesting the presence of an inhibitor in these feedstocks. By using a mixture of polysaccharide-degrading enzymes, over 65% of SBH solids were converted to soluble carbohydrates. FA-Glu titers obtained from SBH hydrolysates containing residual hull solids were still low; however, with the removal of the solids, cell growth improved and FA-Glu yield was 60% higher than with glu- cose. Thus, this low-cost material can be converted to a substrate for production of FA-Glu biosurfactant. Unmodified fiber and skim components of aqueous oil extraction were not beneficial. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Surfactants are amphiphilic chemicals with emulsification and surface tension lowering properties that enable their use as foaming and dispersing agents. There is a growing demand for sur- factants; however, as they are synthesized from petrochemicals or palm oil [1], they are increasingly expensive and can be envi- ronmentally unfriendly. For these reasons, production of “green” biosurfactants from renewable resources has received considerable attention in recent years. Biosurfactants, synthesized by microorganisms, consist of sugars, fatty acids and amino acids [2,3]. Examples of micro- bially derived biosurfactants include rhamnolipids, sophorolipids, Abbreviations: FA-Glu, fatty-acyl-glutamate; SBH, soybean hulls; CHO, carbo- hydrate; EAEP, enzyme-assisted aqueous extraction; PHF, pretreated hydrolysis & fermentation; SHF, simultaneous hydrolysis & fermentation. ∗ Corresponding author at: Chemical Engineering Department, Selc ¸ uk University, Konya 42075, Turkey. Tel.: +90 507 148 9978; fax: +90 332 241 0635. E-mail addresses: marti@selcuk.edu.tr, mustafaesenmarti@gmail.com (M.E. Marti). phospholipids and lipopeptides from various strains of Pseu- domonas, Torulopsis, Candida, and Bacillus [4,5]. Biosurfactants have useful physiological, biocidal, surface activity and physicochemical properties [6]. Potential applications of biosurfactants include use in detergents, pharmaceuticals, oil recovery, cosmetics and food products. Their low toxicity and biodegradability make them envi- ronmentally friendly [7]. Moreover, as they can be produced by fermentation from renewable, biological feedstocks [8,9], they are not linked to uncertain supplies of petrochemicals. Bacillus sp. produces an assortment of cyclic lipopeptide surfac- tants, such as iturins and fengycins [10–16]. A well-known cyclic biosurfactant is surfactin, which consists of a long-chain -hydroxy fatty acid of 12–17 carbons joined via an amide linkage to the amino-terminal glutamic acid residue of a heptapeptide (l-Glu/l- Leu/d-Leu/l-Val/l-Asp/d-Leu/l-Leu) [17,18]. The carboxy-terminal leucine of the peptide is esterified to the -hydroxyl group of the fatty acid to form a lactone. Surfactin is a potent surface active agent that reduces the surface tension of water from 72 to 27 mN/m at 20 M [19–22]. Members of the genus Bacillus grow satisfactorily and pro- duce biosurfactants on a variety of organic waste streams such as potato effluents, orange peels, cassava processing waste, sugarcane http://dx.doi.org/10.1016/j.bej.2014.11.011 1369-703X/© 2014 Elsevier B.V. All rights reserved.