Biosurfactant production by Aureobasidium pullulans in stirred tank bioreactor: New approach to understand the influence of important variables in the process

Highlights

• Aureobasidium pullulans LB 83 is a promising yeast used for biosurfactant production.

• Aeration and sucrose concentration: important variables in biosurfactant production.

• Bioreactor use in this proposal indicates the feasibility aimed to scale-up process.

Abstract

Surfactants are amphiphilic molecules with large industrial applications produced currently by chemical routes mainly derived from oil industry. However, biotechnological process, aimed to develop new sustainable process configurations by using favorable microorganisms, already requires investigations in more details. Thus, we present a novel approach for biosurfactant production using the promising yeast Aureobasidium pullulans LB 83, in stirred tank reactor. A central composite face-centered design was carried out to evaluate the effect of the aeration rate (0.1–1.1 min⁻¹) and sucrose concentration (20–80 g.L⁻¹) in the biosurfactant maximum tensoactivity and productivity. Statistical analysis showed that the use of variables at high levels enhanced tensoactivity, showing 8.05 cm in the oil spread test and productivity of 0.0838 cm.h⁻¹. Also, unprecedented investigation of aeration rate and sucrose concentration relevance in biosurfactant production by A. pullulans in stirred tank reactor was detailed, demonstrating the importance to establish adequate conditions in bioreactors, aimed to scale-up process.

Introduction

Surfactants are an important class of industrial chemicals that presents amphiphilic structure and relevant properties such as emulsification and tensoactivity (Santos et al., 2016). Due to their characteristics, surfactants are widely used in household and industries as food, pharmaceutical, petrochemical and cosmetic (Banat et al., 2000, Bogaert et al., 2016). Although extremely important, currently, most of these compounds are not eco-friendly, once they are chemically synthesized and petroleum-originated as well as their manufacturing processes and byproducts can be toxic and not ease biodegradation (Makkar and Cameotra, 2002). Hence, an intense movement for industrial sustainability has encouraged the interest in biosurfactants as possible substitutes for some of these synthetic surfactants (Marchant and Banat, 2012). For that proposal an improvement in biotechnology has been required for development of novel process for biosurfactant production (Vijayakumar and Saravanan, 2015).

The growth of biosurfactants’ industrial importance is presented in the data reported by Global Market Insights, Inc (2016), which shows that in 2015 the biosurfactant market was estimated at 370,500 tons, which is expected to reach 476,500 tons equivalent to 2.21 billion USD by 2018 and to a further 2.69 billion USD by 2023, with a compound annual growth rate (CAGR) of 4.2%.

The advantages presented by biosurfactants over synthetic surfactants include higher biodegradability, lower toxicity, higher ecological acceptability and greater stability at wide ranges of pH, temperature and salinity. Furthermore, biotechnological production processes allow the use of renewable substrates, making the process even more sustainable (Souza et al., 2014). However, biosurfactants production has already not been able to compete economically with synthetic surfactants produced from petroleum, and hence, reinforcing the need of new technologies aimed to increase productivity and yield process (De et al., 2015). Therefore, the search for new biosurfactant producing microorganisms and renewable and low cost feedstocks should be done. Furthermore, alternative process configurations and studies to understand the influence of important variables in the process are strategies that need to be better investigated aiming to increase the yields and become biosurfactant production viable.

The majority of studies of biosurfactants in literature have been reported in bacteria, however, the pathogenic nature of some products restricts the wide use of these compounds (Amaral et al., 2010, Elshikh et al., 2017). Additionally, bacteria wall and cell membrane are more sensitive to high concentrations of biosurfactants (Cooper and Paddock, 1984, Monteiro et al., 2010). Consequently, studies using yeasts for biosurfactant production has increased in the last years and have highlighted results and promisor characteristics for industrial applications (Amaral et al., 2010, Bogaert et al., 2016, Almeida et al., 2017).

Among yeasts, Aureobasidium pullulans was firstly reported as biosurfactant producer by Kurosawa et al. (1994). This yeast is largely known as source of commercial polysaccharide pullulan, used as water-soluble film in food and pharmaceutical applications, as well as in other valuable bioproducts (Gaur et al., 2010). A. pullulans was classified as Group-I by the World Health Organization (WHO), not presenting risk of infection for the company or laboratory workers (Jadhav and Gawai, 2013, World Health Organization, 1994). This feature facilitates the production process and allows it use in food and pharmaceutical industries.

The typical molecular structure of the biosurfactant produced by A. pullulans reported in literature is classified as polyol lipid, composed by a single polyol head group partially O-acylated with polyester tails usually composed of three or four 3,5-dihydroxydecanoic ester groups. Moreover, some authors also classified them as heavy oil or liamocin (Kim et al., 2015, Kurosawa et al., 1994, Manitchotpisit et al., 2011, Price et al., 2013). The polar moiety of biosurfactant structure may varies as mainly mannitol, glycerol, arabitol but also xylitol, ribitol, threitol, sorbitol and galactitol, according to the strain of A. pullulans and carbon source in the medium (Kim et al., 2015, Leathers et al., 2015, Price et al., 2017). Lipid moiety has similar structure of the lipid Exophilin, excreted by Exophiala psiciphilia, which has proven antimicrobial activity (Bischoff et al., 2015, Doshida et al., 1996). Taking this into account, the activity of this biosurfactant as antimicrobial agent (Bischoff et al., 2015, Leathers et al., 2015, Price et al., 2017) as well as anticancer agent have been studied (Isoda and Nakahara, 1997, Manitchotpisit et al., 2011, Manitchotpisit et al., 2014). These important applications emphasize the need of studies aiming to increase and enable its scale-up production.

Besides the choice of a good biosurfactant producer, the establishment of operating and nutritional conditions for the process are important factors for its industrial production. Several studies have demonstrated the importance of medium composition for the variety and maximum biosurfactant production by A. pullulans (Leathers et al., 2015, Price et al., 2017). Leathers et al. (2015), for example, evaluated cultivation mediums with different composition and used different strains of A. pullulans and observed that sucrose-based medium resulted in higher surfactant production. However, studies focused in the influence of essentials parameters of the process, as aeration, in biosurfactant production by this microorganism have already not been well explored yet.

Therefore, in the present study, a novel proposal for biosurfactant production by Aureobasidium pullulans LB 83 in stirred tank reactor was presented, evaluating the influence of the parameters aeration and carbon source concentration in the biosurfactant production, growing and morphological aspects of this microorganism applied in this process. Biosurfactant production was evaluated based on the observed tensoactivity, and hence, superficial tension reduction, specific characteristic of this compound (Santos et al., 2016, Youssef et al., 2004). As carbon source, sucrose was used due to previous results showing its potential for this bioprocess using A. pullulans (Leathers et al., 2015). The use of reactor in this study is an interesting approach as the related literature is scarce for important parameters in biosurfactants production process by this yeast, allowing generating results for future scale up research work.