Bioprospecting of Microorganism-Based Industrial Molecules. Группа авторов

[81] Glycolipoprotein Aspergillus ustus [82] Glycolipid Ustilago maydis [82]

2.4 Screening for BS Producers

      As shown above, there are diverse microorganisms able to produce BS, but few of them have the kinetic characteristics for large‐scale production since bio‐based product market is appealing [83] for new or better‐producing microorganisms and is a challenge for biotechnology. Microbial screening has been performed on different environments, such as hydrocarbon and oil‐contaminated soil, coastal and offshore [84–86], gas platforms [87], seawater biofilm [88], marine environment [89], mangrove sediments [90], food materials [91], and Amazon rainforest [92]. Many of the isolates are bacteria related to Pseudomona and Bacillus genera. In the case of yeasts, Candida, Starmerella (ex Candida), and Pseudozyma are the most used genera; but more microorganisms are emerging nowadays as BS producers.

Accurate methodologies for isolation and characterization of microorganisms are important to discover specific metabolic capabilities. By traditional methods, microorganisms are isolated from environmental samples and tested for BS production by qualitatively, e.g. CTAB agar assay or hemolysis test as a primary approach. A different strategy limits the screening to oil or hydrocarbon‐contaminated sites, resulting in an increment of isolates related to BS production [93]. In both cases, it is necessary to employ quantitative or semi‐quantitative methods for BS evaluation: drop collapse test, emulsion index, TLC and superficial tension, or techniques such as HPLC/UPLC, LC‐MS, or MS. Nevertheless, this methodology is only for cultivable microorganisms, so there is an undetermined number of potential producers in different environments. At this moment, the metagenomic approach is not a common practice for the screening of BS producers [94].

2.5 A Case Study: SL by Solid‐State Fermentation (SSF), Kinetics, and Reactor Size Estimation

      SL are BS that are produced commonly by liquid fermentation (LF) using glucose as the hydrophilic carbon source and oleic acid (C18:1) as the hydrophobic source [95]. Glucose can be replaced by sugar cane or sugar beet juices, which have high contents of sugars and nitrogen and avoid the use of the expensive yeast extract and urea for the fermentation media [96]. Some potential hydrophobic sources are solid at fermentation temperature (e.g. stearic acid, m.p. = 69.3 °C), complicating their use as substrates in these liquid processes. In such a case, solid‐state fermentation (SSF) is a potentially useful alternative. Furthermore, the SSF avoids the problems that generally occur during the production of SL by liquid fermentation, such as foaming and viscosity increase [96].

      BS production in liquid media is carried out with aeration and forced agitation. However, this causes problems when BS production starts because a large amount of foam is generated. Moreover, there is a tendency for microorganisms to accumulate within the foam, eliminating cells from the culture medium. Also, the presence of foam increases the risk of cross‐contamination and reduces the efficiency of oxygen transfer between the liquid and gas phase in the bioreactor [97]. The use of antifoams has disadvantages. These can be toxic to microorganisms, have high costs, and are extra‐components that must be separated from the BS during the purification processes [98].

      Scientific literature about BS shows a clear tendency in the use of liquid fermentations over SSF, being batch processes at laboratory scale the most used methods. Nonetheless, recent studies, including our group in Mexico, point out that SSF is an advantageous alternative for BS production since oxygen transfers are efficient, and no foaming problems are seen during fermentations, especially when production yields are greater than 0.3 g_BS/kg_dr.

Our research group has some experience with the use of non‐pathogenic microorganisms for producing SL by SSF both at the laboratory and pilot level. Using glucose, vegetable oils, and solid supports of natural origin, production of BS in our hands has been monitored from respirometry studies using an analytical device patented by our group [99] without disturbing the culture. For example, Figure 2.6 shows the carbon dioxide production rate (CDPR) recorded in real‐time during BS production. This measurement is an invaluable tool for the process because it allows us to make decisions in real‐time. In this example, it is seen an imminent increase of CDPR (ca 1.2) followed by a rapid decrease (ca 0.8) that stabilizes in a plateau to drop off subsequently. This is the common behavior observed in many of our productive fermentation. In Figure 2.7, it is observed that O₂ consumption rate is similar to CO₂ production rate during the first days of incubation. After that, greater O₂ consumption is observed with respect to CO₂ production (Figure 2.7). This is reflected in a decrease in respiratory quotient (RQ, Figure 2.8).

Figure 2.6 CO₂ formation rate (empty symbols) and O₂ uptake rate (full symbols) during the production of SL in SSF.

Figure 2.7 Total CO₂ formation (empty symbols) and O₂ uptake (full symbols) during sophorolipid production in SSF.

The RQ (Figure 2.8) is less than 1.0 during the entire cultivation time, and the maximum value of the RQ (0.82 mol of CO₂/mol of O₂) is observed around 36 hours of incubation. These RQ values could be explained by the oxidation of glucose and suggest that, during the first days, fatty acids could be mainly used for synthesizing BS. Literature indicates that fatty acids are hydroxylated and incorporated directly into the synthesis of BS [100], whereas, after glucose depletion, fatty acids can be mainly used as energy source assimilated via β‐oxidation.

Figure 2.8 Respiratory quotient observed during SL production by SSF.

Figure 2.9 Evolution of pH during the production de sophorolipids.

Another parameter that can be easily measured to follow BS production is pH. The change of pH values over time is shown in Figure 2.9. pH values show a significant decrease during the first days of cultivation (from 6 to 3) and then remain. This behavior is similar in liquid fermentation [100]. The addition of alkali has been proposed to control the pH values around 3.5, which are optimal for sophorolipid production [101]. However, for SSF, it is not feasible due to the lack of homogeneity.