## 3 CA Production in _S. clavuligerus_ Submerged Cultivation  
Besides CA, _S. clavuligerus_ produces a plethora of secondary metabolites that can compete with CA biosynthesis and secretion. Those include some \(\beta\)-lactam (penicillin N, cephalincin C, cephalospor, and other clavams) and non-\(\beta\)-lactam (holomycin, \(\beta\)-lactamase inhibitory protein and a tunicamycin-related antibiotic) antibiotics [24; 46; 47; 48; 49; 50; 51; 52; 53; 54]. However, CA is currently the most important specialized metabolite produced by _S. clavuligerus_ considering its clinical and industrial relevance. CA is usually produced in submerged cultivation of _S. clavuligerus_, either wild type or engineered strains. Several operation modes and media compositions have been explored aimed to increase the characteristic low titters of the wild type strain, as [PERSON] et al. [55] reviewed recently. Such strategies might also enhance the CA titers of engineered strains in submerged cultivation. As previously mentioned, glycerol is the most common carbon source used in CA production. When comparing the CA production using glycerol and starch as carbon sources, glycerol promotes CA titers up to 5-fold the observed in cultivations with starch as carbon source [56; 57]. Nevertheless, substrate inhibition can occur at glycerol concentrations higher than 50 g\(\cdot\)L\({}^{-1}\)[56]. As glycerol acts as the C-3 precursor, the biosynthesis of CA is enhanced by amino acid supplementation [42; 58] and rich nitrogen sources like soy protein and soy flour [56; 57] that provide abundant C-5 precursors through the urea cycle. Conversely, the use of dextrose or starch promote the secretion of Cephamycin C [57; 59]. In a media screening study, [PERSON] et al. [60] observed the highest CA production (0.437 g\(\cdot\)L\({}^{-1}\)) with the media having glycerol as the main carbon source and isolated soy protein, while the amino acid supplementation did not enhance the CA productivity. [PERSON] et al. [61] reported CA concentrations in batch, fed batch and continuous cultivations of 0.195, 0.404, and 0.293 g\(\cdot\)L\({}^{-1}\), respectively. The highest CA concentrations obtained in bioreactor cultivations using a wild type strain were reported by [PERSON] et al. [62]. In that study, batch and fed-batch operation modes were tested using a complex medium prepared with glycerol, malt, and yeast extracts, peptone and trace elements. Batch cultivations yielded 0.430-0.530 g\(\cdot\)L\({}^{-1}\) of CA while fed-batch cultivations with ornithine (3.7 g\(\cdot\)L\({}^{-1}\)) led to a CA concentration of 1.560 g\(\cdot\)L\({}^{-1}\)[62; 63]. A complex culture medium provides important amounts of free or hydrolyzed amino acids, which act not only as nitrogen sources, but also as secondary carbon sources. Complex nitrogen sources, such as those containing soy protein, favor considerably the CA accumulation in contrast to the chemically defined media [56]. The use of complex media with vegetable oils supplementation (olive, cotton, palm, and corn) as secondary carbon sources may enhance the CA production up to 2-fold when compared to glycerol as a supplement [64; 65; 66]. However, the use of complex media leads to denser and more viscous fermentation broths due to the high concentration of suspended solids. Those factors may affect the mass transfer in the bioreactor and the downstream CA separation. Phosphate has a potential repressive effect on CA production; thus, culture media are commonly designed as phosphate-limited [6; 42; 55]. In this regard, [PERSON] and [PERSON] [58] found a CA biosynthesis repression when increasing the phosphate concentration in the media over 100 mmol\(\cdot\)L\({}^{-1}\). The studies indicated that CA accumulation is favored in the fed-batch operation under controlled conditions of pH, aeration, and stirring. Those conditions are more easily controllable in stirred tank bioreactors (STR) although the CA production studies are not restricted only to this configuration.