Olive Pomace Oil can be Used as an Alternative Carbon Source for Clavulanic Acid Production by Streptomyces clavuligerus

Clavulanic acid is an important drug, both medically and economically. It is used to combat bacterial resistance to β-lactam antibiotics and is on the World Health Organisation’s List of Essential Medicines in combination with amoxicillin. An olive oil industry waste product, olive pomace oil (OPO), is a potential alternative carbon source for clavulanic acid production by Streptomyces clavuligerus. OPO is six times cheaper than glycerol, which is the current industry standard. The aims of this study were to examine if OPO can be used as a carbon source for clavulanic acid production and to compare the clavulanic acid yield achieved in shake flasks and 1.8 L bioreactors. It was observed that OPO was efficiently utilised as a sole carbon source by S. clavuligerus growing in a P-limited medium. The S. clavuligerus cells grew faster in OPO-containing cultures compared to the glycerol-containing cultures (control) and produced comparable levels of clavulanic acid, but much earlier. In cultures with ISP2 medium that contained glycerol or OPO, higher levels of clavulanic acid were obtained in shake flask cultures with OPO. Interestingly, the same levels of clavulanic acid were observed in oil-containing cultures in bioreactors, but 48 h earlier. Furthermore, the oil-containing cultures did not need addition of an antifoam agent, while higher levels of cell viability were maintained after 72 h in these fermentations compared to the cultures that contained glycerol. Our results suggest that OPO can replace glycerol for clavulanic acid production in S. clavuligerus fermentations, which will significantly increase the productivity and cut the cost for industrial clavulanic acid biosynthesis. The same carbon source can be tested in other similar fermentation approaches for the production of antibiotics or other valuable bioproducts.Graphical Abstract

2 clavulanic acid were obtained in shake flask cultures with olive pomace oil. Interestingly, the same levels of 28 clavulanic acid were observed in oil-containing cultures in bioreactors, but 48 h earlier. Furthermore, the oil-29 containing cultures did not need addition of an antifoam agent, while higher levels of cell viability were 30 maintained after 72 h in these fermentations compared to the cultures that contained glycerol. Our results suggest 31 that olive pomace oil can replace glycerol for clavulanic acid production in S. clavuligerus fermentations, which 32 will significantly increase the productivity and cut the cost for industrial clavulanic acid biosynthesis. The same 33 carbon source can be tested in other similar fermentation approaches for the production of antibiotics or other 34 valuable bioproducts.

43
The most common mechanism by which bacteria acquire resistance to beta-lactam antibiotics is through the 44 production of beta-lactamase enzymes. These enzymes cleave the beta-lactam rings of important antibiotics such 45 as penicillins and cephalosporins, leading to their inactivation. Clavulanic acid, produced by Streptomyces 46 clavuligerus, is used in conjunction with β-lactam antibiotics, acting as a suicide inhibitor that protects these drugs 47 from the β-lactamases of resistant bacteria [1]. The combination of amoxicillin and clavulanic acid is on the World 48 Health Organisation (WHO) list of essential medicines [2]. Due to the importance of this treatment, much research 49 has been done to improve the growth of S. clavuligerus, and the organism's production of clavulanic acid [3].

50
Different from most organisms, S. clavuligerus lacks a glucose transport system [4], so it is unable to utilise this 51 sugar. Instead, glycerol is the most popular carbon source used for industrial production of clavulanic acid [5].

56
Olive mills generate huge waste in short periods of time, which is seen as an environmental problem for 57 Mediterranean countries [7]. These wastes have high phytotoxicity, with studies also showing adverse effects on 58 soil microbial populations [8] and aquatic ecosystems [9]. While olive oil is cheaper than glycerol, and has been 59 shown to work better, it does not seem ethical to add to existing demand, and in turn, increasing waste. One of 60 these wastes from the olive oil industry, olive pomace oil (OPO) [10], has its own potential as a carbon source for 61 S. clavuligerus. Miranda et al. [11] show that there is around 52% carbon composition in the pomace, while the 62 pulp (where regular olive oil is obtained) has around 55%. This suggests that OPO likely has sufficient energy to 63 be a viable carbon source, still outperforming glycerol as olive oil was shown to. The average price of OPO is 64 £1.50 per L, which is six times less expensive than the standard carbon source glycerol (£9 per L). In addition to 65 being cost effective, OPO utilisation would also create a positive reuse for some of the vast amounts of waste 66 found in the olive oil industry. If accepted widely, this could bring huge economic and environmental benefits to 67 Mediterranean countries, and lower costs for clavulanic acid producers. Additionally, oil has natural anti-foaming 68 properties and can enhance secondary metabolism [12].

69
Lots of efforts have been devoted to the development of S. clavuligerus strains with the aim to improve the 70 production of clavulanic acid. These strain improvements, whether they were done through random mutagenesis 71 [13] or targeted modification of metabolic pathways [14], were usually carried out with glycerol in mind as the 72 carbon source. With food oils already yielding higher clavulanic acid production, and in some cases culture 73 growth, strain improvement of S. clavuligerus to increase the utilisation of these oils [15] could greatly improve 74 clavulanic acid production.

75
Although complex media are commonly used for industrial clavulanic acid production, defined media allow us to 76 test varying media ingredients. In this paper, we used defined media to test the growth of S. clavuligerus and the 77 yield of clavulanic acid using OPO as the sole carbon source. To assess the fermentation efficiency of different 78 carbon sources, glycerol was set as control to produce clavulanic acid. Because of the high carbon content, we 79 hypothesise that OPO will prove to be a suitable carbon source, likely more effective than the industry standard 80 of glycerol for clavulanic acid production. In addition, we believe that OPO will continue to be effective at higher 81 volumes as part of complex media. 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63

119
As can be seen in Figure 1A

126
Once it was determined that OPO was able to be utilised in a defined medium, it was then added as a carbon 127 source in a complex media (Figures 2A and 2B), which more accurately replicates the conditions in industrial 128 productions (albeit at a laboratory scale). In the first 24 h of growth in complex media, OD values obtained were 129 very similar between the carbon sources, but OPO cultures achieved higher growth at all time points recorded. In 130 addition, Figure 2B shows that OPO as a carbon source in complex media again led to the production of much 131 larger amounts of clavulanic acid (over double that of glycerol at 48 h). This is similar to previous results obtained 132 comparing clavulanic acid production in olive oil and glycerol [6, 18]. Cell viability was also greater in OPO 133 cultures across all experiments when spread onto plates (data not shown).

139
Using data from the bioreactor cultures, Table 1 shows that OPO outcompetes glycerol in all four aspects 140 measured: growth yield, growth rate, clavulanic acid yield, clavulanic acid rate. Growth yield and growth rate 141 values from OPO cultures were almost double that of glycerol cultures. Clavulanic acid yield between carbon 142 sources was closer, but OPO still appears the more favourable than glycerol (59.96 mg g -1 of carbon source and 143 46.16 mg g -1 of carbon source, respectively). Similarly, the rate of clavulanic acid production was 1.47 times 144 higher in OPO cultures than in glycerol cultures.

145
The effectiveness of OPO also has the potential to drastically increase through strain improvement targeted 146 specifically at the use of this carbon source. Industrial strains reportedly have a clavulanic acid production nearing 147 3 g L -1 which is far higher than wild type strains (around 25-120 mg L -1 ) [3]. Spontaneous mutations with oil as a 148 carbon source have been recorded [19], proving that this is viable. In combination with targeted mutagenesis,

149
there is an even greater potential for OPO. It would be interesting to see the levels of clavulanic acid produced by 150 these industrial strains when OPO is the carbon source.

151
It was also observed in these experiments that OPO acted as a strong antifoam, leading to negligible foaming of 152 the media compared to glycerol. This might partly explain the better results of OPO in the shake flask experiments 153 (antifoam was added to glycerol cultures in the bioreactor experiments). Foam in the glycerol cultures may have 154 impeded on gas exchange, negatively impacting on growth and clavulanic acid production [20]. Foam is generally 155 seen as undesirable in the production of antibiotics [21].

156
All three of these experiments indicate that OPO is a superior carbon source as it led to faster growth, faster 157 clavulanic acid production, and removed the need for an antifoaman additional cost when glycerol is used. In 158 other studies, fed-batch cultures have been shown to further increase the levels of clavulanic acid production [22].