The substrates, selected for enzymatic hydrolysis represent a wide range of CC waste streams regarding composition and purity of material. Substrate H1 is the most simple pre-consumer material, made of pure cotton, non-woven, post-industrial medical waste, with low amounts of contaminants. This substrate was chosen as a starting point to demonstrate that production of terpenes with the glucose juice is possible. As expected this material was efficiently degraded, reaching the maximum concentration of glucose 46 g/L after 30 h of enzymatic hydrolysis, decreasing to 43 g/L final glucose concentration. In contrast the hydrolysis of H2 yielded only about 30 g/L glucose final concentration. Overall the hydrolysis proceeded much slower even though, both H1 and H2 are non-coloured 100% cotton containing materials. Surprisingly the shorter fibre length and less homogeneous structure of H2 seem to have impacted the efficiency of enzymatic hydrolysis. Substrate H3, which represents reality, post-consumer textile waste, woven and knitted, made of 100% cellulose, followed the curve of the RESYNTEX for the first 4h and reached 20 g/L within 24h. The glucose concentration was oscillating strongly with substrate H3, reaching 40 g/L within 50 h of its start, then decreased to 24 g/L and again increased to 45 g/L. This oscillation of the glucose concentration was not expected. The hypothesis here is that glucose might be entrapped in the cellulose matrix or by the Cellic CTec2 enzyme mix, hiding the OH residue of glucose, not allowing it to react with glucose-oxidase (GAGO) and in this manner influencing the measurement (Add ref).
H4 substrate contains 50% polyethileneterephtalate which seems not to have any negative impact on the enzymatic hydrolysis and the production of limonene. This substrate was selected, to test whether the chemical composition (PET/CELL) of the waste material, can reduce the efficacy of the enzymatic hydrolysis, inhibit cell growth or the final glucose concentration. Enzymatic hydrolysis of H4 was also quite slow, 20 g/L within 24 h, experiencing a sudden drop to 8 g/L and a rather slow linear increase of glucose concentration, reaching a concentration of 40 g/L after 8 days of treatment.
Substrates H5 and H6, that both consist of cardboard material, did not degrade well using enzymatic hydrolysis. The reaction was comparable with all the other substrates within the first 24 h of treatment, but did not exceed 25 g/L after 8 days of treatment. The low yield of glucose is probably the consequence of additives that are used in the production of cardboard.
Altogether, all six CC waste streams materials could be solubilized with Cellic CTec2 enzyme blend, reaching a similar final (40g/L) concentration as observed in the RESYNTEX project, except for material H5 and H6. However, only post-industrial waste (H1) could be degraded in a similar time frame as previously observed during the RESYNTEX project, indicating that process optimization is needed for every type of CC waste stream material. Especially enzymatic hydrolysis of post-consumer waste (H2, H3, H4, H5 and H6) is dependent on the waste material composition and will require customised modifications to optimize this process, especially in the pre-treatment phase. In order to make enzymatic hydrolysis economically viable and improve this process a new enzyme bled has been developed - Cellic CTec3, coupled with enzyme recycled 22 and can in this manner lower the overall costs of this process. However, the process of enzymatic hydrolysis will need to undergo customized optimization for every waste stream source. One solution to the problem of substrate diversity could be to mix different CC waste streams into blends that have a steady supply, defined chemical composition and fibre structure. In this way optimizations of enzymatic degradations would have to be done only once for each such blend.
In order to mimic growth during the production process, E. coli_BL21 transformed with pJBEI-6410 was grown in M9 or LB media, supplemented with pure glucose or one of the glucose juices. All cultures were growing under the tested conditions most of them reaching an OD of 2. Interestingly the cell growth during the production process (figure S2), using pure glucose as carbon source, seems to happen in two separate exponential growth phases, which might explain the lower yield of limonene with the CC waste stream derived from glucose juices (Fig. 2). The maximum OD was 2.5 with H4 in LB media, but this did not result in higher limonene titters. On the contrary, the culture grown in LB media with H3 the optical density was only 1.5 indicating growth retardation, when using H3 in combination with LB media, but not in M9 media. These results indicate that higher cell density does not necessarily mean high limonene production. Growth curves for the substrate H5 and H6 could not be established because the addition of those two substrates caused turbidity of the media, which caused inaccurate measurements of OD.
The highest titter of limonene was produced with glucose juice in M9 media using H1 as carbon source. Around 650 mg/L limonene was produced which represents about 50% of the production with pure glucose (1300 mg/L) in M9. Using substrates H2 and H3, only 300 mg/L could be produced in M9 media. While the reduced yield was somehow anticipated in M9 media with the post-consumer cellulose waste H3, it was not for H2, since H2 is also 100% cotton, non–coloured post-industrial similar to H1. The difference in production between H1 and H2 is likely due to additives that are used or due to fewer contaminants that are deposited during the manufacturing process. Since H1 is used as medical material, the production process of H1 must use fewer additives and have a stringent production process that does minimize contamination during the production process. The yield with H4 was about half of that produced with H1 and 25% of the limonene produced with pure glucose. Substrate H4 is of special interest because of its chemical composition, which is made of 1:1 PET : Cellulose. This implies that M9 media supplemented with pre-consumer glucose juice, H1, contains fewer contaminations that impair production, but do not inhibit growth (figure S2) as compared to the post-consumer waste, H2, H3 and H4. Yield with H5 and H6 was higher compared with to yield of H2, H3 and H4, although the M9 production media became turbid after addition of the glucose juice. However the production was not impaired by the developed turbidity and about 500 mg/L limonene could be produced, more than with post-consumer textile waste (H2, H3 and H4).
In general, the yield was lower with LB media, regardless of glucose source. The maximum yield was around 1100 mg/L with pure glucose, while with H1 it was limited to about 200mg/L. The yield of all the other substrates was below 20 mg/L. No limonene was produced with glucose juice H3 in LB-media. Overall higher production levels have been achieved with defined M9 media, especially while using glucose juice as carbon source. Production in LB media seemed to be impacted by compounds that can impair production of limonene in E. coli, but not the growth.
An important factor for the use of glucose juice as substrate for production of limonene is that sterile filtered glucose juice was stable for more the 12 months at 4°C. Similar levels of limonene can be produced from fresh glucose juice and stored glucose juice (Fig. 2). In addition, other terpenes, like linalool, can be produced from glucose juice (data not presented).
In comparison with other efforts to produce limonene via synthetic biology using the plasmid pJBEI-6410 we have reached similar levels of limonene 20. Production can be further increased with optimization efforts like principal component analysis of proteomics (PCAP), increasing the production of limonene by 40% 23. Further improvements were made by integrating the SGC that is encoded on the plasmid pJBEI-6410 into the chromosome of E. coli, in order to improve genetic stability of the production strain and the titters it can produce24.