Initial experiments demonstrated that E. coli could not be cultured under aerobic conditions on pure OMSW fibre hydrolysate. Growth limitation could have been caused by a variety of factors, including insufficient nutrients, nutrient inaccessibility, metal toxicity and/or the presence of unknown organic compounds. To assess the degree to which these factors were affecting growth a series of growth assays were carried out. OMSW fibre hydrolysate was supplemented with a source of nitrogen, sulphate or phosphate, either individually or in combination, and inoculated with E. coli LW06. OD600 was measured over 48 hours as a proxy for growth. Positive control cultures included cells grown on MOPS minimal medium with 5% w/v D-glucose and cells grown on OMSW fibre hydrolysate supplemented with all components of MOPS minimal medium (except a carbon source) to the same final concentration (for detailed methods see 4.2.3.2).
The first series of assays (Figure 4.6-A) showed that OMSW hydrolysate supplemented with all chemical components necessary for growth (i.e. MOPS minimal medium) enabled E. coli to reach an OD600 of ~5.5 (Figure 4.6-A, ‘Hydrolysate + Min. med.’). Cells grown on the positive control medium (MOPS minimal medium + 5% D-glucose) produced ~40% less biomass (Figure 4.6-A, ‘Min. med. + 5% glucose’) and no growth occurred on neat hydrolysate (Figure 4.6-A, ‘Hydrolysate (neat)’). This indicated that growth on neat hydrolysate was primarily constrained by nutrient limitation rather than substrate inhibition.
Growth of E. coli on the OMSW fibre hydrolysate was investigated further by supplementing with a source of sulphate (K2SO4), ammonium (NH4Cl) and phosphate (K2HPO4) at the same concentrations used in MOPS defined medium (Figure 4.6-B). In this series of assays cells grew to a slightly lower OD600 overall, likely due to variations in seed cultures or a difference in the shaker model used. However, when compared to the positive control, the results showed that growth was not significantly increased by the addition of phosphate or sulphate (Figure 4.6-B, ‘Hydrolysate + S’, ‘Hydrolysate + P’). In contrast, ammonium supplementation led to growth comparable with the MOPS minimal medium positive control (Figure 4.6-B, ‘Hydrolysate + N’), although it did not restore growth to the level of hydrolysate supplemented with all components of MOPS minimal medium (Figure
4.6-B, ‘Hydrolysate + Min. med.’). It was concluded that one or more nutrients were
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hydrolysate was supplemented with sulphate, ammonium and phosphate combinatorically (Figure 4.6-C).
Supplementation with both ammonium and phosphate (Figure 4.6-C, ‘Hydrolysate
N & P’) led to growth equivalent to the hydrolysate with minimal medium and hydrolysate
supplemented with all three nutrients (sulphate, ammonium and phosphate). Overall this demonstrated that the OMSW fibre hydrolysate was primarily limited in nitrogen, with a secondary deficiency in phosphate but no significant limitation in sulphate. Nutritional supplements employed in industrial fermentations are typically derived from waste products from other industries, such as corn steep liquor, yeast autolysate or casein hydrolysate, because they are abundant and low cost (Kampen, 2014). Two industrially relevant nutrient supplements were therefore trialled with OMSW fibre hydrolysate: Corn steep liquor (CSL) (a by-product of corn wet-milling) (Liggett & Koffler, 1948) and vitamin- enriched yeast extract (VYE) (a substitute for yeast autolysate, a by-product of the brewing industry)(Kerby & Vriesekoop, 2017). Unfortunately, addition of 1% CSL led to immediate and irreversible precipitation of the hydrolysate which made OD600 measurements impossible. Addition of 1% VYE only produced minimal precipitate formation, therefore this was trialled as an industrial nutrient adjunct.
Nutrients were measured in hydrolysate supplemented with 1% VYE as described in Chapter 3 (3.2.7.5) and are shown in Table 4.9 alongside the nutrient levels measured in neat OMSW fibre (originally presented in Chapter 3, Table 3.4). Addition of 1% VYE increased the microbially available nitrogen (shown as YAN) and total phosphorus approximately 10-fold, while sulphate levels stayed similar, with a slight decrease due to the dilution effect from VYE addition. When E. coli was grown on OMSW fibre hydrolysate supplemented with 1% VYE cells entered exponential phase more rapidly (Figure 4.6-D,
‘Hydrolysate + 1% VYE) and produced almost twice as much biomass as cells cultured on
hydrolysate supplemented with phosphate and ammonium (Figure 4.6-D, ‘Hydrolysate +
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Figure 4.6: Growth of Escherichia coli LW06 on OMSW fibre hydrolysate supplemented with nutrients
All growth curves are averages of three biological replicates. Each plot represents a separate experiment. Error bars show standard deviation from the mean for triplicate fermentations. A: OMSW fibre hydrolysate supplemented with MOPS minimal medium components (‘Hydrolysate + Min. med.’) or 40 mM MOPS buffer (‘Hydrolysate (neat)’). Positive control fermentation: MOPS defined medium with 5% D-glucose (‘Min. med. + 5% glucose’).
B: OMSW fibre hydrolysate supplemented with 0.3 mM K2SO4 (‘Hydrolysate + S’), 10 mM NH4Cl
(‘Hydrolysate + N’) or 0.5 mM K2HPO4 (‘Hydrolysate + P’). ‘Hydrolysate + Min. med.’ and ‘Min.
med. + 5% glucose’ as in A.
C: OMSW fibre hydrolysate supplemented with 10 mM NH4Cl and 0.5 mM K2HPO4 (‘Hydrolysate
+ N & P), 0.5 mM K2HPO4 and 0.3 mM K2SO4 (‘Hydrolysate + P & S), or 0.5 mM K2HPO4, 10 mM NH4Cl and 0.3 mM K2SO4 (‘Hydrolysate + P, N & S’). ‘Hydrolysate + Min. med.’ as in A.
D: OMSW fibre hydrolysate supplemented with 1% vitamin-enriched yeast extract (‘Hydrolysate
+ 1% VYE’) or excess ammonium and phosphate (20 mM NH4Cl and 1 mM K2HPO4) (‘Hydrolysate
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E. coli attained a final OD600 of ~8.0 on hydrolysate with 1% VYE, which contains
approximately 7% w/v D-glucose and D-xylose (the major metabolically accessible sugars for E. coli). This level of biomass production is proportional to cultures grown on MOPS minimal medium containing 5% D-glucose, which entered stationary phase at an OD600 of ~5.5 (Figure 4.5-A, ‘Min. med. + 5% glucose’). It is also greater than the average growth of
E. coli on the standard rich medium LB, which usually culminates at an OD600 of ~7.0
(Sezonov et al, 2007). In fact, the high level of growth observed with VYE could only be recapitulated by culturing E. coli on hydrolysate supplemented with an excess of ammonium and phosphate (20 mM NH4Cl2 and 1 mM K2HPO4, respectively) (Figure 4.5-D,
‘Hydrolysate + N & P (excess)’). However, these cells exhibited a longer lag phase
compared to cells grown with VYE. This was surprising as ammonia is the preferred nitrogen source of E. coli and amino acids (the primary nitrogen source in VYE) are used less efficiently so slower growth would be expected (Wang et al, 2016).
Overall, the model fermentative microorganism E. coli demonstrated efficient and unrestricted growth on nutrient-supplemented OMSW fibre hydrolysate. The biomass levels attained were commensurate with the available sugars and no notable substrate inhibition from metals or inhibitors was evident. Furthermore, VYE was shown to have potential as an industrially relevant adjunct for supplementing microbially accessible nitrogen and phosphate in OMSW fibre hydrolysate fermentations.
Nutrient
Concentration (mM)
Neat Hydrolysate Hydrolysate +1% VYE
mM ±SD mM ±SD
Yeast Available Nitrogen (YAN)* 4.85 ±0.14 46.63 ±4.06
Of which: Ammonia (NH3) 2.27 ±0.09 3.48 ±0.10
L-Arginine 0.03 ±0.01 0.82 ±0.03
Primary Amino Nitrogen (PAN) 0.32 ±0.01 5.28 ±0.51
Phosphate (PO4 as phosphorus) 0.44 ±0.003 3.83 ±0.17
Orthophosphate (PO43-) 0.11 ±0.001 0.63 ±0.01
Sulphate (SO42-) 15.24 ±1.21 14.85 ±0.52
Table 4.9: Levels of phosphorus, orthophosphate, sulphate, nitrogenous compounds and calculated yeast available nitrogen (YAN) in OMSW fibre hydrolysate and OMSW
fibre hydrolysate supplemented with 1% vitamin-enriched yeast extract (VYE).
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4.3.2 Time-Course Kinetics of Eight Species Grown on OMSW Fibre Hydrolysate