Revalorisation of brewer's spent grain for biotechnological production of hydrogen with Escherichia coli

Abstract

Introduction: Agro-industrial wastes are generated in huge amounts triggering damages to the environment and human health. Therefore, there is an urgent necessity for its revalorisation into high-value compounds, including biofuels. One such wastes is the brewer's spent grain (BSG), a by-product of the beer industry, which is produced in vast quantities worldwide. The rich-fibre and protein content of BSG makes this waste a valuable resource for biotechnological applications, although the main challenge of this approach is to make the carbohydrates and proteins available for bacterial metabolisation into high-value products. This work aims to optimise a thermal-hydrolysis process to revalorise BSG by bacterial conversion into hydrogen (H₂), as a clean energy that can replace fossil fuels.

Methods: A 2k full factorial design method was employed hydrolysation of BSG and showed that temperature and acid concentration are significant factors that affect the extraction of reducing sugars (RS) and proteins. Subsequently, steepest ascent and central composite design (CCD) statistical methods were applied to determine the optimal conditions for hydrolysis.

Results: The optimised hydrolysis condition were 0.047 M H₂SO₄, 150°C, 30 min and 15% BSG, leading to the theoretical concentrations of 54.8 g RS/L and 20 g/L proteins. However, 5'-hydroxymethylfurfural (HMF) was generated in thermal-hydrolysis conditions at higher temperatures exceeding 132°C. Therefore, a screening of HBSGs fermentation using Escherichia coli was conducted in order to identify the most suitable conditions for maximizing H₂, as well as the production of volatile fatty acids (succinate and acetate) and ethanol. Among the tested conditions, HBSG A17 (117°C, 20 min, and 0.1 M H₂SO₄) yielded the highest H₂ production of 48 mmol/L in this work.

Conclusion: This study provides valuable insights into the optimisation of BSG pre-treatment for biotechnological applications, which may help in the selection of the most appropriate hydrolysis conditions based on the desired end product.

Keywords: Escherichia coli; biohydrogen; brewer’s spent grain; dark fermentation; design of experiment.

FIGURE 1

Scheme and description of pretreatment conditions and the design of experiment (DoE) methodology.

FIGURE 2

Effect of hydrolysis pre-treatment of BSG. Concentration before and after the pre-treatment in g/L: reducing sugar (RS) and total protein. S1, S2, and S3: 0.1 M, 0.2 M, and 0.3 M H₂SO₄, respectively; C1, C2, and C3: 0.1 M, 0.2 M, and 0.3 M HCl; N1, N2, and N3: 0.1 M, 0.2 M, and 0.3 M NaOH; K1, K2, and K3: 0.1 M, 0.2 M, and 0.3 M KOH and A: water. The data from each condition are obtained from one replicate and are shown in Supplementary Table S3.

FIGURE 3

Curves of reducing sugar (RS) and proteins (g/L); box plot of RS yield with respect to BSG and acid used. All results were obtained under 17 thermochemical conditions using one replicate, as described in Table 2 and Supplementary Table S5. A17, A18, and A19 are plotted independently but are considered as three replicates.

FIGURE 4

Standardised Pareto chart of RS yields as response variables: g RS/g BSG (A,B) and g RS/(g BSG mL acid) (C,D).

FIGURE 5

Curves of RS, proteins, and total carbohydrates analysed by HPLC (g/L). Box plots of temperature and sulphuric acid concentration obtained from HBSG samples in seven different thermochemical conditions using one replicate, as indicated in Supplementary Table S8.

FIGURE 6

Response surface curve for the optimisation of RS concentrations as a function of sulphuric acid and temperature.

FIGURE 7

Specific H₂ production (Y_H2/X) and volumetric H₂ production (Y_VH2/X) in Escherichia coli wild-type strain using 20% (A) and 40% (v/v) (B) from HBSG samples obtained under 17 different conditions, as described in Table 2, at 70 h after inoculation. Plots represent the average, and error bars represent the standard deviation using two biological replicates.

FIGURE 8

Analysis of the fermentative HBSG samples (A1–A17) in Escherichia coli using 40% (v/v) at initial (0 h) and at 70 h post-inoculum of carbohydrates and RS (g/L) (A) and VFA (acetate and succinate), ethanol, and pH at 70 h (B). Plots represent the average, and error bars represent the standard deviation using two biological replicates.