Development of a novel, robust and cost-efficient process for valorizing dairy waste exemplified by ethanol production

Abstract

Background: Delactosed whey permeate (DWP) is a side stream of whey processing, which often is discarded as waste, despite of its high residual content of lactose, typically 10-20%. Microbial fermentation is one of the most promising approaches for valorizing nutrient rich industrial waste streams, including those generated by the dairies. Here we present a novel microbial platform specifically designed to generate useful compounds from dairy waste. As a starting point we use Corynebacterium glutamicum, an important workhorse used for production of amino acids and other important compounds, which we have rewired and complemented with genes needed for lactose utilization. To demonstrate the potential of this novel platform we produce ethanol from lactose in DWP.

Results: First, we introduced the lacSZ operon from Streptococcus thermophilus, encoding a lactose transporter and a β-galactosidase, and achieved slow growth on lactose. The strain could metabolize the glucose moiety of lactose, and galactose accumulated in the medium. After complementing with the Leloir pathway (galMKTE) from Lactococcus lactis, co-metabolization of galactose and glucose was accomplished. To further improve the growth and increase the sugar utilization rate, the strain underwent adaptive evolution in lactose minimal medium for 100 generations. The outcome was strain JS95 that grew fast in lactose mineral medium. Nevertheless, JS95 still grew poorly in DWP. The growth and final biomass accumulation were greatly stimulated after supplementation with NH₄⁺, Mn²⁺, Fe²⁺ and trace minerals. In only 24 h of cultivation, a high cell density (OD₆₀₀ of 56.8 ± 1.3) was attained. To demonstrate the usefulness of the platform, we introduced a plasmid expressing pyruvate decarboxylase and alcohol dehydrogenase, and managed to channel the metabolic flux towards ethanol. Under oxygen-deprived conditions, non-growing suspended cells could convert 100 g/L lactose into 46.1 ± 1.4 g/L ethanol in DWP, a yield of 88% of the theoretical. The resting cells could be re-used at least three times, and the ethanol productivities obtained were 0.96 g/L/h, 2.2 g/L/h, and 1.6 g/L/h, respectively.

Conclusions: An efficient process for producing ethanol from DWP, based on C. glutamicum, was demonstrated. The results obtained clearly show a great potential for this newly developed platform for producing value-added chemicals from dairy waste.

Keywords: Corynebacterium glutamicum; Ethanol production; Lactose-utilization; Medium optimization.

Fig. 1

Construction of the lactose-metabolizing C. glutamicum strain. Aa Introduction of the lacSZ operon into the chromosome of C. glutamicum; Ab lactose catabolism in the C. glutamicum strain with integrated lacSZ operon; Ba additional chromosomal introduction of the galMKTE operon; Bb lactose catabolism in the C. glutamicum strain harboring both the lacSZ and galMKTE operons

Fig. 2

Growth comparisons of JS95, JS93 and JS34 on the BMCG medium with different carbon sources. The experiments were performed using a microbioreactor (Biolector). The standard deviations were calculated from three independent experiments. AU arbitrary units, which is the parameter indicating cell density for the Biolector

Fig. 3

Ethanol batch fermentation by JS122 on DWP under oxygen deprivation conditions. The cell density was 24 g/L CDW. The standard deviations were calculated from three independent experiments

Fig. 4

One representative ethanol fed-batch fermentation by JS122 on DWP under oxygen deprivation conditions. DWP contains 95 g/L lactose and 5 g/L galactose, and after 24 h and 48 h, 50 g/L of lactose was added. The cell density was 24 g/L CDW

Fig. 5

Ethanol production by recycled cells under oxygen deprivation. The left column shows the titer (g/L) of ethanol achieved by each recycle batch reaction. The right column shows the average time (h) of each recycle batch. The standard deviations were calculated from three independent experiments