Boosting d-lactate production in engineered cyanobacteria using sterilized anaerobic digestion effluents

Bioresour Technol. 2014 Oct;169:462-7.

Hollinshead WD1, Varman AM2, You L1, Hembree Z1, Tang YJ3.

1Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130, USA.

2Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130, USA; Biological and Materials Science Center, Sandia National Laboratories, Livermore, CA 94550, USA.  Electronic address: [email protected]

3Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130, USA. Electronic address: [email protected]

 

Abstract

Anaerobic digestion (AD) is an environmentally friendly approach to waste treatment, which can generate N and P-rich effluents that can be used as nutrient sources for microalgal cultivations. Modifications of AD processes to inhibit methanogenesis leads to the accumulation of acetic acid, a carbon source that can promote microalgal biosynthesis. This study tested different AD effluents from municipal wastes on their effect on D-lactate production by an engineered Synechocystis sp. PCC 6803 (carrying a novel lactate dehydrogenase). The results indicate that: (1) AD effluents can be supplemented into the modified BG-11 culture medium (up to 1:4 volume ratio) to reduce N and P cost; (2) acetate-rich AD effluents enhance D-lactate synthesis by ∼ 40% (1.2g/L of D-lactate in 20 days); and (3) neutral or acidic medium had a deleterious effect on lactate secretion and biomass growth by the engineered strain. This study demonstrates the advantages and guidelines in employing wastewater for photomixotrophic biosynthesis using engineered microalgae.

Copyright © 2014 Elsevier Ltd. All rights reserved.

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Significance Statement

“Milking” D-lactate from cyanobacteria

Microalgal photo-biorefineries may replace sugar-based microbial fermentations for the production of commodity chemicals and biofuels. In this study, we have genetically engineered a cyanobacterium (blue-green algae), Synechocystis 6803, for the synthesis of optically pure D-lactic acid (used for the manufacture of biodegradable plastics) from CO2. This strain carries a novel D-lactate dehydrogenase (enzyme for lactate synthesis) and a transhydrogenase (enzyme to power lactate synthesis), producing about 1 g/L D-lactate from CO2. To reduce cyanobacterial cultivation costs, we have integrated lactate biosynthesis with anaerobic digestion of organic wastes. The effluents accumulated from this waste treatment process contain nitrogen, phosphorus, and acetate. We monitored the cyanobacterial growth in wastewater via the absorbance measurements of extracted chlorophyll a (using a biomass correlation of OD730=3.3 x OD663, OD: optical density). A series of tests indicated that the use of acetate-rich wastewater can help reduce culture medium costs and promote D-lactate synthesis.  More importantly, we have found that an alkaline cultivation pH is crucial for D-lactate secretion. Typically, cyanobacteria grow best in the neutral pH.  But, in order to “milk” lactate out of cell (intracellular pH~7), we have to gradually raise the culture medium pH (up to ~10). This is because cell membrane only allows the free diffusion of non-charged lactic acid. Taken during the peak of lactate production phase, our most recent intracellular lactate measurement (using 13C-lactate as the internal standard) found that intracellular lactate is around 20 mg/g dry cell. Increase of culture medium pH can reduce the presence of non-charged lactic acid in the medium and thus facilitate cyanobacterial lactate secretion.  Under optimal control of light, carbon/nutrient sources, and pH conditions, engineered cyanobacteria can accumulate up to 2g/L extracellular lactate (much higher than intracellular lactate level).

In addition, our study provides several guidelines for using wastewater to culture engineered cyanobacteria: 1) waste effluents may inhibit photosynthesis, therefore requiring dilution with normal culture media (1:4 volume ratio) to achieve favorable cell growth; 2) the effluents should be sterilized to avoid the bacterial consumption of lactate; 3) the effluents’ pH should be adjusted, as the engineered strain is sensitive to acidic pH conditions. Although there are roadblocks for effective photobiorefineries, cyanobacterial bioprocesses are promising alternatives for both the economical synthesis of commodity chemicals and waste management (by stripping the wastewater of soluble C/N/P and by reducing the emission of greenhouse gases).

 

Figure: Comparison of a cyanobacterial bioprocess to that of a traditional sugar-fermentation process.

Boosting D-lactate production in engineered cyanobacteria using sterilized anaerobic digestion effluents .Renewable Energy Global Innovations

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