Description
Our project aims to remove heavy metals, in particular lead and cadmium, from sludge of water treatment plants and to produce ethanol from the cellulose it contain using an engineered Cupriavidus metallidurans. If the sludge used as fertilizer contains too much heavy metal, these elements can go into the crops. These metals are likely to cause various pathologies and their presence in the diet is a major problem. In order to reduce their concentrations in the sludge, we use a bacterium called Cupriavidus metallidurans. It is known for its ability to develop in extremely high concentrations of heavy metals, as it has a wide range of dedicated molecular resistance mechanisms. These mechanisms may consist of a flow or an aggregation/precipitation system. By inhibiting the efflux part of the mechanisms, we aim to increase the aggregation of metals by bacteria. Then the bacteria are removed from the sludge, allowing to extract the metals. Sludge also contains significant amounts of cellulose that can be used to produce ethanol. However, Cupriavidus metallidurans is not capable of degrading cellulose or using glucose to produce ethanol. However, by giving our bacteria genes for alcoholic fermentation and glucose uptake, and by applying pre-treatment of sludge with cellulase to degrade cellulose, we have made this possible.
Inspiration
Beauce (France), near the University of Orléans, is considered as the France’s attic. Moreover, right next to the University, there is a water plant treatment owned by the City of Orléans. This inking in the territory and this proximity make us concerned about the quality of the way sewage water is treated and the quality of the food produced by our lands. The spreading of sewage sludge on farmland, the main disposal route, is part of a circular economic strategy and aims to recover this waste.
Figure 1. Destination of sewage sludge in France.
Figure 2. Evolution of sewage sludge production in France.
Figure 3. Heavy metals accumulation in fruits and vegetables.
In 2011, 70% of sewage sludge in France is spread (Figure 1). Sludge has fertilizing properties because it contains a significant amount of organic matter, which is of agronomic interest. But they also contain undesirable elements such as metallic trace elements.
A decree of January 8, 1998 sets specific conditions for the spreading of sludge, which must comply with limit values for heavy metals and organic trace compounds. The reduction of the concentration of heavy metals in sludge in a biological way, using bacteria, will allow it to be used safely as a fertilizer.
We found it interesting to recycle sewage sludge in order to preserve its fertilizing effects and minimize the risk of contamination by heavy metals.
All these heavy metals can come from different sources such as industry (Cadmium, Chromium), automobile pollution (Lead) or rainwater runoff on roofs and roads (zinc, copper). These metals have a high toxicity, even at low doses.
A meta-analysis published in the British Medical Journal evaluated the effect of several heavy metals on our health (https://www.bmj.com/content/362/bmj.k3310) and shown the association of metals such as cadmium or lead with an increased cardiovascular disease risk.
A study published in 2011 in the journal Toxicology highlighted the oxidative effect of these metals which, at too high a dose, can damage DNA and cellular metabolism.
(https://www.sciencedirect.com/science/article/pii/S0300483X11000886?via%3Dihub)
We chose Cupriavidus metallidurans CH34 for its natural resistance to heavy metals. It has several heavy metal resistance operons on its two mega-plasmids pMOL30 and pMOL28[1]. Among all these mechanisms, Cadmium - based on the Czc operon - and Lead - based on the Pbr operon - have aroused our interest because they are well characterized and the former can be optimized for Cadmium aggregation. Its genetic material has been fully sequenced and annotated to facilitate its use for our purpose.
When we visited the water treatment plant, we noticed that cellulose is a major problem in purification machines, because it was present in very large quantities and blocks one of the stages of water treatment. We wanted to transform cellulose into bioethanol, a biofuel that can be used in gasoline engines. This process generally requires a first step of degradation of cellulose in glucose, followed by a second step of glucose degradation in ethanol, usually in alcoholic fermentation. However, Cupriavidus metallidurans is not able to do both tasks. The expression of the genes of alcohol dehydrogenase and pyruvate decarboxylase can answer this problem (link prot fusion BioBricks).
We then chose to use commercial purified cellulase to degrade cellulose to glucose and to have Cupriavidus Metallidurans express the glucose assimilation genes.
Our ideas were forged and refined in September, October and November. We wanted to have a project on ecology because it is a huge challenge for our generation.
References
[1] - Monchy S, Benotmane MA, Janssen P, et al. Plasmids pMOL28 and pMOL30 of Cupriavidus metallidurans are specialized in the maximal viable response to heavy metals. J Bacteriol. 2007;189(20):7417–7425. doi:10.1128/JB.00375-07
[2] – Janssen PJ et al. “The complete genome sequence of Cupriavidus metalliduans CH34, a master in harsh and anthropogenic environnemens.” PLoS One, may 5,
[3] - "Activation of Ethanol Production by Combination of Recombinant Ralstonia eutropha and Electrochemical Reducing Power" written by Bo Young Jeon, Jun Yeong Yi, Il Lae Jung, Doo Hyun Park, published by Advances in Microbiology, Vol.3 No.1, 2013 https://www.scirp.org/journal/PaperInformation.aspx?PaperID=29122
Part:BBa K1122673 - parts.igem.org. Available at: http://parts.igem.org/wiki/index.php?title=Part:BBa_K1122673 (Accessed: 28th June 2019)