Technology

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Bioprocess

Electronic equipment is the fastest growing waste stream in many countries. E waste grows rapidly because markets in which these products are produced cross the other side of the ‘Digital Divide’. We stand in front of the following problem: what we are we going to do with all those computers, smartphones which we buy? All these products become obsolete or unwanted within 1-3 years of purchase. Where will we find a new source of metals necessary to fabricate electronic equipment?
Using old and broken WEEE in our project not only gives us a source of metals to produce new smartphones or computers, but also solves problem of storing WEEE.

This figure shows a view on the context of re-use of EEE or its components. At the end of the use EEE returns to producers or to companies, which specialize in reuse of e-waste. The next step is preparation for re-use. On this step waste electronic equipment is deconstructed. Not only computer memories or other parts which contains valuable metals are re-use. We also want to recover such materials as plastic or glass. That step requires labor work or special machines which will prepare WEE for another point of the plan. Some parts of the TVs or computers may not be useful in another manufactory. Sometimes we need to remarked parts which we need and work in place with specialist equipment. Later we can use our bacteria with lanthanides binding sequences to re-use lanthanides.
1) Removing from WEEE plastic and stirring parts which are abundant of rare earth metals such as lanthanides, especially computer memories. Stirring increase availability WEEE with acid which is produced by Thiobacillus ferrooxidans. It display terbium from alloy.
2) Effluent from our bioreactor has low pH (about 2,5) what is a lethal for E.coli. That is why we add Ca(OH)₂ to effluent. It increases our pH to a level which is optimal to E.coli (about 7). Ca(OH)₂ is also cheap and it is not increasing cost of the process very much.
3) Effluent goes to packed column bioreactor in which E.coli is immobilized. It gets across whole column. Ions of terbium binds and senses trough periplasmic domain.
4) Column (with no initial effluent) is flushed by NaCl. PmrB can easily denaturate in NaCl aq , losing shape. Ions of terbium do not precipitate with NaCl aq and do not make insoluble components with it.
5) Ions of terbium are recovered from solution by electrolysis

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Discussion

Our process finds not only a way to recovery the lanthanides, but also other metals such as copper ( it takes place at first point of it and other rare metals.
Our way of recovery lanthanides does not produce pollution but it also is a safe way of managing with WEE.

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Challenges

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Alternative methods

Our final system was of course not the only possibility. There were some points where we had to decide...

Reporter protein

Finally we decided for GFP protein because of it's versality, simplicity and fact that GFP does not require any additional reagents (eg. IPTG).
We could have used other fluorescent proteins, for instance superfolder fluorescent proteins constructed by iGEM Warsaw 2013 Team, but regular GFP was the simpliest choice.

Binding agent

Although, unfortunately, we could not have implemented lanthanide binding system because of lack of time, we had several ideas how to accomplish this.
Poly-LBT peptide
First was to construct a poly-LBT peptide which would be then anchored in the outer membrane of E. coli or transported to periplasm. We discarded this idea due to problems with modelling of behaviour of this polymer and problems with wet-lab design.
PmrB over-expression
Another idea consisted of PmrB dependent over-expression of PmrB(LBT) protein. In such system we would have pmr^C promoter - some logical device to boost the signal - PmrB(LBT), so in presence of lanthanides amount of PmrB(LBT) protein per cell would rise sharply, which should allow effective binding of lanthanides.
Small peptide fused with LBT
Our final and probably best idea was to create a construct peptide of such composition:
BBa_J32015(E. coli periplasm signal peptide)-structure peptide(ubiquitin or 1L2Y [BBa_K1459015]) - lanthanide binding tag.
The plan was to create a small, 'rubbish' protein which would only bind lanthanides without having any physiological function in cell (since we were afraid whether over-expression of PmrB would be cytotoxic).

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