ARALEX

Autocatalyst Recycling via Advanced Leaching Extraction using oXalate

The current problem

Too important to Waste

Platinum Group Metals also known as PGMs are rare and valuable elements found in countless of our everyday technologies, from automobile catalytic converters to all kind of electronics they are essential in everyday life. They also play a critical role in our path to a green and circular economy composing main elements in fuel cells, hydrogen electrolysis and sustainable chemistry. Yet every year, about 10 billion dollars’ worth of PGMS are discarded worldwide, with less than 25% ever being recovered [1, 2].

The scarcity of these metals is striking, Platinum occurs at only 0,005ppm in the Earth´s crust (5 parts per billion), that is equal to an amount of 5 nanograms per gram of earth’s crust [3]. To extract only 31 grams of Platinum, up to 500 tons of rock are needed to be mined and processed. This comes at a staggering cost of 26 to 39€ per Gram in South African mines [4]. PGM mining also leaves an enormous ecological footprint, the production of only 1kg of Platinum emits around 30.000 kg of CO2 together with a variety of heavy metals and toxic waste [5].

The fragility of PGM supply chains is also a known problem. More than 80% of our global PGM reserves are located in South Africa, making the market highly vulnerable to any kind of strikes, floods or political instabilities [6]. This fragility drives extreme price volatility, Rhodium being a perfect example, reaching a price of over 450€ per gram in the year 2021, that is estimated to be more than 15 times the price of gold [7].

We burn money daily in our cars’ exhaust pipes. A single automobile catalytic converter contains between 1-3g of Platinum, 2-7g of palladium and about 0,2-0,5g of Rhodium, which all together are valued at 300-1000€ per car [8, 9]. Without an effective recycling system most of these metals are simply lost and respectively discarded.

If humanity wants a sustainable future on this planet, the choice is clear, recycling our PGMs is not just an economic necessity, but an imperative for our planet’s environment an PGM reserves [1, 5, 10].

The idea

There are currently two main methods for recycling PGMs:

Pyrometallurgy:

Is often used industrially due to high recovery rates
Requires special equipment and high temperatues (up to 2000°C), leading to high energy demands
Oven needs to be operational continuously, so secondary resources are fed, leading to impurities
Leads to volatile waste and slag production, like sulfur dioxide emissions, as well as CO and Cl₂ during separation

Hydrometallurgy:

Milder temperatures and lower energy costs
Uses chemical leaching with reagents like aqua regia (HCl/HNO₃), cyanides or chloride-based systems, leading to hazardous waste and gas emissions
Washing stages produce a lot of wastewater [Yakoumis et al, 2021]

Bioleaching offers an environmentally friendly alternative for recycling PGMs. It does not use corrosive chemicals, require high temperatures, nor does it involve emitting hazardous gases. Normally, fungi like A. niger are used to produce organic acids with the ability of forming complexes with PGMs, but we wanted to try using genetically modified bacteria to produce this acid [Pathak et al, 2022].

Organism

Corynebacterium glutamicum

Although in literature Aspergillus niger is often used as a natural oxalic acid producer [Cameselle, 1998], we chose Corynebacterium glutamicum as our production platform. This gram-positive bacterium is a well-established host organism and easy to manipulate genetically. Another reason for our decision towards a bacterial system is the undesirable ability of fungi like A. niger to form spores [Lyu et al, 2025].

In industrial biotechnology, C. glutamicum is well-known for producing amino acids [Bramkamp, 2025] and was recently named „Microbe of the Year 2025“ [VAAM, 2025]. Its significance is also evident in our own institution - many researchers here work with this strain, and their expertise helped us in creating our own production strain C. glutamicum-oahA.

Metabolic Engineering

A common substrate like glucose (yellow) is added to the bacterial suspension. This substrate is consumed by the bacteria. With the help of our inserted gene fragments (red), this uptake is partially converted into oxalic acid by modified metabolic pathways. Corynebacteria are known to store large amounts of metabolic products in the extracellular lumen as an additional substrate source. So as oxalic acid is produced, it is exported into the lumen where it can be detected and further harvested for following leaching of PGMs (blue).

Plasmid

pGiga_oahA

Since C. glutamicum does not produce oxalic acid in its metabolism on its own, the necessary gene needed to be transferred into the bacteria via a vector:

The Gene for oxalic acid production (encoding oxaloacetate hydrolase/oxaloacetate acetylhydrolase) from Aspergillus niger was synthesized -> oahA-gene
The gene was amplified and subsequently cloned into pEKEx2 and pPBEx2 shuttle vectors using Gibson Assembly.

pEKEx2 and pPBEx2

pEKEx2 and pPBEx2 are shuttle vectors for E. coli and C. glutamicum used for the production of proteins with the help of IPTG-induction (isopropyl-beta-D-thiogalactopyranoside). The plasmids of our bacteria include the following:

oahA: encoding oxaloacetate hydrolase/oxaloacetate acetylhydrolase
KanR: Kanamycin resistance was used for the selection of successfully transformed cells
LacIq + Tacl promotor: The lacIq gene encodes a variant of the LacI repressor that is expressed at elevated levels, resulting in enhanced repression of the tac promoter. This tight regulation minimizes leaky expression of the target gene under non-induced conditions and enables robust transcriptional activation upon the addition of the inducer IPTG
M13 fwd / M13 rev: primer binding sites
pEKEx2 contains the C. glutamicum origin of replication (ori) from pBL1 and the E. coli ColE1 replicon

In comparison

Weakness of pEKEx2: Leaky gene expression in pEKEx2-derived plasmids due to a less functional LacI repressor with a modified C-terminus and duplicate DNA sequences in the plasmid backbone -> plasmid instability.
pPBEx2 is a pEKEx2-derivate -> contains a restored lacI gene and has no longer the unnecessary duplicate DNA sequences

To account for possible leakiness in pEKEx2, experiments were always carried out with both variants.

Optimization

In order to optimize the oxalate production, we followed two different approaches:

Changing the cultivation parameters
Engineering the strain itself

Since we used a inducible plasmid as carrier for the oxalacetate hydrolysate gene, we tested different induction times and inducer concentrations in our cultivations. This way, we were able to determine the optimal production conditions. The experiments were carried out in BioLector or shake flask cultivations and oxalate production was measured using an enzyme assay.

To obtain a more stable production strain without the need for antibiotic selection, we are planning to integrate the oahA gene into the C. glutamicum genome. For this, we will introduce the gene via NEB Hifi DNA Assembly into the integration vector pK19mobsacB, which will then be electroporated into the bacteria. After selection by antibiotic resistance markers, the confirmed mutants can be tested for oxalate production.

Integration of oahA_CG with lacI repressor system into the genome of *C. glutamicum*.

Other not yet realized optimization strategies include the cultivation under nitrogen limitation as well as supplementing e.g. ethanol, since this has been proved to elavate organic acid production in some cases. Another consideration would be transporter engineering to enhance oxalate export. [Zhou et al, 2023; Guillaume et al, 2021]

Chemistry

Coordination chemistry

The current process follows a simple experimental setup:

Leaching: Oxalic acid and PGM dust are solved in water and the solution is heated up in an oil bath. The duration of the experiment and temperature are varied to find the optimum.
Distillation: After filtering the solution to get rid of excess PGM dust, the solution is distilled to boil away the water.
Analysis: Analysis of the samples is performed via Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES).

For the future, we did some literature research on how we could improve the leaching rates. One approach we want to try is pre-treating the PGM dust via ultrasound following a Box-Behnken DoE to minimize experimental runs. We'll then perform response surface methodology (RSM) to find the optimum across the parameter space [Karim et al, 2020].

What a final product could look like

References

1. CME Group. Platinum recycling and the circular economy [Internet]. 2023 [cited: 19. Aug. 2025]. URL: https://www.cmegroup.com/articles/2023/platinum-recycling-and-the-circular-economy.html

2. Thermo Fisher Scientific. Platinum group metal recovery from spent catalytic converters using XRF [Internet]. 2020 [cited: 19. Aug. 2025]. URL: https://www.thermofisher.com/blog/metals/platinum-group-metal-recovery-from-spent-catalytic-converters-using-xrf/

3. Wikipedia. Platinum [Internet]. 2025 [cited: 19. Aug. 2025]. URL: https://en.wikipedia.org/wiki/Platinum

4. Mining Weekly. PGM recycling and low-grade resources offer opportunities in low price environment [Internet]. 2024 Apr 15 [cited: 19. Aug. 2025]. URL: https://www.miningweekly.com/article/pgm-recycling-lower-grade-resources-offer-opportunities-in-low-price-environment-2024-04-15

5. The Chemical Engineer. Worth its weight: Recycling platinum group metals [Internet]. 2020 [cited: 19. Aug. 2025]. URL: https://www.thechemicalengineer.com/features/worth-its-weight/

6. MDPI Crystals. PGM resources and recycling potentials. Crystals [Internet]. 2023;13(4):550 [cited: 19. Aug. 2025]. URL: https://www.mdpi.com/2073-4352/13/4/550

7. Wikipedia. 2000s commodities boom – Rhodium [Internet]. 2025 [cited: 19. Aug. 2025]. URL: https://en.wikipedia.org/wiki/2000s_commodities_boom

8. ResearchGate. Removal of platinum group metals from the used auto catalytic converter [Internet]. 2008 [cited: 19. Aug. 2025]. URL: https://www.researchgate.net/publication/26583964_Removal_of_platinum_group_metals_from_the_used_auto_catalytic_converter

9. RoadSumo. How much platinum, rhodium and palladium is in a catalytic converter? [Internet]. 2024 [cited: 19. Aug. 2025]. URL: https://roadsumo.com/how-much-platinum-rhodium-and-palladium-is-in-a-catalytic-converter/

10. MDPI Minerals. Recycling rates of PGMs in industry vs. electronics. Minerals [Internet]. 2021;11(7):673 [cited: 19. Aug. 2025]. URL: https://www.mdpi.com/2075-163X/11/7/673

11. Yakoumis et al. (2021): Recovery of platinum group metals from spent automotive catalysts: A review, Cleaner Engineering and Technology, Volume 3. 100-112. URL: https://doi.org/10.1016/j.clet.2021.100112

12. Pathak et al. (2022): Recycling of platinum group metals from exhausted petroleum and automobile catalysts using bioleaching approach: a critical review on potential, challenges, and outlook, Reviews in Environmental Science and Bio/Technology, Volume 21. 1035-1059 URL: https://doi.org/10.1007/s11157-022-09636-x

13. Cameselle et al. (1998): Oxalic acid production by Aspergillus niger, Bioprocess and Biosystems Engineering, Volume 19. 247-252. URL: https://doi.org/10.1007/PL00009017

14. Lyu et al. (2023): “Heterogeneity in Spore Aggregation and Germination Results in Different Sized, Cooperative Microcolonies in an Aspergillus niger Culture.” mBio vol. 14,1 (2023): e0087022. URL: https://doi.org/10.1128/mbio.00870-22

15. Bramkamp, M. Corynebacterium glutamicum: Modellorganismus der bakteriellen Zellbiologie. Biospektrum 31, 9–13 (2025). URL: https://doi.org/10.1007/s12268-025-2373-4

16. VAAM: Microbe of the Year 2025: Corynebacterium glutamicum – a hidden champion. URL: https://vaam.de/en/microbiology-portal/microbe-of-the-year/microbe-of-the-year-2025/

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18. Billerach, Guillaume et al. (2021): Impact of nitrogen deficiency on succinic acid production by engineered strains of Yarrowia lipolytica, Journal of biotechnology vol. 336. 30-40, https://doi.org/10.1016/j.jbiotec.2021.06.001.

19. Karim et al. (2020): Ultrasound-assisted nitric acid pretreatment for enhanced biorecovery of platinum group metals from spent automotive catalyst, Journal of Cleaner Production vol. 225. 120-199, https://doi.org/10.1016/j.jclepro.2020.120199.