SymBioLoop

SYMBIOLOOP was born at the end of our value chain. We develop multi-purpose plastics specifically designed for a circular economy, replacing harmful fossil plastics. These materials are bio-based, biodegradable, high-performance and recyclable in closed LOOPs. But a key hurdle remains: our monomers—long-chain dicarboxylic acids (DCAs)—essential to unlock the full potential of our materials, are not available at competitive prices, even from fossil fuels.
Our interdisciplinary consortium of five partners aims to disrupt the status quo. Our mission is to enable bio-based production of long-chain DCAs at ultra-low costs. We have access to genetically engineered yeast strains capable of converting natural fatty acids to DCAs through an enzymatic ω-oxidation bioconversion, outperforming traditional chemical methods. Although our bioconversion has been validated at TRL 7 in large-scale fermenters, high costs due to glucose demand, fatty acid substrate expenses, and costly downstream processing remain barriers to competitive pricing for commodity plastics.
We recognized the need for a disruptive change to overcome these limitations. Our goal is to integrate our yeasts into algal biofilms through SYMBIOtic co-cultivation using only waste streams as feed. A biofilm enables effective yeast immobilization, keeping biomass out of the product stream, drastically reducing downstreaming costs, enabling a continuous process, and reducing carbon source requirements.

Feedstock

• Our bioconversion directly converts fatty acids to DCAs via enzymatic ω-oxidation, preserving the entire fatty acid carbon chain. DCA product costs dependent on the fatty acid feedstock, glucose and downstream processing. Currently used palm kernel oil esters—a major cost driver pre-SymbioLoop—should be replaced by three local waste streams:
  • Black soldier fly larvae fat (BSLF) from food waste conversion (MS1)
  • Used cooking oil (UCO; MS2)
  • „Plastic fat“ – byproduct of plastic waste upcycling with oxygen (MS3)
• These waste streams complement each other in their fatty acid profiles: BSLF is rich in lauric acid (C12), UCO provides mostly C16 and C18, and plastic fat provides C10–C30, giving us a versatile substrate library. The different chain lengths of the fatty acids and DCAs strongly influence the properties, i.e. bioconversion and downstream process conditions are different for each intermediate.
• BSLF and UCO were received from different suppliers in kg-quantities as crude, partly unsaturated triglycerides with solid impurities and off-colors. Our performance polymers require saturated carbon chains, so we had to hydrogenate the waste streams. To minimize costs, we used established fat hardening (think margarine production) with NysofactⓇ 120 (BASF) and hydrogen, which quantitatively converted BSLF and UCO to saturated fats on a kg scale.
  We transesterified the saturated triglycerides with cat. H2SO4 to fatty acid ethyl esters (FAEEs), as ethyl moieties are most readily bioconverted (vide infra). FAEEs can be used directly as the natural mixtures or distilled to obtain FAEEs of desired chain lengths. From BSLF, we isolated pure ethyl laurate (C12; 99% GC), the major component (40–45%), to produce pure C12 DCA.
• Plastic fat (MS3) is a byproduct of oxidatively fragmenting waste polyolefins with heat, air, and MnO₂ catalyst to produce long-chain DCAs. Pre-SymbioLoop, fatty acids have been major side products available for bioconversion to DCAs. After our initial failed scale up attempts, we found that adding steel balls to mimic a fluidized bed reactor drastically improved yield and selectivity by forming thin polymer films, increasing oxygen uptake and mixing. This serendipitous finding boosted our process yielding C10–C30 DCAs with > 80% and very high selectivity for DCAs of 78%. However, selectivity has so greatly improved that fatty acids are now a minor side product, limiting our access to samples for bioconversion testing at scale in Stage 1. Due to the very promising reaction design, aevoloop has secured funding to scale up oxidation and will provide more plastic fat side products in Stage 2 to complete MS3.

Symbiotic co-culture

• SymbioLoop’s individual Stage 1 goal was to find at least one viable option out of > 15,000 yeast/algae combinations; a yeast strain capable of converting our waste feedstocks and algae able to provide a suitable biofilm environment for effective yeast immobilization.
• Yeast: From our collection of several hundred yeast strains with varying productivity for different feedstocks, we shortlisted seven based on overall productivity and robustness. These seven strains were tested with 27 different fatty acid derivatives—varying in chain lengths, ester moieties, functionalities, and mixtures—in over 200 experiments for a comprehensive overview. The best conversions were achieved with C12 to C18 ethyl esters; ethyl ester moieties are preferred, while functionalities like double bonds or -OH groups are less favored. Strain sAA3041 demonstrated the widest substrate conversion range and productivity and was selected for further investigation.
• Algae: We accessed the Koethen Algae Strain Collection (KASC) of ~350 strains. To shortlist candidates, we focused on their ability to form biofilms and produce EPS, to effectively host yeasts. Based on the literature and our KASC experience, we screened 22 strains from 16 genera, including cyanobacteria, red and green algae, both self-isolates and commercial strains for EPS production using methylene blue staining. This resulted in 12 promising strains for EPS sugar profiling. After developing the analytical methods, we found major differences in EPS sugar profiles among the strains. Due to limited data on yeast growth on EPS sugars, we screened growth rates on the major sugars present in EPS. Glucose, galactose, and xylose resulted in similar maximum yeast growth rates (μmax ~0.29 h-¹), whereas arabinose, glucuronic acid, and fucose resulted in much lower rates (μmax 0.04-0.09 h-¹). Notably, natural EPS were converted (~μmax 0.10), suggesting that our yeast possesses enzymes to hydrolyze algal carbohydrates, critical for symbiotic potential. To evaluate biofilm stability, we inoculated the algal strains onto different porous plastic or glass substrates. Microscopic analysis and dry biomass measurements of the biofilms led us to focus on 2 strains.
• To combine yeast and algae in symbiotic co-culture biofilms, we needed a medium suitable for both organisms. The two algae strains could not grow in yeast media, but vice-versa with ammonium nitrate as the nitrogen source. With the selected algae strains, we focused on growing larger biofilms as modules for two different bioreactor setups. Surprisingly, a 3D printable polypropylene grade was the most suited substrate, allowing us to 3D-print complex geometries for our biofilms. We printed cylinders with a 3D gyroidal lattice to serve as inserts for a trickle-bed reactor and porous helical stirrers for bench-top fermenters. Both geometries supported stable biofilms that could be inoculated with yeast cells. Microscopic images and significantly increased algal growth rates in co-cultures confirmed the successful formation of symbiotic co-cultures between KASC I-001 and KASC 09-011 with sAA2693.

Continuous Process Design

• As a first crucial step toward a continuous symbiotic algae/yeast bioreactor and to meet the MS4 in Stage 1, we validated the ability of our production yeast strain to continuously bioconvert waste-derived FAEEs using only glycerol as a carbon source. In a 3 L benchtop fermenter equipped with a sedimentation tank for continuous removal of solid DCA product, we converted FAEEs from BSLF (C12–C18 mix) for 57 days (still running), achieving titers of up to 92 g/L and full conversion of all fatty acids to DCAs. In addition, we demonstrated that the medium could be recycled by removing accumulated harmful phenols and PAHs using bespoke upcycled water filters from consortium member PolymerActive. With our 3D-printed biofilm stirrer module, we can replace the standard stirrer of a fermenter and turn it into a continuous symbiotic bioconversion in Stage 2. An initial experiment showed that yeasts incorporated into the biofilm on the stirrer can convert FAEEs to DCAs, achieving our most important project-specific milestone on the biotech side.
• To mitigate risks in the design of our novel symbiotic biofilm process, we developed a second reactor concept based on a trickle-bed. In this setup, inoculated porous inserts are exposed to a constant trickle feed of substrate, glycerol and medium in a tubular glass reactor. Similar to the biofilm stirrer approach, we successfully observed conversion of FAEEs to DCAs. As a risk mitigation strategy, we plan to continue investigating both reactor designs in Stage 2 and commit to one once proven.

Polymer Products

• Long-chain DCAs are excellent monomers for making fully recyclable, biodegradable plastics with properties close to fossil polyolefins, as shown in a consortium Nature paper.1 However, typical polymerization with diols such as glycol yields polyesters with lower melting points than polyethylene. Mixing different DCAs, which is common in natural or waste feedstocks, further reduces the melting points to niche application levels.2
• In Stage 1, we developed a new polymer class that tolerates DCA blends, provides higher melting points, and ensures recyclability and degradability. We combined DCAs via a novel short-long polyesteramide (PEA) motif using ethanolamine as a co-monomer. We designed a self-accelerated chain end mechanism to generate polymers without catalyst, achieving mechanical properties of engineering grade polymers. The resulting melting points were about 30–40°C higher than comparable polyesters, even with blends in our DCA sweet spot range (C12–C18), allowing full utilization of our waste streams.
• Within weeks, we scaled up the reaction to the kg-level, produced compounds, and processed them into prototypes. Beyond injection molding, film extrusion and fiber spinning, we successfully 3D printed our materials using a pellet printer and achieved a proof-of-concept for SLS powder printing – the pinnacle of polymer processing. Remarkably, our polymers remain enzymatically degradable, allowing for low-energy, closed-loop recycling but also representing a prerequisite for biodegradability, which we will explore more deeply in Stage 2.