GreenZyme in the Literature: Khusainova and Shapiro's Enzyme EOR Research at DTU

The enzyme-EOR research referenced throughout our GreenZyme literature posts didn't come from a single study; it came from a research line at the Technical University of Denmark (DTU), carried out in the Center for Energy Resources Engineering, Department of Chemical and Biochemical Engineering. Below is that lineage in order, with the GreenZyme-specific findings pulled directly from the papers.

Where GreenZyme Is Formally Introduced

In Khusainova's 2016 thesis, GreenZyme® gets a formal introduction in Chapter 1, Section 1.2 (“Enzyme Products Designed for EOR Applications Available on the Market”), where it's listed as the third of five commercially available enzyme EOR products surveyed. The thesis describes it plainly: “GreenZyme® is manufactured by Apollo Separation Technologies Inc… The enzyme product is believed to bind to the surface of the oil-particle complex in the reservoir rock, triggering breakdown of the complex. Then the enzyme covers the surface of the released particle. Thereby the particle is prevented from re-agglomeration with the oil, which enables the oil to be carried away with the flow. The product is claimed to be the first bio-enzyme used in EEOR.” GreenZyme is then tested directly, alongside a competing commercial enzyme mixture and fifteen individually supplied enzyme samples, in the wettability-screening work covered below.

2012 — Khusainova, Shapiro, Stenby & Woodley: Wettability Improvement with Enzymes

Presented at the 33rd IEA EOR Symposium (August 2012), this is the first DTU test of GreenZyme. The design was a head-to-head comparison: fifteen individually supplied enzyme samples (esterases/lipases, carbohydrases, proteases, oxidoreductases) against two commercial enzyme mixtures — GreenZyme and one other competing product — using North Sea dead oil, synthetic seawater, and calcite crystals standing in for carbonate rock. The comparison is where GreenZyme's specific profile becomes clear:

The competing commercial mixture showed no ability to change the wettability of the mineral surface at all. GreenZyme, by contrast, demonstrated absolute non-adhesion behavior with the contact angle declining by an average of 60% (about 15°), for every calcite mineral tested, at every concentration tested — the most consistent result of any product in the study.

But the mechanism was different from the winning “pure” enzyme group. The best-performing esterases/lipases behaved like the reference protein (BSA) — consistent with an enzyme adsorbing onto the mineral and/or forming additional interfacially active oil compounds. GreenZyme's behavior instead resembled the reference surfactant (SDS): its performance was attributed to a drastic drop in interfacial tension, the surfactant mechanism, rather than the enzyme-adsorption mechanism the pure enzymes relied on. The authors' conclusion is direct: “for GreenZyme's mixture containing the surfactant, wettability improvement may be both due to enzyme and surfactant that is difficult to distinguish.”

2015 — Khusainova, Nielsen, Pedersen, Woodley & Shapiro: Wettability of Calcite Surfaces

This is where Sidsel Marie Nielsen joins as a co-author alongside Khusainova, with Hanne Høst Pedersen — a co-author from the enzyme-supply industry — joining as well. “Study of wettability of calcite surfaces using oil–brine–enzyme systems for enhanced oil recovery applications” ran the same GreenZyme-vs-competing-product-vs-individual-enzyme comparison as the 2012 conference paper, but as the full peer-reviewed study, with reference experiments added to isolate the mechanism. Two GreenZyme-specific results stand out:

First, at a concentration equivalent to 1% enzyme product, GreenZyme drove interfacial tension down to 5.9 mN/m — close to SDS surfactant's 0.8 mN/m, and far beyond anything the individually tested enzyme samples achieved. Second, because the composition of GreenZyme's stabilizer was undisclosed (proprietary), the DTU team could not run the same isolation test they ran on the other enzyme samples — where they proved the effect came from the enzyme itself, not the stabilizer — on GreenZyme. That's an important caveat the paper states outright: for GreenZyme, whether the enzyme or an undisclosed surfactant component drives the wettability change “was not possible to check.” The paper's summary line, later repeated in Khusainova's thesis, is the one most often quoted: GreenZyme is “the only sample for which there was no correlation between contact angle measurements and adhesion behaviour” — a way of saying its performance didn't fit the pattern the individually tested enzymes followed.

2016 — Alsu Khusainova: Enhanced Oil Recovery with Application of Enzymes (PhD Thesis)

The thesis folds the 2012 and 2015 wettability results into Chapter 3 and adds the line most often quoted in our own posts: “An exception is the commercial mixture GreenZyme exhibiting a surfactant-like action, indeed most likely explained by presence of surfactant in the product.” The thesis also adds an adsorption study (Chapter 4) that GreenZyme itself was not directly re-tested in — it used other individually supplied enzyme samples and BSA protein as the study substances — which found that chalk retained protein at a far higher, largely irreversible rate compared to sandstone.

Field Takeaway: Chalk Is a Hard Setting, and Tight Formations Get a Dilution Adjustment

Khusainova's adsorption chapter is worth reading with an eye toward field application, not just mineral chemistry: irreversible protein retention in chalk, far exceeding sandstone, is a real obstacle for any enzyme-based product in that rock type — not a defect in the enzyme itself, but a mismatch with chalk's very high internal surface area. Our own position follows from that evidence: GreenZyme is most likely not the right fit for chalk, and we would not recommend it there unless an operator first runs a filtration test to confirm the formation can actually accept the product without the retention/clogging problem the DTU data describes.

That's a separate point from tight formations more broadly. Outside of a sandstone setting — in low-permeability, carbonate-leaning, or shale formations that are tight but not chalk — we've learned to adjust dilution rather than avoid the product. The adaptation is simple: keep the same total amount of GreenZyme an application calls for, but dilute it further, to a 5% solution at the time of surface application, instead of the standard 10%. It's a field-tested adjustment for tight-but-workable formations, aimed at reducing how much organic material is available to adsorb and build up in the tightest pore throats before it ever reaches the target zone.

The Common Thread: Professor Alexander Shapiro

Alexander A. Shapiro appears across the entire lineage — as a co-author on the 2012 conference paper and the 2015 journal paper, and as Khusainova's supervisor for the 2016 thesis. Across four years of published work, his name is the constant, consistent with his role heading this line of EOR research at DTU's Center for Energy Resources Engineering.

Sources

Khusainova, A., Shapiro, A., Stenby, E.H., & Woodley, J.M. (2012). Wettability Improvement with Enzymes: Application to Enhanced Oil Recovery under Conditions of the North Sea Reservoirs. 33rd IEA EOR Symposium. (Held in BTP's internal research archive; digital link to be verified.)

Khusainova, A., Nielsen, S.M., Pedersen, H.H., Woodley, J.M., & Shapiro, A. (2015). Study of wettability of calcite surfaces using oil–brine–enzyme systems for enhanced oil recovery applications. Journal of Petroleum Science and Engineering, 127, 53–64. DOI: 10.1016/j.petrol.2014.12.014.

Khusainova, A. (2016). Enhanced Oil Recovery with Application of Enzymes. PhD Thesis, Technical University of Denmark. (Held in BTP's internal research archive; digital link to be verified.)