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Sustainable Benzene Degradation – A Community Affair

The summary

  • Benzene is toxic, mobile, and does not degrade easily, which is why we are so concerned with it.
  • Many microbes are thought to degrade benzene. In the real world, benzene does not degrade easily.
  • Recent research explains that out of 111 different bacteria, only 2 directly degrade benzene and the rest live on the metabolites and dead biomass (colorfully: necromass).
  • The research also explains why nitrate is such a great way to biostimulate biodegradation, but also why it is so dangerous. Nitrate is great because it stimulates the upper and lower benzene degradation pathways. It’s dangerous because too much nitrate causes nitrite to accumulate. Nitrite poisons bacteria and humans.

The Benzene Conundrum

Often benzene is our primary concern when we consider land or water contaminated with hydrocarbons. We worry about benzene for good reason. It is mobile in groundwater, which means it can rapidly spread throughout a city or region. It causes cancer at very low concentrations, and thus we often need to try to remediate it in groundwater to low parts per billion level. If there is little oxygen, benzene degrades slowly, if at all. Thus, benzene has been a topic of research and technology for decades.

But, pardon the pun, the mechanism by which benzene degrades has proven recalcitrant to understanding. Initially, researchers isolated organisms that could degrade benzene by coupling benzene degradation to nitrate, then sulfate, and then iron. Further, researchers began to understand how benzene degradation occurred biochemically under all of these conditions. However, the more we learned about benzene degradation, the more puzzling it began. If all of these different organisms could degrade organisms, why does benzene persist in so many soils?

One wrong answer is “well, there are not enough terminal electron acceptors”. For example, bacteria that consume nitrate to degrade benzene are plentiful, and there are many other bacteria that consume sulfate to degrade benzene. Yet in soils with lots of nitrate and sulfate, benzene persisted. In other words, benzene was not being degraded even though degradation conditions were prime.

This led to three major competing theories (an aside, theories don’t really compete, as normally each theory holds a facet of correctness, but is incomplete in some fashion, and scientists gradually fill in the incompleteness until the theory is whole):

  1. Inoculants. Or… the reason the benzene does not degrade is that we are missing a key bacterium, and if we add that bacteria in sufficient quantities, benzene will degrade.
  2. Nutrients. Or… the reason the benzene does not degrade is that we are missing a key nutrient, like phosphorus or maybe iron, and if we add bioavailable phosphorus, benzene will degrade.
  3. Syntrophy. Or… the sum of the whole is greater than the parts, and the entire microbial community contributes towards benzene degradation in some fashion.

A recent, and quite frankly, amazing paper1 led by Chrats Melkonian has filled in major parts of the Inoculant and the Syntrophy theory. But first some background. The research group had a microbial community, which they had been feeding benzene to for 15 years. The microbial community lived inside a bioreactor, which means that if a microbe was not growing, it would get washed away as new water was added and old water flowed away. The research group then got money, lots and lots of money, and did everything that a scientist would ever want to do with this community by sequencing the community, constructing virtual genomes, advanced metabolite analysis, and then manipulative experiments.

I have to say in my 30 years of research, this paper is a tour de force in microbial ecology.

Here is how they have improved each theory:

  1. Inoculants. There are between 47 and 111 different bacteria in this community, but it looks like only 2 of the bacteria (maybe 7, but 2 for sure) are involved in direct benzene degradation. They are from the Peptococcaceae and Rhodocyclaceaea families. So, if your soil doesn’t have these organisms, this means you are not likely going to be degrading benzene.
  2. Nutrients. The nutrient hypothesis was not the focus of this story, but their work is congruent with the idea that most other organisms are feeding off the necromass of the benzene degraders, and the nutrient cycling here might be an essential limiter of the benzene degraders.
  3. Syntrophy. They found that members of the community were denitrifying nitrate to nitric oxide (NO) or nitrous oxide (N2O), and that these oxygen containing compounds were being used by other members of the community to complete benzene degradation. In other words, the initial attack on benzene (called the upper pathway) was by the Peptococcaceae and Rhodocyclaceaea organisms, but the final degradation (lower pathway) occurred by organisms that were using oxygen stripped from NO and N2O. Cool, eh?

1) https://www.nature.com/articles/s42003-021-01948-y

Improved Solutions to Benzene

The improvements to the theories help us organize our approaches to cleaning benzene from soils. First, it suggests that if you are adding nitrate to biostimulate benzene degradation you do not need to inoculate. The Peptococcaceae and Rhodocyclaceaea organisms are common in soils, and if you provide available nitrate, then they will likely commence benzene degradation.

But the paper highlights the extreme risk you take when adding nitrate to biostimulate. Most of this community had no capacity to degrade nitrite. A quick reminder, nitrate gets converted to nitrite by many organisms. Thus, if you add too much nitrate, it creates nitrite. Nitrite is toxic to humans and bacteria. In the paper, and in real life, nitrite poisons bioremediation activity. In the paper, benzene degradation quickly plummeted to zero once nitrite exceeded 0.6 mM (27 mg/L). Thus, if you do add nitrate, add it slowly. This is a great example of how syntrophic communities can be good because they help you degrade but also can serve as a weak point by either, (i) producing a toxic metabolite like nitrite, or (ii) consuming nutrients that the degraders need.

A few limitations to this work. If your site has no nitrate, and is using sulfate or fermentation for benzene, this work does not speak to this site type. Hence, inoculants might still be needed. But for many sites in which benzene is in the top 5 to 8m of the surface, this work helps explain why what works and what doesn’t work.