Soluble vs. insoluble for anaerobic substrates

In anaerobic bioremediation treatment (chlorinated solvent remediation, for example), a food source (electron donor) is provided to support microbial activity and growth. A healthy debate continues in the industry regarding which form of substrate is ideal for delivering this electron donor in bioremediation applications: soluble (carbohydrate-based) or insoluble (oil-based). Here, we provide an overview of the key differences, our preferred approach, and some common objections that we often hear.

Comparison Overview

A primary point of comparison is utilization speed. Utilization speeds of soluble substrates and emulsified oils, such as emulsified soybean oil, can differ significantly due to their bioavailability, distinct chemical structures, and required metabolic pathways. Note, that temperature, pH, and nutrient availability can significantly influence the degradation rates of both soluble substrates and soybean oil.

Soluble Substrate Utilization

Characteristics:

• Simple Sugars: These low-molecular-weight compounds are quickly and easily metabolized by anaerobic bacteria.

• Quick Metabolism: Anaerobic bacteria can rapidly metabolize soluble substrates through pathways such as glycolysis, followed by fermentation or anaerobic respiration.

• Fermentation Products: The end products of soluble substrate fermentation can include organic acids (like lactic acid), alcohols, and gases (such as hydrogen and carbon dioxide).

Utilization Rate:

• Fast: Due to their simple structure, soluble substrates are typically metabolized quickly and completely by anaerobic bacteria. For example, in optimal conditions, certain fermentative bacteria can start utilizing soluble substrates almost immediately, with doubling times ranging from 20 to 30 hours.

Emulsified Oil Utilization

Characteristics:

• Bioavailability: Oil is a LNAPL unless temporarily emulsified. Bacteria consume food sources within the dissolved phase or at the oil/water interface. Very small amounts of oil are dissolved and the larger mass either in emulsified droplets or in separated LNAPL is largely unavailable for use by bacteria.

• Complex Lipids: Emulsified oil is primarily composed of triglycerides, which are complex molecules consisting of glycerol bound to three fatty acid chains.

• Slower Metabolism: The metabolism of triglycerides involves initial hydrolysis to free fatty acids and glycerol, which are then further metabolized through beta-oxidation and other pathways.

• Metabolic Pathways: Fat breakdown is more energy-intensive and slower than carbohydrate breakdown due to multiple metabolic steps involved. Also, anaerobic degradation of lipids typically involves multiple microbial communities, including hydrolytic bacteria, syntrophic bacteria, and methanogens.

Utilization:

• Slow: Due to the complexity of triglycerides and the multi-step process required for their degradation, the utilization rate of soybean oil by anaerobic bacteria is generally slower than that of carbohydrates. The initial hydrolysis step can be particularly rate-limiting. The complete degradation of soybean oil might take several days to weeks, depending on environmental conditions and microbial community composition. Note that the presence of specialized bacteria (e.g., lipolytic bacteria for oil degradation) can enhance the utilization rates of more complex substrates like soybean oil.

Comparative Summary

Two Key Benefits of Soluble

#1: Better distribution and utilization

Soluble substrates mimic water properties closely, enhancing their distribution capabilities. As a result, whether delivered by direct push, well injection, or recirculation system, the radius of influence will be improved. It is rapidly consumed, creating highly reductive conditions, and resulting in a rapid population growth and rapid dechlorination. In comparison, emulsified oil can eventually separate into a NAPL layer, limiting its ability to be utilized by bacteria.

#2: Ease of application

From a fieldwork perspective, the application could not be easier. Just mix with water and inject.

Two Key Objections

Objection #1: “But you need slower utilization to maintain long-term reductive conditions and avoid rebound.”

Thanks to the large biomass generated by a fast-growth carbohydrate substrate, the microbes themselves become a long-term food source for ongoing reductive conditions. This will help if geology and lithology contribute to a rebound – which is also less common with a soluble substrate due to better distribution.

But it is also worthwhile to consider a general challenge to this conventional thinking: If all the substrate can be utilized quickly, and therefore the necessary respiratory source (i.e. the contamination) is also quickly consumed, in what scenario would that not be preferred? The desirability of a long-term substrate presumes that the site remediation must be a long, slow process. It doesn’t have to be that way!

Objection #2: “But emulsified oil substrates have greater electron donation potential pound for pound.”

This is true. However, despite their theoretical energy potential, emulsified oils’ actual utilization often remains incomplete due to limited distribution and complexity. Carbohydrate substrates achieve near-complete utilization, thereby often requiring smaller quantities overall.

A) Utilization percentage: While utilization rate is often discussed, utilization percentage is less often considered but equally important. The bioavailability limits, distribution, and complexity of emulsified oil limits its ability to be utilized completely.

B) Biomass: Since the utilization rate is slower, the population of bacteria will be limited. In comparison, a fast-growth carbohydrate-based substrate will have the multiplying effect of a microbial biomass used as a long-term electron donor.

Conclusion

In summary, carbohydrates are metabolized much more quickly by anaerobic bacteria compared to soybean oil due to its simpler structure and the fewer metabolic steps required for its breakdown.

What does this mean for remediation?

Soluble substrates, because of their simple structures and solubility, have much higher utilization rates than complex insoluble substrates.  This allows the bacteria using the soluble substrates for a carbon source to quickly deplete competing electron acceptors, create reductive conditions, and achieve rapid dechlorination of the target COCs.  Since the utilization of soluble substrates is near 100%, less substrate may be needed compared to lesser utilized substrates.

Emulsified oil is insoluble in water and must be emulsified to place droplets into the groundwater.  Utilization rates while the oil is emulsified are a slower, more complex process.  The slower process requires more time to deplete competing electron acceptors and create very reductive conditions.  Also, emulsified organics will not stay emulsified indefinitely, forming LNAPL at the water table or within the soil pore space.  Once the separate NAPL layer is formed, the unutilized portion of the product injected would be similar in persistence to free-product diesel or other petroleum products.

At ETEC, we’ve been successfully using a soluble substrate for decades. Check out our CarBstrate^TM product page for more information. Our Case Study page includes many examples of the positive results we’ve experienced.