Mini-coreflood experiments validate in vitro community
control
To evaluate the effect of our top-down engineering approach on oil
recovery, we applied the same treatments to a miniature coreflood
system. The mini-coreflood (MCF) design (Figure 5A-C ) was used
to simulate microbial enhanced secondary oil recovery from
representative core material to evaluate the effects of the carbon and
micronutrient supplements in parallel. In addition to pressure generated
by the microbes in the MCF, we evaluated the composition of the
microbial community and the produced oil. After initial oil recovery
from brine flooding was completed, 9:1 mixtures of effluent and
molasses-nutrient supplements were injected into the MCFs and allowed to
sit for 10 days in an anaerobic environment to seed the core and
incubate the growing microbes. Similar to our in vitromicrocosms, we found that molasses was able to induce microbial growth
and activity, generating up to 26 PSI. Nitrate-treated floods generated
the most pressure while the molybdate and combination treatment
generated less pressure (Figure 5D ). We suspect that the
suppression of H2S and the limitation of
sulfate-reducing bacteria account for the differences in pressure
generation between the molybdate-treated cultures and the untreated or
nitrate-treated cultures. Because increased pressure applies more force
on the trapped oil deposits, the amounts of oil recovered trended with
the pressure produced (Figure 5E/F ). We also found that the
microbial community in the MCF was distinct from our in vitroscreening, reflecting the contribution of the high petroleum hydrocarbon
concentrations in these samples. MCF microbial communities drastically
shifted toward Bacteroides under the molybdate and combination
treatment whereas the untreated and nitrate-treated MCFs were dominated
by Lachnoclostridium (Figure 5G ). As at the end of thein vitro microcosms, both Bacteroides andLachnoclostridium were prominent members of the final MCF
microbiome (Figure 4, Figure 5G ), but these MCF trials contain
significant amounts of the oil hydrocarbons that can be used as an
additional carbon source. While the V4-V5 16S rRNA gene region amplified
here is not specific enough to identify the species of these genera,
microbes belonging to the Lachnoclostridium group have been found
to degrade some hydrocarbons and some reduce sulfate to
H2S.47–49 Similarly, we observed
increases in the abundance of organisms belonging to the genusPseudomonas which is not a prominent producer of gases and has
been linked to naphthalene,50 normal
paraffin,51 and polycyclic hydrocarbon
degradation.52,53 As shown in (Figure 5E/G )
the proportion of n-paraffin dropped from ~35 %w/w to
~25 %w/w of the relative abundance whilePseudomonas sp. increased in abundance in the molybdate and
combination MCFs. This condition suggests that Pseudomonas sp.degraded around 30% of the long chain n-paraffins (C14 – C29) in a
period of 14 days (Supplemental Figure 7A, Supplemental Table
1 ). Although isolates were not identified here, Pseudomonas sp.,
such as P. proteolytica, P. xanthomarina, and P. aeruginosa , are
expected to induce n-paraffin degradation starting from n-tetradecane
(C14) by alkane hydroxylases systems.53–55 Certainly,
biodegradation of long chain n-paraffins is recognized to have a
positive influence in oil recovery processes by means of reducing the
viscosity of the crude oil while increasing its
fluidity.56 The relative abundance of
monocyclo-paraffins increased from ~30 %w/w to
~40 %w/w in total corresponding to the increment of the
C9 – C17 fraction for all treatments (Supplemental Figure 7B,
Supplemental Table 1 ), likely due to degradation of the n-paraffins.
However, no specific trend was observed for the abundance of
iso-paraffins. Aromatics such as naphthalene also decreased in abundance
with treatment and the presence of Pseudomonas sp.
(Figure 5F/G ). Species such as P. mendocina, P. putida,
P. fluorescens, P. paucimobilis, P. vesicularis, P. cepacia, P.
testosteronei, P. aeruginosa, and P. stutzeri have been reported
to induce naphthalene biodegradation.52,57,58 In
contrast, the triaromatic fraction (C17) increased as a response of the
supplements here applied. The relative abundance of dicyclo- and
monocyclo-paraffins increased when these treatments were applied, likely
because of n-paraffin degradation, and thus, changed the overall
composition of the produced oil (Figure 5E/G, Supplemental Table
1 ). While the results presented in Figure 5 represent one trial of
these treatments, an additional replicate treatment at a different time
(Supplemental Figure 8A-C ) showed n-paraffin degradation for
all treatments. However, the same trend for the aromatic polycyclic
hydrocarbon fraction is not seen in the replicate likely because the
microbiomes of the produced water used to seed these wells were
different (Supplemental Figure 8D ). Regardless of these
specific differences, we observed reproducible control of the oil well
microbiome as a function of nutrient supplementation to alter produced
oil paraffin profiles while limiting H2S production and
pressurizing the reserve.
Our results demonstrate a clear correlation between pressure generation
and oil recovery in MEOR processes. While the
H2S-limiting treatments did not substantially enhance
pressure generation in the presence of hydrocarbons and confined
geometry, we were able to validate the role that molybdate plays in
controlling souring and corrosion by oil well microbes. However, field
trials and economic analysis are needed to determine the viability of
our process relative to current surfactant-based oil recovery processes.
The modest improvements in oil recovery may be offset by the increased
operating costs needed for corrosion maintenance, surfactant production,
and microbial control via more expensive ammonium quaternary
disinfectants currently used.19 Moreover, we
demonstrated that the specific oil compositions were a strong function
of microbial activity, which could be controlled via top-down
engineering. Further optimization of the nutrient formulation, such as
increased amounts of molasses or other nutrients, may enhance pressure
generation or alter the microbial dynamics so that more oil can be
recovered and specific hydrocarbon compounds can be enriched or
depleted. In agreement with the in vitro findings, these results
suggested that we can control the microbial population in small scale
oil recovery settings and can use that to concurrently modulate oil
composition.