### Biological Upgrading of Heavy Oil for Viscosity Reduction

***William T. Stringfellow\
Biological Processing Laboraotory\
Lawrence Berkeley National Laboratory\
Berkeley, CA\
wstringfellow@lbl.gov***

#### August 29, 2001

### Introduction

The United States petroleum industry is increasingly dependent on heavy
crude oil to meet the domestic demand for gasoline and distillate fuels.
Owing to their high viscosity, heavy oils are more expensive to
transport and process than less viscous oils. It has been proposed that
"bioprocessing" may offer an economical approach for treating heavy oil
at the wellhead, before pipeline transport.

Biocatalytic viscosity reduction (bioprocessing) uses bacteria to
partially transform less valuable crude oil components to surface-active
compounds (alcohols and carboxylic acids) that reduce crude oil
viscosity. In this research, we have targeted the terminal oxidation of
alkanes to alcohols as a specific mechanism for development. Research
conducted at Texaco has demonstrated the efficacy of linear alcohols in
reducing crude oil viscosity. Biological conversion of alkanes to
alcohols is carried out by alkane monooxygenase, an enzyme found in
bacteria able to grow on alkanes. The development of biocatalysts that
convert alkanes to alcohols has application both in refining and in bulk
chemical manufacturing and is therefore an attractive target for
research.

A major challenge to implementing crude oil bioprocessing is to develop
and control biocatalytic agents so that they target specific alkanes in
complex mixtures. We have defined the specific challenge of being able
to oxidize alkanes of C~8~ and above without oxidizing alkanes of C~7~
or less. We are further interested in understanding the difference
between bacteria that oxidize *iso*-pentane from those that do not.
*Iso*-pentane is a major component of gasoline and is highly valued in
crude oil.

For biocatalytic viscosity reduction to be economical, the process will
have to be operated under non-sterile conditions using whole-cell
biocatalysts. Non-sterile operation precludes the use of genetically
engineered organisms and requires that the process be controlled to
maintain desirable biocatalytic agents and favorable kinetics in the
presence of competing bacteria. A successful process will depend on a
complete understanding of bacterial alkane oxidation, on the
physiologic, genetic, and kinetic level.

The objective of this research was to evaluate diversity among alkane
monooxygenases and to develop methods for selecting and evaluating
biocatalytic agents for use in viscosity reduction and chemical
manufacture. We investigated differences between well-characterized
alkane oxidizing bacteria and novel environmental isolates. Our study
has shown that there is significant variation among alkane oxidizing
microorganisms in terms of their phylogeny, physiology, genetics,
substrate specificity, and alkane oxidation kinetics. Some alkane
oxidizing microorganisms are considerably different from
well-characterized strains and deserve further study. As a result of
this research, we have developed methods for screening and evaluating
bacteria as potential biocatalytic agents. We have identified
microorganisms that have significantly different genetic profiles than
known alkane oxidizing organisms and selected promising bacterial
strains for further development.

### Research statement

A controlled process under non-sterile conditions will require a
thorough understanding of both the substrate specificity and the kinetic
activity of proposed biocatalysts. Understanding of the enzymatic
diversity of alkane oxidizing bacteria is the first step in the
selection and application of bacterial catalytic agents (biocatalysts)
for the processing of specific alkanes in complex mixtures.

### Research approach

In this research, we isolated bacteria from petroleum-contaminated
environments based on their ability to utilize octane as a growth
substrate. We compared our bacteria to a well-characterized alkane
degrading bacteria, *Pseudomonas putida (oleovorans)* GPo1 (kindly
provided to us by Bernard Witholt of the ETH, Zurich). Strain GPo1
contains a well-characterized alkane monooxygenase enzyme (AlkB) that
oxidizes linear alkanes to 1-alcohols (van Beilen *et al.* 1994). The
*alkB* gene codes for AlkB enzyme (van Beilen *et al.* 2001). GPo1 is a
benchmark organism, and most of what is known about the genetics and
enzymology of alkane oxidation has been discovered using AlkB system or
closely related proteins (Marin et al. 2001).

### Research results

*Bacterial isolation and phylogenic analysis*

Isolation of microorganisms containing an appropriate enzyme system is
the first step in the development of a biocatalyst. Bacteria were
enriched from petroleum contaminated environments using octane as a sole
carbon and energy source. Bacteria were isolated on Luria-Bertani agar
in an octane atmosphere (LB-OCT). Isolated bacteria were sorted
according to source of isolation and colony type on LB-OCT. Specific
strains were selected as 'type-strains' for each colony type. Fatty acid
methyl ester (FAME) and 16s ribosomal-DNA (rDNA) analyses were used to
determine the phylogenic category of the type-strains.

Thirty strains from three different alkane contaminated environments
were isolated for testing. Environmental isolates were compared to the
well-characterized bacterial culture strain GPo1 and a negative control
strain GPo12 (Smits *et al.* 1999). Results from twenty-seven strains
and the controls are included in this report. The bacteria were sorted
into 15 groups based on their colony morphology or other
characteristics. From each group, one strain was selected as the
"type-strain" and characterized by FAME and 16s rDNA analysis (Table 1).
The type-strains were subject to the most complete analysis. Except
where otherwise noted, the results gathered with the type-strains were
found to be representative of their entire group.

The majority of organisms isolated in this study were Gram positive
actinomycetes bacteria (family Actinomycetaceae). This group includes
*Rhodococcus*, *Nocardioides*, *Nocardia*, *Mycobacterium*, and
*Ochrobactrum*. These bacteria are well known for their ability to grown
on alkanes and produce surfactants (Hommel 1990). However, the alkane
oxidase activity of actinomycetes has not been extensively studied and
the isolation of new alkane oxidizing strains in this group presents
opportunities for the development of novel biocatalysts.

*Substrate specificity*

The substrate range of all bacteria was tested using a plate assay.
Results from the type-strains are presented in Table 2. Bacteria were
plated on a mineral salts (SSC) agar and placed in chambers containing
alkane vapors. Growth of the bacteria on alkanes was evaluated after
seven, fourteen, and twenty-one days and scored on a scale of zero to
four, with four being the most abundant growth. Alkane oxidase activity
was also confirmed using a respirometric assay (Stringfellow *et. al.*
2001a, Stringfellow *et al.* 2001b).

Most of the organisms isolated were able to grow on and oxidize a wide
variety of alkanes (Table 2). *Rhodococcus globerulus* OT15DY,
*Nocardioides simplex* OT13D1, and *Rhodococcus globerulus* OT18A were
able to grown on all alkanes tested. *Nocardia nova* OT32AA and
*Mycobacterium fortuitum* OT32DG were able to transform all the alkanes
except *iso*-pentane. Bacterial strain OT32AB (not yet identified) was
limited to the metabolism of alkanes of C~10~ or less. The reference
strain, *Pseudomonas putida* GPo1, was not able to grow on *iso*-pentane
or alkanes above C~10~ in length. *Alcaligenes xylosoxydans* OT6A
demonstrated an unusual metabolic pattern, exhibiting poor growth on
only on three of the hydrocarbons tested. These results suggest that
there is a significant diversity in alkane metabolizing ability among
bacteria. This diversity offers opportunities for the development of
catalysts targeted toward specific alkanes, if the mechanism of
specificity can be better understood.

*Genetic evaluation*

As part of our biocatalyst development, the type-strains were tested for
the presence of genes that were homologous with AlkB type monooxygenases
(van Beilen et al. 2001, Marin et al. 2001). The potential biocatalytic
agents were screened for the presence of the *alkB* gene using a
non-specific primer set developed to probe for *alkB* in diverse
organisms (Smits *et al.* 1999). Homology with *alkB* of strain GPo1 was
indicated by the presence of a PCR product that was approximately 550 bp
in size.

All strains that were negative for alkane oxidation were negative for
the presence of *alkB* (Table 3). Four of the seven alkane metabolizing
strains were positive for the presence of an *alkB* gene of the same
size as the positive control (GPo1). OT6A had some homology with *alkB*,
but on a smaller PCR product (approximately 220 bp). Two strains, OT32AA
and OT32DG, did not demonstrate homology with *alkB* using these primer
sets (Table 3). There was no clear correlation between homology with
*alkB* and biocatalyst substrate specificity. These results suggest that
there are alkane oxidizing enzymes that are not AlkB type enzymes in
some microorganisms. Further investigation will determine if these
strains contain truly novel enzymes.

*Kinetic evaluation*

The development and application of biocatalysis for alkane oxidation
requires a complete understanding of the kinetic properties of the
catalysts. Alkane oxidation rate as a function of alkane concentration
was measured using mixed and pure cultures. From these measurements, the
kinetic parameters K~m~ and V~max~ were estimated by fitting data to the
Henri-Michaelis-Menton equation (Segal 1975). Results from measurements
made with octane for the type-strains are presented in Table 4. These
results demonstrate a variability among biocatalysts that is not
apparent from the substrate specificity assay.

Kinetic measurements are being made for a full range of alkanes that
will allow the prediction of product formation in different mixtures of
alkanes. Mixed cultures were tested for their ability to oxidize a
variety of alkanes. Competitive inhibition models (Segal 1975) were used
to predict the fate of *iso*-pentane in the presence of other alkanes,
such as octane. The higher affinity of an alkane oxidase for octane than
*iso*-pentane suggested that it would be possible to selectively oxidize
octane in the presence of iso-pentane (Stringfellow 2001).

### Research summary and conclusion

The alkane oxidizing bacteria isolated as part of this study were
compared to the well-characterized AlkB system of *Pseudomonas putida*
GPo1 for differences and similarities in genetics, kinetics, substrate
specificity, and phylogeny. This study has shown that there is
considerable diversity among alkane oxidizing bacteria and opportunities
for the development of novel biocatalysts. Five bacterial strains
characterized in this study have been selected for further development.
The bacteria are considered to have potential as biocatalytic agents due
to their substrate specificity, kinetic properties, or their probability
of harboring novel enzyme systems.

As currently conceptualized, crude oil bioprocessing will be operated
under non-sterile conditions and the directed oxidation of long-chain
alkanes will be effected by a combination of biocatalyst selection and
engineering controls. The results of substrate specificity and kinetic
analysis suggest that there is significant potential for the development
of a biocatalytic process that can oxidize less valuable, longer chain
alkanes, without damaging valuable, gasoline range alkanes. Further
research is needed to better characterize the action of biocatalysts in
alkane mixtures. The factors that determine substrate specificity need
to be better understood and new methods of biological process control
need to be developed before bioprocessing can be implemented.

### References

Hommel, R. K. 1990. Formation and physiological role of biosurfactants
produced by hydrocarbon-utilizing microorganisms: biosurfactants in
hydrocarbon utilization. *Biodegradation* 1: 107-19.

Marin, M. M., T. H. M. Smits, J. B. Van Beilen, and F. Rojo. 2001. The
alkane hydroxylase gene of *Burkholderia cepacia* RR10 is under
catabolite repression control. *J. Bacteriology* 183: 14: 4202-9.

Segel, I. H. 1975. *Enzyme Kinetics: Behavior and Analysis of Rapid
Equilibrium and Steady-State Enzyme Systems*. New York: John Wiley and
Sons.

Smits, T. H. M., M. Rothlisberger, B. Witholt, and J. B. Van Beilen.
1999. Molecular screening for alkane hydroxylase genes in Gram-negative
and Gram-positive strains. *Environ. Microbiol.* 1: 4: 307-17.

Stringfellow, W. T. 2001. Evaluation of bacterial biocatalysts for
terminal alkane oxidation. Abstracts of the 221st American Chemical
Society National Meeting, San Diego, CA, April 1 -- 5.

Stringfellow, W. T., S. Smriga, K. C. Oh, and T. Letain. 2001a.
Evaluating novel alkane-oxidizing bacteria for the biological up grading
of crude oil. Abstracts of the 101st Annual Meeting of the American
Society for Microbiology, Orlando, FL, May 20 - 24.

Stringfellow, W. T., S. Smriga, K. C. Oh, and T. Letain. 2001b.
Evaluation of metabolic diversity among potential bacterial catalysts.
Twenty-Third Symposium on Biotechnology for Fuels and Chemicals,
Breckenridge, CO, May 6 -- 9.

Van Beilen, J. B., J. Kingma, and B. Witholt. 1994. Substrate
specificity of the alkane hydroxylase system of *Pseudomonas oleovorans*
GPo1. *Enz. Microbial Technology* 16: 10: 904-11.

Van Beilen, J. B., S. Panke, S. Lucchini, A. G. Franchini, M.
Rothlisberger, and B. Witholt. 2001. Analysis of *Pseudomonas putida*
alkane-degradation gene clusters and flanking insertion sequences:
evolution and regulation of the *alk* genes. *Microbiology-SGM* 147:
PT6: 1621-30.

Table 1. Phylogenic analysis of potential biocatalytic agents. Strains
were selected for further analysis based on their diversity.

+-----+---+-----------------------+---------------+-----------------+
| ##  | * | **Colony Description  | # **FAME**    | **16s rDNA**    |
|  St | * | (LB-OCT.)**           |               |                 |
| rai | S |                       |               |                 |
| n N | t |                       |               |                 |
| ame | r |                       |               |                 |
|     | a |                       |               |                 |
|     | i |                       |               |                 |
|     | n |                       |               |                 |
|     | C |                       |               |                 |
|     | a |                       |               |                 |
|     | t |                       |               |                 |
|     | e |                       |               |                 |
|     | g |                       |               |                 |
|     | o |                       |               |                 |
|     | r |                       |               |                 |
|     | y |                       |               |                 |
|     | * |                       |               |                 |
|     | * |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      | *Rhodococcus  | *Rhodococcus    |
| OT1 |   | entire, convex        | erythropolis* | globerulus*     |
| 5DY |   |                       | (0.696)       |                 |
| \   |   |                       |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               |                 |
| OT1 |   | entire, convex        |               |                 |
| 5DY |   |                       |               |                 |
| 1** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               |                 |
| OT1 |   | entire, convex        |               |                 |
| 5DY |   |                       |               |                 |
| 2** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               |                 |
| OT1 |   | entire, convex        |               |                 |
| 6** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               |                 |
| OT1 |   | entire, convex        |               |                 |
| 6AD |   |                       |               |                 |
| 1** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **O | 1 | circular, cream,      | *Rhodococcus  |                 |
| T16 |   | entire, convex        | erythropolis* |                 |
| B** |   |                       | (0.666)       |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               |                 |
| OT1 |   | entire, convex        |               |                 |
| 6BD |   |                       |               |                 |
| 1** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               |                 |
| OT1 |   | entire, convex        |               |                 |
| 7** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| *   | 1 | circular, cream,      |               |                 |
| *OT |   | entire, convex        |               |                 |
| 18H |   |                       |               |                 |
| 2** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               |                 |
| OT1 |   | entire, convex        |               |                 |
| 8HD |   |                       |               |                 |
| 2** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               | *Rhodococcus    |
| OT2 |   | entire, convex        |               | globerulus*     |
| 5BD |   |                       |               |                 |
| 1** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               | *Rhodococcus    |
| OT2 |   | entire, convex        |               | globerulus*     |
| 5BD |   |                       |               |                 |
| 2** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | circular, cream,      |               |                 |
| OT2 |   | entire, convex        |               |                 |
| A** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **O | 2 | circular, blue        | *Alcaligenes  |                 |
| T6A |   | halo/diffuse cream    | xylosoxydans* |                 |
| \   |   | center, entire        | (0.827)       |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 2 | circular, blue        |               |                 |
| OT2 |   | halo/diffuse cream    |               |                 |
| B** |   | center, entire        |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **O | 3 | circular, pink,       |               | *Staphylococcus |
| T6B |   | entire, pinpoint,     |               | saprophyticus*  |
| \   |   | pulvinate             |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **O | 3 | circular, pink,       |               |                 |
| T33 |   | entire, pinpoint,     |               |                 |
| A** |   | pulvinate             |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **O | 3 | circular, pink,       |               |                 |
| T33 |   | entire, pinpoint,     |               |                 |
| B** |   | pulvinate             |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 4 | circular, beige,      | no match      | *Nocardioides   |
| OT1 |   | undulate, flat        |               | simplex*        |
| 3D1 |   |                       |               |                 |
| \   |   |                       |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| *   | 5 | punctiform, light     | *Strept       | *Streptomyces   |
| *OT |   | cream, irregular,     | overticillium | sp.*            |
| 26A |   | raised                | sp.* (0.392)  |                 |
| \   |   |                       |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| *   | 6 | circular, blue        | *Rhodococcus  | *Rhodococcus    |
| *OT |   | halo/cream center,    | erythropolis* | globerulus*     |
| 18A |   | filamentous, convex   | (0.763)       |                 |
| \   |   |                       |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 6 | circular, blue        |               |                 |
| OT1 |   | halo/cream center,    |               |                 |
| 8AD |   | filamentous, convex   |               |                 |
| 1** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 7 | circular, light       | *Nocardia     | *Nocardia nova* |
| OT3 |   | orange, entire,       | sp.* (0.336)  |                 |
| 2AA |   | convex, pinpoint      |               |                 |
| \   |   |                       |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| *   | 7 | circular, light       |               |                 |
| *OT |   | orange, entire,       |               |                 |
| 32D |   | convex, pinpoint      |               |                 |
| O** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 8 | punctiform, white,    | *Rhodococcus  | *Mycobacterium  |
| OT3 |   | entire, flat, small   | equi* (0.521) | fortuitum*      |
| 2DG |   | pinpoint              |               |                 |
| \   |   |                       |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| *   | 9 | circular, blue        | *Ochrobactrum | *Ochrobactrum   |
| *OT |   | halo/solid cream      | sp.* (0.568)  | anthropi*       |
| 18F |   | center, entire,       |               |                 |
| \   |   | convex                |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **  | 1 | beige, whitish small  |               |                 |
| OT3 | 2 | round                 |               |                 |
| 2AB |   |                       |               |                 |
| \   |   |                       |               |                 |
| *** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| **G | 1 | circular, beige,      | *Pseudomonas  | *Pseudomonas    |
| Po1 | 0 | entire, diffused      | sp.* (0.648)  | pse             |
| ^†  |   | green pigment         |               | udoalcaligenes* |
| ^** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+
| *   | 1 | circular, beige,      |               | *Pseudomonas    |
| *GP | 0 | entire, diffused      |               | pse             |
| o12 |   | green pigment         |               | udoalcaligenes* |
| ^‡  |   |                       |               |                 |
| ^** |   |                       |               |                 |
+-----+---+-----------------------+---------------+-----------------+

\* Type-strain for strain category, ^†^ Positive control, ^‡^ Negative
control

Table 2: Substrate specificity of potential biocatalytic agents. Results
demonstrate difference among alkane oxidizing bacteria that is important
to their utility as biocatalytic agents.

  ------------ ------------------- ------------- ------------ ------------ ------------ -------------- ----------------- ----------------- ----------------
  **Strain     ***Iso*-pentane**   **Pentane**   **Hexane**   **Octane**   **Decane**   **Dodecane**   **Tetradecane**   **Pentadecane**   **Hexadecane**
  Name**                                                                                                                                   

  **OT15DY**   3                   2             2            3            3            3              3                 3                 3

  **OT6A**     0                   0             0            1            0            1              0                 1                 0

  **OT6B**     0                   0             0            0            0            0              0                 0                 0

  **OT13D1**   2                   2             1            2            2            3              2                 2                 2

  **OT26A**    0                   0             0            0            0            0              0                 0                 0

  **OT18A**    2                   2             2            3            2            3              3                 3                 2

  **OT32AA**   0                   2             2            3            3            3              3                 3                 3

  **OT32DG**   0                   2             0            3            3            3              3                 3                 3

  **OT18F**    0                   0             0            0            0            0              0                 0                 0

  **OT32AB**   1                   2             2            2            2            2              0                 0                 0

  **GPo1**     0                   3             2            3            2            3              0                 0                 0

  **GPo12**    0                   0             0            0            0            0              0                 0                 0
  ------------ ------------------- ------------- ------------ ------------ ------------ -------------- ----------------- ----------------- ----------------

0 = absolutely no growth, 1 = no significant growth away from primary
streak; micro-colony, 2 = very faint colony formation, 3 = very
definitive growth, 4 = maximum growth on plate

Table 3: Strain homology with *alkB*, a well-characterized gene coding
alkane monooxygenase. Four different primer sets yielded the same
results. Homology with known *alkB* genes did not correlate with
hydrocarbon utilization patterns. Alkane oxidizing strains that do not
have homology with alkB may harbor a novel mechanism for alkane
oxidation.

+------+-----------+-----------+-----------+-----------+-----------+
| **St | ***alkB*\ | ***alkB*  | ***alkB*  | ***alkB*  | **Hy      |
| rain | (TS2S/    | (TS2S/d   | (TS2Smod/ | (         | drocarbon |
| Na   | deg1RE)** | eg1RE2)** | deg1RE)** | TS2Smod/d | ut        |
| me** |           |           |           | eg1RE2)** | ilization |
|      |           |           |           |           | pattern** |
+------+-----------+-----------+-----------+-----------+-----------+
| #    | yes       | yes       | yes       | yes       | All HC    |
| # OT |           |           |           |           |           |
| 15DY |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| **OT | no        | no        | no        | no        | C~8~,     |
| 6A** |           |           |           |           | C~10~,    |
|      |           |           |           |           | C~15~     |
+------+-----------+-----------+-----------+-----------+-----------+
| **OT | no        |           |           |           | None      |
| 6B** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| **   | yes       | yes       | yes       | yes       | All HC    |
| OT13 |           |           |           |           |           |
| D1** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| *    | no        |           |           |           | None      |
| *OT2 |           |           |           |           |           |
| 6A** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| *    | yes       | yes       | yes       | yes       | All HC    |
| *OT1 |           |           |           |           |           |
| 8A** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| **   | no        |           |           |           | n-C~5~ -  |
| OT32 |           |           |           |           | C~16~     |
| AA** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| **   | no        |           |           |           | n-C~5~ -  |
| OT32 |           |           |           |           | C~16~     |
| DG** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| *    | no        | no        | no        | no        | None      |
| *OT1 |           |           |           |           |           |
| 8F** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| **   |           |           |           |           | n-C~5~ -  |
| OT32 |           |           |           |           | C~12~     |
| AB** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+
| **GP | yes       | yes       | yes       | yes       | n-C~5~ -  |
| o1** |           |           |           |           | C~12~     |
+------+-----------+-----------+-----------+-----------+-----------+
| *    | no        | no        | no        | no        | None      |
| *GPo |           |           |           |           |           |
| 12** |           |           |           |           |           |
+------+-----------+-----------+-----------+-----------+-----------+

Table 4: Kinetic evaluation of potential biocatalytic agents. Results
demonstrate that biocatalysts have different levels of alkane
monooxygenase expression (V~max~) and harbor enzymes with different
affinities for the oxidation of octane (K~m~). Understanding kinetic
differences between biocatalysts is important to the development of an
engineered process.

  ------------ ------------------------------- --------------------------
  **Strain     **V~max~ for octane\            **K~m~ for octane\
  Name**       (μmoles O~2~/min/g biomass)**   (μM)**

  **OT15DY**   30                              1.28

  **OT6A**     32                              2.14

  **OT13D1**   13                              0.43

  **OT18A**    82                              2.76

  **OT32AA**   29                              0.74

  **OT32DG**   9                               0.81

  **GPo1**     28                              1.57
  ------------ ------------------------------- --------------------------