Research Proposal: Diesel Bioremediation Project with Rhamnolipids

This research proposal outlines a project focused on the bioremediation of diesel-contaminated soil using rhamnolipids. The project aims to assess the effectiveness of rhamnolipids, potentially in conjunction with a commercial bacterial consortium, in accelerating the degradation of total petroleum hydrocarbons (TPH) in soil. The study will involve site sampling, soil preparation, and TPH analysis using gas chromatography over a 12-week period, with pH and moisture level monitoring. The methodology includes laboratory testing to identify effective treatments and optimize conditions. The research seeks to compare the efficacy of rhamnolipids, natural attenuation, and a rhamnolipid-bacterial consortium approach to determine the most efficient and cost-effective method for diesel remediation. The proposal reviews existing literature, including studies on bioremediation techniques, microbial activity, and the impact of various factors on the degradation process. The project anticipates benefits such as increased knowledge and efficiency of rhamnolipids in bioremediation and cost-effective remediation strategies.

RESEARCH PROPOSAL FORM
Professional Scientist
1. Project Title
Diesel Bioremediation Project with the use of rhamnolipids
2. Project Start Date 3. Project Mentor
Semester Study Period 2
6. Project Background and Summary
Project Summary
Diesel is generally used as fuel which is not referred as very common source of contamination
for environment with context leaks in pipe and storage container and accidental spills. The diesel
contaminated soil gives major impact on microbial community, biodiversity and plant community
in severity aspect. It had gained capability for finding its own way for contamination in
groundwater systems which provides long term effect. It is considered as very important for trial
and remediation of soil which is returned to very healthy biome. There are different means for
utilising hydrocarbon degrading microorganism such as natural attenuation, bio-augmentation,
bioremediation strategies and bio stimulus with context of phytoremediation for the purpose of
re-mediating contaminated sites. It had been argued that bio augmentation is considered as best
for specific recalcitrant contaminants where auto chyhonous population might be missing. Its
selection had gained capability for producing microbial diversity which is essential for cleaning
less recalcitrant contaminant. Usually, biodegradable contaminants which would persist outcome
of conditions of sub-optimal environment like pH, temperature, availability of electron acceptor
along with biotic factors like predation through protozoa. In this specific review, comparative
study would be performed in scientific literature with reference to bioremediation of soil which is
polluted through diesel along with objective of optimising very efficient and cost effective method
for resolving this major issue. In the same series, it had also articulated various risk with its
duration.
Background and review
Comparative study
Study 1
Balseiro-Romero, M. and et. al., 2017. Characterization and degradation potential of diesel-
degrading bacterial strains for application in bioremediation
According to Balseiro-Romero and et.al. (2017), Bioremediation of diesel oil could be
occurred via natural attenuation and treated via bio-augmentation and bio-stimulation. It had
been evaluated via different technologies for degradation of numerous petroleum hydrocarbons
in soil. In the same series, presence of various diesel degradation microorganisms and microbial
activity for dehydrogenase check is also monitored. According to Er and et.al., (2018), natural
attenuation is referred as very effective as compared to bio-stimulation. However, there was
observance of huge microbial activity along with bio-augmentation on natural attenuation. The
microorganism from diesel degradation and population which is heterotrophic population is not
directly influenced through bioremediation treatments. It had been justified that identification of

remediation strategy provides effects on each soil type nutrients release rate. The effect of bio-
stimulation always decrease over time with rapid increase on pre effect on remediation.
Study 2
Chen, M. and et. al., 2015. Bioremediation of soils contaminated with polycyclic aromatic
hydrocarbons, petroleum, pesticides, chlorophenols and heavy metals by
composting: applications, microbes and future research needs
According to Chen and et.al. (2015), there is brief comparison on natural attenuation and
bio-stimulation with application of fertilizer which is directly based on NPL in an Alpine area
which is polluted in chronic aspect. It had been articulated that temperature gives major effect on
rate of bio degradation. On the contrary, bio stimulation would be giving effect which is
decreasing over the time but it had attained direct reduction of 13.9 micrograms with context of
hydrocarbons per gram. There is rapid remediation effect via bio stimulation.
Colla, T. S. And et.al., 2014. Bioremediation assessment of diesel–biodiesel-contaminated soil
using an alternative bioaugmentation strategy
Fuentes, S. and et. al., 2016. From rare to dominant: a fine-tuned soil bacterial bloom during
petroleum hydrocarbon bioremediation.
According to Colla and et.al. (2014), there are various factors which are considered for
application of microorganisms for purpose of bioremediation. It consists of numerous microbial
and environmental properties for hydrocarbon pollutant. There are various aerobic
microorganisms like species of Pseudomonas and Azotobacter which are most applicable on
bioremediation microorganism as it generates surfactants and helps in enabling emulsification of
diesel droplets allowing in new areas for availability of metabolism through microorganisms.
According to Fuentes and et.al. (2016), petroleum hydrocarbon was considered as very
important factor for contamination of soil in various parts of world. However, the possibility had
been indicated for degradation of fuel in various anaerobic conditions. It has gained outcome of
decrement in diesel fuel in its all conditions for comparing it with absence of electron
supplemented oil pillars. The rate of degradation of diesel is higher than compared to mixed
electron acceptor.
Study 3
Jung, J., Philippot, L. and Park, W., 2016. Metagenomic and functional analyses of the
consequences of reduction of bacterial diversity on soil functions and bioremediation
in diesel-contaminated microcosms.
According to Jung, Philippot and Park (2016), there is specific analysis of techniques for
monitoring its degradation of its contaminant degradation and time-frames of bioremediation.
Different hydrocarbon pollutant of petrol’s along with alkanes and aromatic compounds are
contained in diesel for posing specific health risks to organisms. Similarly, the bacterial
community evaluation had been monitored with both molecular culture independent methods and
culturable techniques which could be employed. However, there is absence of time frames and
experiments and assessment of bioremediation which could be observed for more than 2 – 3
months on basis of specific range. The time frame and its effectiveness is dependent on various
processes with application of material.
Kuppusamy, S., Thavamani, P., Megharaj, M. and Naidu, R., 2016. Bioaugmentation with novel
microbial formula vs. natural attenuation of a long-term mixed contaminated soil—
treatability studies in solid-and slurry-phase microcosms
As per views of Kuppusamy and et.al. (2016), time frame would be improved for purpose
of remediation is referred as rhizoremediation. It is performed through combination of bacterial
microorganisms and plant. The roots of specific plant had been spread on bacteria for assisting
to penetrate in different parts of soil which is normally very easy.

7. Industry Benefits
 It would be providing additional knowledge along with effeciency of rhanmnolipids in
bioremediation.
 The triple bottom line would be affecting success which consist of cost effectiveness and
speed with reference to re-mediation.
PROJECT PLANNING
8. Project Objectives
The main objective of this specific project is to remediate diesel contaminated soil with in
25 squares meter along with concentration of 100 mg/kg. It is concentrated in low aspect as it
would be laying special focus on application of rhamnolipids with context of diesel contaminated
soil in bioremediation and in raising its effectiveness in its process.
Research question
 Will the addition of rhamnolipids to a commercially available bacterial consortium
increase the speed of bioremediation and reduction of TPH (Total petroleum
hydrocarbons) from diesel contaminated soil?
Hypotheses
The application of commercial grade rhamnolipid biosurfactant along with microbial
culture would be re-mediating with context of diesel contribution in low aspect which is
contaminated in soil in rapid manner for lowering final concentration as compared to microbial
culture treatment. This particular research would be assisting various companies and different
bodies for the decision which is informed, dependening on objective of bioremediation and for
cost effectiveness. There is also application of rhamnolipid along with bacterial consortium would
be more costly as it might consider raise in process. There is presence of various studies on
basis of rhamnolipid with its application in bioremediation but this specific proposal provides
direct comparison on rhamnolipid, natural attenuation and rhamnolipid along with bacterial
consortium for assessing the fastest process and cost effectiveness. This study would be directly
laying special emphasis on hydrocarbon concentration and weekly testing would be done to
determine level with context of removal of hydrocarbon, which had been gained over duration of
12 weeks. Its implementation of rhamnolipid has reflected the highest degree of efficacy with
application of commercial bacterial consortium Global Clean up.
9. Research Methodology

Methods for investigating problem
The effectiveness of rhamnolipids could be determined by raising up process of
bioremediating diesel for contaminating soils could be performed in two steps:
 Identifying very effective treatment
 Identification of effective treatment through laboratory testing
Identification of effective treatment
 Site sampling: It had been performed for determining treatments in effective aspect for
various optimal conditions which could be performed in such aspect. The site would be
divided in 25 * 1 square meter numbered plots and there would be collection of 2 kg
contaminated from every plot. Its sum of total sample would be weighting 50 kg.
 Sample preparation: The sample of soil would be similar at initial level for total
petroleum hydrocarbons which are tested with method of EPA as it is solvent extraction
for preparation of sample for purpose of Gas Chromatography for identification of
hydrocarbons.
 Data analysis: It would be on context of total petroleum hydrocarbons on daily aspect for
2 weeks with application of gas chromatography and method had been trapped for
identification of effective treatment.
Identification of effective treatment with Laboratory testing
 There would be identification of effectiveness of specific treatment with context of
conditions of laboratory testing has to be undertaken. The optimal condition should be
ensured by commencement of pH and moisture levels with testing of 25 Square meter
plots.
 The site would be vaccinated along with rhamnolipids and Global clean-up at determining
its specific concentration.
 The testing of TPH would be on daily aspect for first week and pH and moisture testing
would be performed in duration of 3 days. The CFU bacterial plating and ecotoxicology
test through daphnia and earthworm assays.
 TPH testing would be initiated on 2nd week as site would be tiled on every third days with
requirement of pH and moisture tested and adjusted. It would be determining bacterial
viability through CFU bacterial plating.
 In 12th week there would be ensured of its TPH level which had reduced through 90%.
In the same series, sample could be undertaken on daily aspect from 3 sites in every plot
in first week and it should be tested weekly, for 12 weeks. The level of TPH has to be analysed
from every sample and its outcome should be averaged in every plot. pH and soil moisture must
be tested and changes if there is any requirement. Every plot would be tiled in duration of 3 days
for providing surety on mix of microorganisms along with distribution of nutrients, moisture and
oxygen.
10. Project Data, Analysis & Evaluation
Equipment, materials and techniques required
Bacterial Consortium - The bacterial consortium we will be utilizing is a well-established
bioremedial blend of naturally occurring indigenous bacterial and fungal microorganisms
called Global Clean Up. A 5 litre drum will be sufficient to remediate the 25 square
metres and for the ex situ testing. Global Clean Up has proven effective for diesel
degradation. For bioremediation we need to ensure optimum conditions in the soil for
bacterial growth including pH ranges (5.5 to 9.5) adjusted if required, temperature (15 to
45 degrees celsius) and moisture. Additionally, it is important to have as much oxygen
distributed throughout the soil as possible, as the more oxygen in the soil, the greater
efficacy and greater rate of clean up. Regular tilling of the soil by hand hoeing will be
instrumental in ensuring this.
Gas Chromatography is the preferred method of testing for quantitative analysis of TPH
contamination in soil or water. We will be using the purge and trap method as this is an
efficient technique to determine TPH concentration of the diesel range organics (DROs).
Microbial testing will be conducted weekly to determine the levels of bacteria present.
The analysis methods to be used are plate counts, respirometric analysis and the most-

probable-number (MPN) technique. This will give us insight into microbial activity and
growth.
Description of data generation & analysis
The results of the soil subsamples will be taken from the gas chromatography tests.
From this, TPH concentration will be determined and analysed from across the 12 week
period. By further inferring data from DROs, the ideal levels of pH, temperature, moisture
and soil oxygenation will be able to be deduced with regards to the presence or absence
of rhamnolipids. Furthermore, the data generated from the results will validate or negate
our hypothesis, as well as establish the speediest and cost-effective methods of
bioremediation with respect to rhamnolipids. The results will also be analysed by the
PCA (principal component analysis) in order to determine the most intensively altered
bacterial consortium. Results from the sample will be analysed by a Pearson chi test to
provide further levels of analysis.
11. Identification of Background Intellectual Property (IP) & Potential Project IP
The existing IP is based on the hypothesis of the use of rhamanolipids to speed up the
bioremediation process with a tried and tested bacterial consortium. If the hypothesis
proves successful, a combination bioremediation rhamnolipid/bacterial product could be
developed.
12. Dissemination of Results
Stakeholder Key Message Communication Action Who
RMIT university Convey detail
about project
timeline, financial
requirements.
Send research proposal
to university.
Local council
Community
consultation
event
Convey
significance and
limitation of
project.
Local council meeting,
send fax.
Research project
Scientific
community
Send detail of
entire project with
estimated results
(Szulc and et.al,
2014)
Writing article and
discuss success of
entire project.
Research project
13. Project Delivery Risks
Risk Management Action
Breaching of code of conduct
It might be possible that while working on this
project, team members do not follow code of
standards. This is responsibility of researcher
that employees follow each code of conduct in
appropriate manner, otherwise it might create
issue in entire project
Researchers has to ensure that each
regulation is followed properly and
proper training of these standards
given to team members (Saborimanesh
and Mulligan, 2015).
Plagiarism
It is another risk that might impact negative on
entire proposal. Project manager will have to
take support of other scientists work but
individual has to ensure that their material
Work of others properly paraphrased
so such issue do not take place.

does not get copied (Ma and et.al, 2016).
Plagiarisms against the research ethic and if it
is found in any kind of project then this might
impact negatively on entire project.
Financial risk
It is responsibility of project manager that to
allocate resources efficiently, individual due to
lack of funds researcher might get trouble to
complete this task appropriately.
Effective allocation is done and
effective monitoring is required to
minimise this risk (Szulc and et.al,
2014).
Timing risk
Individual has short duration to finish this
project, due to lack of time length person may
face trouble and might compromise with quality
(Satti and et.al, 2018).
Proper scheduling and monitoring
Confidentiality risk
There is high risk of confidentiality, it might be
possible that data is hacked by other and
misuse this data.
Passwords for computers and security
checking ensured (Safdari and et.al,
2018)
Human resource risk
Project manager can face issue of human
resource, it might be possible that team
members do not understand the concept and
they fail to perform their duties well (Safdari
and et.al, 2018).
Training to staff members and give
them reward so that they feel motivated
and perform well.
Intellectual property disputes Reviewing contract properly and
following terms and conditions
effectively.
Community perception Individual should ensure that
community members give their
concerns for the project
14. Project Schedule– See Table 1.
15. Project Expenditure Summary –See Table 2.
Project manager will have to recruit staff members those who can work on this project
effectively for 15 weeks. Diesel Bioremediation is considered as most essential project
that can help in improving environmental condition and protecting natural resources to
great extent. In order to accomplish this task in efficient manner, project manager will
require various resources (Ma and et.al, 2016).
Manger will require many tools and techniques in order to execute this project in
efficient manner. Individual will need 5 litre drum for the ex situ testing. Furthermore,
manager will have to test pH range of soil and its temperature as well (Saborimanesh
and Mulligan, 2015). For that pH testing machine will be mandatory. Furthermore, it is
very important to have adequacy of Oxygen. Manager of Diesel Bioremediation Project
will have to apply probable number of technique, respirometric analysis method etc. In
order to perform these activities individual will need pH probe and relevant meters.
Furthermore moisture probe will be required to test the moisture level of soil.
In addition, other physical and technical resources will be needed in this project
are such as water tanks, sprinkler system, gas chromatographs, incubator, personal
protective equipment, hand spray equipment, CO2 respirometer analysis kit etc (Safdari
and et.al, 2018). Furthermore, individual will need human resources, including those who
can perform all these methods and can do testing. Transportation sources will be
required for examining different soil and their nature. In addition, manager will need

financial resources in order to buy these equipment and machineries, so that soil testing
can be done in appropriate manner. All these technical, physical, human resources will
be needed in order to accomplish this proposal (Risk assessment in project
management, 2018).
In order to complete Diesel Bioremediation Project, manager will have to spend huge
amount in various activities). Personnel expenditures include research associated cost
and fee taken by environmental consultant. This will require spending of $41700.
Furthermore, equipment cost for TPH testing would be 28.6 per sample. Hand tool for
tilling soil cost $150. Furthermore, transportation will cost $500 and other miscellaneous
expenses of entire project would of worth $700. Total cost of this project would be
$88018.6.
APPLICATION ACKNOWLEDGMENT:
I have undertaken this research proposal development in
consultation with my project group. This is my documentation of
the group work.
Date
REFLECTIVE COMMENT
Comment on:
 It was such as great experience for me to work on Diesel Bioremediation Project with the
use of rhamnolipids. I have learnt about various system and techniques that can help in
improving environmental condition. I learnt about soil pH and temperature aspect that is
very essential for such type of projects. It was such an amazing experience for me because
I got chance to work with highly talented people. Team working plays significant role in any
task, if all team members perform their duties well, then it would support in completing
project in efficient manner. My group has worked so good, I have conducted meeting with
them every week. In this meeting, we discussed about progress and risks. This has
supported in getting innovative ideas of completing this proposal in effective manner.
Conducting meeting has developed trusts and we started communicating with each other
properly. All team members have given equal chance to others to show their talent and
accomplish the task properly.
 All members have participated equally that has helped me in completing the entire project
within stipulated period of time. Team members were researching about Diesel
Bioremediation Project with the use of rhamnolipids by taking support of other researchers.
They share their learning and ideas in every meeting. This thing has motivated all team
members and encourages them to work more effectively.
 I have learnt many technical things while working on this project. I have developed my
understanding about framing hypothesis in quantitative researches. I learnt about how to
use this hypothesis in statistical calculations in order to generate valid results. I have also
learnt that if all group members work to achieve common purpose then it helps in
completing task efficiently and meeting with this objective. I have learnt importance of time
management, if individual is unable to manage time well then person will get failed to
complete this project within set period.
 This Diesel Bioremediation Project has improved my knowledge and now I am able to work
more effectively on such kinds of proposals. I have improved my communication,
leadership and research skills to great extent. I have also improved my knowledge about
environmental issues and tactics to improve condition of surroundings. I can add this trait in
my CV for getting better job opportunities. I acquired technical skill; I can add this thing into
my CV so that I can create good impression on employer.

Table 1. Project Schedule

Table 2. Expenses Summary