Microbial biodegradation potential of hydrocarbons evaluated by

colorimetric technique: a case study

E. D. Bidoia, R. N. Montagnolli and P. R. M. Lopes

Departamento de Bioquímica e Microbiologia, Instituto de Biociências, UNESP – Univ Estadual Paulista, Av. 24A, 1515,

13506-900 Rio Claro-SP, Brazil

The increasing industrial development promotes serious environmental damage due to pollution of the environment.

Regarding the petrochemical industry, contamination by oil and its derivatives causes the degradation of terrestrial and

aquatic ecosystems. Thus, control and treatment strategies to combat the hazardous effects of oil pollution are needed.

However, conventional physical-chemical treatments have high costs and can generate residues that are toxic to the biota.

Allying high efficiency and low cost, bioremediation processes represent an extremely important way of recovering

contaminated areas among several other cleaning-up techniques. These strategies involve microorganisms and their

metabolism in biodegrading organic compounds. Also, the use of nutrients, aeration, pH and temperature adjustments or

the addition of substances could make the biodegradation process easier. In order to accomplish this, screening and

evaluation methods adapted to a potentially biodegrading microbiota in different types of contaminants have been

established. Viable methods in biodegradation data generation during biotechnological process application are

fundamental in the elaboration of original references about the biodegradability of certain substances. There are many

techniques capable of precisely evaluating biodegradation processes, including colorimetric methods. The isolation,

characterization and profile of specific bacteria in petrol derived oil biodegradation capacity studies are important when

deciding the correct bioremediation strategy. Different microorganism species have different biodegradation capabilities.

Due to this fact, the elaboration of different types of oil biodegradation profiles by different bacteria is an important task

for selecting microorganisms in bioremediation processes. In order to accomplish this, screening and evaluation methods

adapted to potentially biodegrading bacteria have been established. One of these methods adapted to biodegradation

evaluation is colorimetry, which is a technique used to evaluate the biodegradation of some substances. DCPIP based

colorimetric technique provides enough data on hydrocarbons used as metabolic substrates by microorganisms. The

concentration detection is possible due to the absorbance determination in a specified light specter. The 2,6-dichlorophenol

indophenol (DCPIP, redox potential +0.217 V) indicator is widely used in colorimetric processes. Its property is the color

change from blue to transparent when subjected to chemical reduction. The indicator, when oxidized is blue and when

reduced is transparent. The color change occurs due to a structural change in the molecule, in which the double bond

between nitrogen and carbon passes to a simple bond. This insaturation changes the entire molecule, resulting in a

macroscopic change in the overall color of the biodegraded substance. The DCPIP indicator is applied in a series of

electron transfer reactions, including biodegradable substances. Colorimetric methodology applied to oil biodegradation

promotes a better handling of different oil microbial biodegrading profiles. Moreover, such rapid and simple colorimetric

methodology provides resources on the development of new techniques in effluent treatments, not only during petrol

derived oils, but also on other contaminated organic polymeric compounds.

Keywords oil pollution; bioremediation; microbial metabolism; colorimetry; 2,6-dichlorophenol indophenol

1. Remediation of impacted environments

1.1 Oil pollution

Petroleum products are extensively widespread all over the world and their intensive use is strongly connected to the

anthropogeneous discharge of hydrocarbons into the environment [1]. Environmental contamination by petrol

derivatives has been a subject of study over the past four decades. The leakage of these derivative oils, such as lubricant

oils, is capable of harming the environment in many ways [2].

A major concern for petroleum hydrocarbon bioremediation is the presence of heavy compounds such as polycyclic

aromatic hydrocarbons (PAHs), asphaltenes and many branched compounds with 20 or more carbon atoms. These

heavy hydrocarbon constituents are not easily metabolized by microorganisms and are considered potential health risks

due to their possible carcinogenic and mutagenic actions [3].

Furthermore, 1% of all oil consumption is used to produce lubricants [4] and all procedures in lubricant oil

production and transport can generate environmental impacts [5]. Hamblin [6] emphasized the percentage of used oil

discharged into ecosystems without any treatment as 13% for Europe and 32% for U.S.A. Moreover, lubricant oil can

persist for more than six years in some ecosystems, resulting in chronic problems to the biota [7]. Even under laboratory

conditions, the complete metabolism of oil by microorganisms takes weeks to months [2].

Lubricant discharge in nature causes continuous concern due to its non-quantified impact and its potential chronic

damage [8]. In this context, residual lubricant oil contained in commercial bottles causes significant contamination,

because one ton of this automotive oil corresponds to the waste produced from the domestic sewers of 40,000

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inhabitants per day. Besides this, only 1.0 L of lubricant is able to deplete the oxygen of 1,000,000 L of water, therefore

a city discharge of 200,000 inhabitants is the same as 47 L of residual oil, which affects 47 million L of water [9].

1.2 Remediation processes

Physical and chemical processes are usually used in remediation of contaminated areas. Dispersion, dilution, sorption,

volatilization and abiotic transformations are important means of hydrocarbon elimination. However, this type of

treatment system requires heavy machinery and the environmental consequences of this pollutant removal may result in

massive air pollution [10]. Allying high efficiency and low costs, bioremediation processes represent an extremely

important way of recovering oil contaminated areas among several other cleaning-up techniques [11].

Remediation by physical treatments revealed that, even though the soil is unaffected or unmodified by these

treatments, the costs are high, and thus unviable in a large scale.

In spite of this, biodegradation is most often the primary mechanism for contaminant clean-up [12, 13]. Also, for the

remediation of hydrocarbon contaminated sites, biological technologies are a widely used, cost efficient and sustainable

approach [14].

2. Biological treatment

2.1 Degradation by microorganisms

Bioremediation exploits the ability of some microorganisms to degrade organic contaminants and has been established

as an efficient, cost-effective, and environmentally friendly treatment [13; 15]. This process depends on environment

conditions and the microbial community structure and, in unfavorable conditions, is generally achieved via

bioaugmentation, biostimulation or both [16].

Microbial biotransformation is considered a major environmental technique in treating hydrocarbon pollution in both

terrestrial and aquatic ecosystems [17; 18]. This suggests that the biodegradative contribution of the indigenous

microorganisms is often significant [13; 19].

Therefore, in order to eliminate or minimize the effects of such contaminations, bioremediation shows up as an

effective alternative, which can be used by potentially biodegrading microorganisms [2]. Microorganisms are capable of

performing the mineralization of organic chemicals, transforming substances ultimately into CO2, water and biomass

[20]. This transformation capacity is of main importance in bioremediation processes, which relies on biodegradation.

The activation of natural degradation potentials in environmental media is the challenge for environmental research

that addresses remediation methods. Notwithstanding the widespread application, in situ bioremediation is a site-

specific process and feasibility studies are required before full-scale remediation can be applied successfully [21].

Moreover, degradation rates and in particular residual concentrations derived from preliminary studies in the laboratory

can be used to predict achievable rates and concentrations in the field [22].

Hence, feasibility studies are a prerequisite for any planned strategy in bioremediation contaminated environments in

order to identify limitations towards biodegradation and to predict remediation performance and thereby rule out

technologies that may be inappropriate for the clean-up of the site of concern [23; 24]. Additionally, the environmental

behavior and chemical-physical properties of lubricant oils are the basis for new fluid developments and for ecological

treatment strategies [25].

2.2 Bioremediation of hydrocarbons

Viable methods in biodegradation data generation during biotechnological process application are fundamental in the

elaboration of original references about the biodegradability of certain substances [26]. There are many techniques

capable of precisely evaluating biodegradation processes, including colorimetric methods. The isolation,

characterization and profile of specific bacteria for petrol derived oils biodegradation capacity studies are important

when deciding the correct bioremediation strategy.

According to Torstensson [27] and Aichberger et al. [23], preliminary assessments account for three critical

prerequisites in bioremediation, namely: (i) the availability of chemicals for the biodegrading microorganisms; (ii) the

quantity of these microorganisms; and (iii) their activity level. Other factors are also important in moderating and

driving biodegradation, such as temperature, pH, aeration, organic matter and nutrients.

Due to hydrocarbons being widespread pollutants, their hydrophobicity causes a low bioavailability and therefore a

particular persistence against bioremediation measures [28; 29]. However, numerous terrestrial and aquatic species of

microorganisms possess the ability to degrade these hydrocarbons. In order to increase the bioavailability of these

otherwise hardly accessible substrates, microorganisms use strategies like increasing the hydrophobicity of their

surfaces or the production of biosurfactants [30].

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Long chain hydrocarbons, specifically cyclic alkanes present in petrol derived oils, are difficult for bacteria to

degrade [31]. In order to efficiently promote biodegradation, many hydrocarbon degrading bacteria were investigated

after isolation in an oil polluted environment [32-35].

Referring to automotive lubricants, biodegradation studies demonstrated that used oil showed considerable

assimilation by microbial metabolism when compared to new fluids such as mineral, semi-synthetic and synthetic ones.

Automotive lubricant processing in engines modifies its physical-chemical properties due to high pressure and

temperature that promote breaks in the hydrocarbon chains. Thus, shorter carbon molecules facilitate microorganism

degradation [36; 37].

Lopes et al. [38] studied the toxicity of different lubricant oils (mineral, semi-synthetic and used) before and after

biodegradation using Eruca sativa seeds (arugula) and Eisenia foetida (earthworm) as test-organisms. It was observed

that although used oil was the most biodegradable, it was more toxic than new lubricants. Also, as biodegradation

proceeded the toxic potential was reduced, but a substantial toxicity remained as hydrocarbons persisted in the soil [39].

Thus, the used lubricant oil hazards are harder to quantify because of its complex composition. Many compounds

contribute to high toxicity, but the PAHs formed in service transform used oil into a potential carcinogen. PAHs derive

from fuel combustion and lubricant decomposition, therefore their concentration increases in used oils [40].

2.3 Biostimulation

Microbial efficiency in degrading organic compounds is a function of the ability of the microbial degraders to remain

active in the natural environment. Hence, increasing the ability of the inoculated microorganisms by bioaugmentation or

promoting the activity of indigenous microbial degraders by biostimulation could improve bioremediation [12].

It has been reported that the addition of nutrients has a beneficiary effect on hydrocarbon degradation in the soil by

enhancing microbial activity in organic compounds metabolization [39; 41-43]. According to this, a

carbon:nitrogen:phosphorous (C:N:P) ratio of 100:10:1 is commonly proposed [44; 45].

2.4 Bioaugmentation

Individual isolates have lower ability than microbial consortia in biodegradation, especially for complex mixtures of

compounds such as diesel oil and crude oil [43].

Vecchiolli et al. [46] demonstrated that in addition to the natural presence of hydrocarbon degrading bacteria in soils,

exogenous microbial inoculation is able to accelerate the biodegradation whenever conditions are appropriate. They

conclude that biodegradation of polluted soils with petroleum hydrocarbons such as accidental spills may be enhanced

by indigenous bacteria inoculation. In the same way, Goldstein et al. [47] observed that when species capable of

destroying organic compounds are added to the natural environment there is an enhanced biodegradation of these

substances. The addition of microorganisms in polluted environments is a technique called bioaugmentation.

Bioaugmentation provides specific microbes in sufficient number to complete the biodegradation process. Over the

last few years, contaminating compounds such as insecticides, petroleum compounds and a growing number of toxic

organic chemicals have been successfully remediated using bioaugmentation [48-50].

Several advantages are present in bioaugmentation when compared to other techniques: the specific inoculum

addition starts the degradation process immediately whereas, in biostimulation, after the nutrients are injected there is a

delay in beginning the major biodegradation process [51].

It must be noted that in nature biodegradation capacity is not due to a single microorganism within oils. Metabolic

processes in hydrocarbon breaks occur with a succession of species within a microbial consortium. Even non-petrol

degrading microorganisms may play an important role in environment oil removal, as the degradation of petroleum

leads to a progressive chain of reactions, which leads to intermediate compounds handled by other types of

microorganisms [52].

Moreover, different microorganism species have different biodegradation capabilities. Due to this fact, the

elaboration of different types of oil biodegradation profiles by different bacteria is an important task for selecting

microorganisms in bioremediation processes. In order to accomplish this, screening and evaluation methods adapted to

potentially biodegrading bacteria have been established.

3. Redox indicator 2,6-dichlorophenol indophenol

3.1 Redox reactions

Alternative means of determining when reducing conditions exist are provided from redox indicators. Generally, it is

observed that oxidized forms are colored and reduced forms are colorless. Due to this fact, color changes can be

monitored with a spectrophotometer, evaluating changes in redox indicator absorbance value.

The redox half reaction of a redox indicator is described by Equation 1 below. As the oxidized indicator reacts with a

reductant, the absorbance decreases and the reducing power of the sample can be estimated.

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(1)

where:

Indox is the oxidized form of the indicator, Indred is the reduced form, ne- is the number of electrons transferred, and m

is the number of protons transferred - pH dependant [53].

Also, many redox indicators are reversible and couple to a Pt electrode [54]. The redox potential for an indicator

(Eind) is determined by the relative concentrations of both oxidized and reduced species and the Nernst Equation 2

below.

(2)

where:

Eo’ind,pH is the formal potential of the indicator at the specified pH.

The potential can vary between various indicators and is often a complex function of pH, since many different

indicators have groups (i.e. amines) that can be protonated or deprotonated.

3.2 Redox within microorganisms

Metabolic pathways within microbial cells are concisely regulated and extremely sensitive to the cell’s need. As a rule,

redox reactions and specific redox couples, that cycle between oxidized and reduced states, occur in all of the major

metabolic pathways in cells. The concentrations of the redox couples help to regulate the flow of metabolites through

these pathways. These intracellular concentrations of redox couples might, likewise, be responsive to receptor agonists,

as well as cell nutrients [55]. According to this fact, by using chemical analysis, such concentration changes can be

evaluated.

As an example from Rabinowitz et al. [56], some biochemical techniques are used substantially for the direct

quantization of NAD+/NADH, NADP+/NADPH, cystine/cysteine, and oxidized/reduced glutathione (γ-

glutamylcysteinylglycine, GSSG/GSH), thus, developing probes of cellular redox reactions permit the screening of

chemical libraries for compounds affecting redox metabolism. Moreover, for any one redox pair, different potentials

may be maintained in different subcellular compartments [57].

3.3 Redox indicator DCPIP as an electron acceptor

Hydrocarbon oxidation processes by microorganisms involve redox reactions, in which electrons are transferred to

electron acceptors, such as O2 nitrates and sulfate [58], as seen in Fig. 1. The microbial sulfate-reducing redox is one of

the most reducing redox environmental systems. In terms of formal reduction potentials, sulfate reduction to sulfide

occurs at about 220mV at pH 7 [59]. A low redox level is achieved in an environment where conditions are completely

anaerobic and the terminal electron acceptors (TA) such as NO3, Mn(IV) and Fe(III) have been fully depleted [60].

Sulfate-reducers use sulfate as the TA and commonly lactate, acetate, or H2 as the electron donor in the process of

creating ATP, and producing sulfide [59].

Fig. 1 DCPIP redox scheme, for the catalytic oxidation of sulphide

(adapted from Florou [61]).

Common means of measuring sulfate include ion chromatography with a conductivity detector and gravimetric or

turbidimetric methods based on Ba(II) as a precipitating agent [62]. Redox environments, including the sulfate-reducing

and/or nitrate-reducing conditions in environmental systems, are usually determined by monitoring decreases or

increases in sulfate concentration. This way, as seen in Fig. 1, microbial metabolism can also be commonly measured

with some colorimetric methods including methylene blue method, iodometric titrimetry or the 2,6-dichlorophenol-

indophenol indicator (referred as from this point as DCPIP).

Colorimetric methods are referred to as a low cost and rapid procedure in detecting microbial metabolism

occurrence, both in aerobic or anaerobic instances. These methods represent a rapid catalytic microbial based method in

which the natural co-substrate (which can be oxygen, sulfates or nitrates) is substituted by a synthetic mediator [63].

Colorimetry is characterized by high levels of microorganisms and mediators, facilitating the fast reaction. Mediators

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basically act as chemical electron carriers that are able to pass through the cell walls of microorganisms, movingbetween intracellular and extracellular spaces. Once inside the microbial cell, an interaction between mediators andtheir bioelectrochemical counterparts, such as NAD, NADP, flavoproteins, iron–sulfur proteins, quinones andcytochrome, happens by their interaction with the metabolic process and current redox reactions [64]. Microbial cellscatalyze mediator reduction with certain ease when in a very favorable environment. The reduction activity is asensitive indicator directly coupled to respiration via the electron transport chain, and thus reflects the metabolic statusof the cell [65]. DCPIP indicator is included in this category of lypofilic mediator and electron acceptor, capable ofprobing microbial metabolism.

Thus, by incorporating an electron acceptor such as DCPIP, it is possible to ascertain the ability of a microorganismto utilize a hydrocarbon substrate by simply observing the color change, in this case, from blue (oxidized) to colorless(reduced) according to Fig. 2.

Fig. 2 DCPIP reduction reaction.

This technique is aimed at the need to reach a selection of sensitive biocatalysts and mediators capable of detectingthe metabolic perturbations caused by various substrates, including toxic ones [66] or biodegrading processes. It is wellknown that mediators fundamentally work by interacting with the metabolic pathways of the microbial cell. Eventhough the facts regarding interaction of microbial metabolic pathways with electron transfer mediators is quite animportant study, with some important practical usage, few publications are devoted to finding the solution to such animportant question as the search for the interaction mechanism between microbial and mediator, and also the origin ofthe electrons. Thus, more studies are necessary in order to elucidate completely this subject. According to Heiskanen etal. [67], once the interaction mechanism between microbial and mediator is made clear, the mediator will assume thefunction of an electrochemical probe, as the functionalized mediators would be useful for a better comprehension of theeffects of physiochemical reactions occurring within microorganisms in the redox state.

3.4 DCPIP characteristics

DCPIP is an enzyme-catalyzed oxidation electron acceptor that is blue in its oxidized form and colorless in its reducedform [68]. Its loss of color is monitored at a wavelength of 600 nm. A peak in the absorbance is observed at 600 nm, asreported by Yoshida et al. [69]. Some studies affirm that DCPIP is unstable in darkness or when exposed to light for along time [70]. Moreover, the color of the DCPIP indicator in the presence of light is reasonably stable and can be usedreliably for some time, especially when adding NAHCO3 to the dye solution [71].

Additionally, higher concentrations make the color become too dark and the color change induced by reduction withredox reactions is not easily apparent [72]. On the other hand, at lower DCPIP concentrations, the reagent amount isinsufficient to signal any perceptible decreases. So, 1g L-1 is the concentration usually selected in many studies [73-75].

A pH value of 7.25 is chosen as satisfactory working stability with DCPIP, since the redox mediator is notsufficiently stable for higher pH values. Moreover, DCPIP’s reactivity is reduced because of its exposure to a veryalkaline medium (0.07 M KOH, pH 12). Regarding the temperature effects, the response of the indicator is alsoestablished to a maximum response ranging from 297 to 303 K [76]. A calibration curve for DCPIP was obtained from0.2 to 140 μM with the same spectrophotometer and showed an excellent correlation coefficient (r=0.9997) according toNakamura et al. [73]. In an earlier report, Yari et al. [71] obtained a similar curve and similar correlation coefficient.

DCPIP is a pH dependant redox indicator, and not only does it change from blue to colorless, it can also turn into ared color at low pH systems, and also in the presence of some other cations e.g. Al3+, Fe3+, Pb2+ and Cu2+, which changethe pH of the system and act as Lewis’s [53]. Therefore, DCPIP can act as an acid-base indicator, which is reddish atpH< 5.7. Furthermore, at pH >5.7 in alkali and neutral aqueous solutions its color is blue, acting as the electrochemicalresponse of DCPIP [77]. Below a particular potential, it is capable of accepting two electrons from its environment,becoming reduced and giving up its color [71].

4. DCPIP Applications

4.1 Discovery and original utilization

The DCPIP redox reaction was originally used and discovered by Vernon and Zaugg in 1960 [78]. They found out thatthe DCPIP, when ascorbate was present, formed an efficient electron donor couple for photosystem 1. Studies involving

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.)

©FORMATEX 2010 1281

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individual photoreactions in plant chloroplasts were improved by proper assay systems available and specific

photosystems operating in normal photosynthesis. DCPIP brought more details to the knowledge regarding the

mechanism of NADP photoreduction by photosystem 1.

Subsequent studies by Yamashita et al. [79] and Nakamoto et al. [80] showed that following the procedure of

washing chloroplasts with 0.8 M tris buffer at pH 8.0 prevented any ability of chloroplasts to produce oxygen, however

in the presence of DCPIP, along with an excess of ascorbate, photophosphorylation of the non-cyclic type could be

restored, which indicated that electrons were entering into the system at the level of photosystem 2. Vernon and Shawl

[81] presented some other studies using DCPIP and produced some great achievements in botany regarding oxygen

evolution activity within chloroplasts.

4.2 DCPIP related studies

The redox indicator capabilities from DCPIP chemical compound were reported in a series of various studies. In their

study regarding the use of Gluconobacter oxydans bacteria cells as a biocatalyst at an anode of a biofuel cell with air-

based cathode, Alferov et al. [82], used the anode and cathode of the cell made of graphite; DCPIP as an electron

transport mediator; and glucose as the substrate to be oxidized. Other uses for DCPIP are: the measurement of

mitochondria complex activities, which determined the activity of succinate–ubiquinone oxidoreductase by the

reduction of DCPIP [83]; as the reactions of thiocholine with DCPIP, which is a simple, reliable and easy method for

automatable measurement of acetylthiocholine during clinical laboratory routines [84]; measurements of the zero

current potential of a platinum electrode immersed in solutions of tannins or in wines of various origins with a solution

of DCPIP, in order to obtain a global indication for the resistance of some wines to oxidation [85]; the application of an

electrochemical sensor, based on a glassy carbon electrode, modified with a cellulose acetate polymeric film bearing

DCPIP, for flow injection analysis of sulfide [76]; the kinetic parameters of both human liver and rat purified

recombinant PDH E1 proteins were determined utilizing DCPIP [86]; a simple screening protocol using DCPIP to

examine the prevalence and the molecular basis of thalassemia in pregnant Lao women [87] and many other original

references ranging from botany to medicine [75; 88-90]. A study informed that altered redox homeostasis in control of

cancer cell survival and proliferative signaling are also a chemical vulnerability that could be targeted by pro-oxidant

redox intervention [91].

5. Biodegradation screening with DCPIP indicator

Pollution by petroleum and its derivates caused by accidental spillage is a concerning problem in the environment. A

well known fact is that there is a group of main microorganisms able to consume petroleum hydrocarbons, which are

bacteria and fungi [92]. Microorganisms are considered degrading agents of a great variety of substances, corroborating

that biological treatment is the most promising alternative for reducing the environmental impact of oil spills [93].

Product composition when released into the environment begins to change immediately because of various

biochemical processes. The characteristics of discharged oil pollutants may be changed by physical, chemical and

biological mechanisms after large periods in altered environmental conditions. Mixed-function oxidase systems present

a great catalytic versatility and are also widely distributed in the environment, taking part in the biotransformation of a

wide variety of both exogenous and endogenous organic compounds [94]. Biodegradation involves a list of

monooxygenation reactions that are catalyzed, including heteroatom oxygenation and dealkylation, aliphatic

hydroxylation, desaturation, oxidative group migration and many other modes of inactivation [95]. Thus, the use of

DCPIP redox indicator presents a rapid, simple and low cost tool for evaluating capability of different microorganisms

to degrade different oils [74; 96].

The biodegradability of petrol derived oils can be verified using the technique based on the DCPIP redox indicator

according to what was discussed up to this point. It is important to remember that the principle of this technique is that,

during the microbial oxidation of the carbon source, electrons are transferred to electron acceptors such as O2, nitrates

and sulfate. Most petroleum-related hydrocarbons are readily degraded via aerobic microorganisms although a number

of studies have shown that in the absence of oxygen alternate electron acceptors such as nitrate, manganese (IV), iron

(III), sulfate and carbon dioxide are used [21; 97-101]. By incorporating an electron acceptor such as DCPIP to the

culture medium, it is possible to ascertain the ability of the microorganism to utilize the substrate by observing the color

change of DCPIP from blue (oxidized) to colorless (reduced) in a screening technology first reported to be applied in oil

biodegradation by Hanson et al. [96]. This technique had also been successfully employed in several studies presented

below.

Enzyme induction DCPIP activity and the study of the mycelium of Aspergillus ochraceus in a kerosene

biodegradation were also evaluated. The subject extract was used as an enzyme source for investigation of the induction

of NADPH-DCPIP reductase, aminopyrine N-demethylase and kerosene degradation activity, therefore evaluating

biodegradation. [102].

A survey of petroleum-degrading bacteria was carried out in India to evaluate distribution of naturally occurring

petroleum-degrading aerobic bacteria. Some genera, such as Pseudomonas, Mycobacterium, Klebsiella, Acinetobacter,

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Micrococcus, and Nocardia were found to be the most common petroleum degraders. Other heterotrophic bacteria

included several species of Escherichia, Klebsiella, non-oil-degrading Pseudomonas, Vibrio, Streptococcus,

Staphylococcus and Bacillus. Following preliminary selection, five strains, showing best growth in medium with oil

fraction as the sole carbon source, were chosen for estimation of the efficiency of crude oil biodegradation using DCPIP

as one of the bioindicators [103]. The use of this method proved to be reliable enough in experiments to quantify

biodegradation kinetics, taking into account the time taken for decolorization of the blue DCPIP to the colorless form.

The strains, which decolorized the DCPIP in the quickest time, were chosen as the best oil degraders [103], but the

absorbance values in 600 nm could also be used to precisely collect data regarding the biodegradation process through

time.

6. Automotive lubricant oils biodegradation: a case study

6.1 Purpose

Alternative means of determining when reducing conditions exist are provided from redox indicators. Generally, it is

observed that oxidized forms are colored and reduced forms are colorless. Due to this fact, such color change can be

monitored with a spectrophotometer, evaluating changes in redox indicator absorbance value.

The redox half reaction of a redox indicator is described by Equation 1 below. As the oxidized indicator reacts with a

reductant, the absorbance decreases and this way the reducing power of the sample can be estimated.

6.2 Material and methods

The biodegradability of automotive lubricant oil (synthetic, semi-synthetic, mineral and used) was evaluated using the

technique based on the redox indicator DCPIP. Hanson et al. [96] technique has been employed in other studies [74;

103; 104]. However, it has never been used in automotive lubricant oils and vegetable oils biodegradation evaluation

before.

Colorimetric assays were made using Bacillus subtilis CCT 2576 in NA medium cultures conserved at 10 C. The

cells were transferred from storage culture tubes and streaked onto a Petri dish with PCA medium. Then, microbial

culture reactivated after 48 h were inoculated to 50 mL of BH medium at 35°C for biomass growth. No agitation or

darkness conditions were applied. The BH medium contents are, g L-1

: MgSO4, 0.2; CaCl2, 0.02; KH2PO4, 1.0;

K2HPO4, 1.0; NH4NO3, 1.0; FeCl3, 0.05; according to Difco [105].

After 48 h in BH medium, B. subtilis culture was inoculated to tubes along with DCPIP indicator and the oil under

analysis. Tube contents followed Table 1 and the experiment was conducted in triplicate.

Table 1 Colorimetric assays content.

DCPIP BH B. subtilis inoculum Oil

Control 0 400 µL 7.5 mL - -

Control 1 400 µL 7.5 mL 100 µL -

Water 1 400 µL 7.5 mL - 50 µL of Synthetic Oil

Water 2 400 µL 7.5 mL - 50 µL of Semi-Synthetic Oil

Water 3 400 µL 7.5 mL - 50 µL of Mineral Oil

Water 4 400 µL 7.5 mL - 50 µL of Used Oil

Inoculum 1 400 µL 7.5 mL 100 µL 50 µL of Synthetic Oil

Inoculum 2 400 µL 7.5 mL 100 µL 50 µL of Semi-Synthetic Oil

Inoculum 3 400 µL 7.5 mL 100 µL 50 µL of Mineral Oil

Inoculum 4 400 µL 7.5 mL 100 µL 50 µL of Used Oil

Control assays (C0-1) evaluated interactions of the assay components and DCPIP indicator. C0 contained only

DCPIP indicator and BH medium. On the other hand, C1 presented DCPIP indicator, BH medium and B. subtilis

inoculum; and was done to determinate inoculum influences in DCPIP through time. Another series of blank assays,

named “Water” contained DCPIP indicator, BH medium and different types of oils (W1-4). Finally, “Inoculum” assays

were made up of DCPIP indicator, BH medium, B. subitilis inoculum and the different oils (I1-4).

The absorbance of all the assays was measured by Hach® DR 2500 spectrophotometer. Data was collected three

times per day, totalizing 230 h. Then, absorbance values were compared between different assays.

6.3 Results

The colorimetric analysis demonstrated that the biodegradation was successful. It occurred in all the oil containing

tubes, due to the general pattern whereby DCPIP loses of color.

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The control assays (C0 and C1) kept the blue color, whereas the oil containing assays passed through the

biodegradation process (W1-4 and I1-4). However, it was noticeable that the biodegradation time necessary for the blue

color to completely vanish was different between the different types of oils.

In the oil containing assays without B. subtilis inoculum, total biodegradation times for synthetic (W1), semi-

synthetic (W2), mineral (W3) and used (W4) were 210 h, 162 h, 138 h and 89 h, respectively. For the “Inoculum”

assays, the DCPIP color changes were 138 h, 125 h, 75 h and 87 h for synthetic (I1), semi-synthetic (I2), mineral (I3)

and used oils (I4). For example, Fig. 3 is representing the DCPIP decolorization process during the incubation time for

control and synthetic oil assays.

Fig. 3 DCPIP decolorization process through time (3, 137 and 210 hours, respectively) as observed in synthetic lubricant oil

biodegradation. From left to right, the five test tubes are: distilled water; C0; C1; Water1 and Inoculum1.

According to results and Fig.3, B. subtilis inoculum assays demonstrated a faster biodegradation compared to assays

without the bacteria. Even though no biomass was inoculated, biodegradation occurred in “Water” assays probably due

to previous microorganism presence in oils. Moreover, the total biodegradation time observed generally followed an

ascendant order: synthetic oil, semi-synthetic oil, mineral oil and used oil.

Regarding quantitative data, the absorbance readings of the DCPIP indicator are represented in Fig. 4.

In relation to Fig. 4a, a small decrease in DCPIP concentration can be noticed even in control assays. This decrease

indicates an overall pattern of natural disintegration of DCPIP chemical structure through time. Therefore, it cannot be

due to the Bacillus subtilis inoculum, since the DCPIP concentration decreases equally in both C0 and C1. Considering

this, even though the DCPIP concentration tends to naturally decrease, the tubes containing oils reduce the DCPIP

concentration even further because of biodegradation.

The results of Water and Inoculum assays for the different oils are presented in Figs. 4b, 4c, 4d and 4e.

Fig. 4 DCPIP concentration quantified at 600 nm absorbance (a) in C0 and C1 assays - without oils; and in oil containing assays

without (Water) and with (Inoculum) Bacillus subtilis (b) synthetic - 1; (c) semi-synthetic -2; (d) mineral - 3 and (e) used - 4.

_______________________________________________________________________________________

At the outset, the complete loss of color happened in the last measurement point plotted. So, the DCPIP was

considered reduced as a result of the biodegradation when the absorbance value led to a DCPIP concentration below

0.030 g L-1

. Values below this concentration could not be measured by this spectrophotometric method.

Another consideration regarding oils containing assays (Figs. 4b, 4c, 4d and 4e) is that all the decreasing curves

approach a linear model, except Fig. 4b and Fig. 4c, which are better fit to an exponential decay model.

As a pattern observed in the assays, DCPIP quantification demonstrated a tendency through time (Fig. 4a) but it was

not as significant as the DCPIP concentration reduction attributable to oil biodegradation by B. subtilis. These assays

presented a DCPIP absorbance decrease, which indicates a positive interaction of the bacteria in oil degradation, since

all “Inoculum” assays yielded a faster biodegradation. Also, “Water” assays presented a DCPIP concentration reduction

as they already previously contained microorganisms in oils.

6.4 Discussion

As a general pattern, used oils tend to be more easily biodegraded, biodegrading faster than the non-used retail available

samples. On the other hand, the synthetic and semi-synthetic automotive lubricant oils took the longest time (125 and

138 h, respectively) to completely change the color of DCPIP from blue to transparent, even with B. subtilis inoculum.

The mineral oil had an intermediate biodegradation value between the automotive lubricant oils.

Concerning the biodegradation of automotive lubricant oils, the process had been previously reported using different

methodologies, such as respirometric studies. In Lopes and Bidoia [36], the synthetic lubricant oil presented a better

biodegradation performance than the mineral and semi-synthetic lubricant oils.

Eisentraeger et al. [25] observed that used oils were more biodegraded than new ones, and proposed that this is due to

changes in the chemical structure of the hydrocarbons, facilitating the interactions between the oil and the

microorganisms. Also, the use of lubricant oil in engines, with high pressure and temperature, break the long

hydrocarbon chains, thus supporting microbial activity.

In conclusion, this rapid and simple colorimetric methodology applied to oils biodegradation promotes some

discussion leading to a better handling of contaminated water medium by oils and their respective biodegrading profile.

Thus, it promotes the development of new techniques in water treatment. From this data, the automotive lubricant oils

biodegradation occurs because hydrocarbons and organic polymers are used as metabolic substrates by microorganisms.

Also, some lubricant oil compounds such as cyclic alkanes are not biodegraded by only one microorganism. Hence, a

microorganism consortium allied to a known potentially oil metabolizing microorganism such as B. subtilis will also

perform an important role in degrading these compounds [106]. Therefore, a Bacillus subtilis inoculum yields a better

oil biodegradation.

7. Final considerations

Microorganisms represent an extremely reliable way to biodegrade petrol derived substances. This ability is highly

exploitable during bioremediation processes to ultimately free the environment from subsequent pollutants derived from

these hydrocarbons. The development of efficient techniques in order to obtain biodegradation data is a fundamental

tool when proposing different strategies for bioremediation of polluted areas. DCPIP redox indicator colorimetric

technique proved itself to be a rapid, easy and low cost method when detecting microbial metabolism from carbon and

other nutrients sources, including hydrocarbons. To ascertain microbial ability to utilize hydrocarbon substrates by

simply observing the color change of DCPIP, in which the quickest decolorization time represents the best oil

degradation, is a major breakthrough in biodegradation studies. Further studies in the applicability of this method and

other different redox indicators are recommended in order to reach a wide and precise selection of mediators capable of

detecting the metabolic perturbations caused by many types of substrates, even though DCPIP usage has already been

successfully reported for a wide range of situations.

Acknowledgements The authors thank FUNDUNESP; FAPESP; CAPES and CNPq for support.

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