**2. Materials and methods**

#### **2.1 The choice of sunflower oil is explained based on the following criteria**


The chemical composition of sunflower oil is shown in **Table 1**.


**Table 1.**

*Fatty acids that make up sunflower oil and specific stereo analysis [18]. Results in % moles.*

#### **2.2 Physicochemical parameters and analytical methods**

#### *2.2.1 Chemical oxygen demand (COD)*

The potassium dichromate method was used to evaluate COD levels. The method used is a variation of the standard method [28]; however, it maintains its basis. The variation used has the advantage it uses a significantly smaller sample and reagents. The sample is chemically oxidized through the action of potassium dichromate at 150°C for 2 h. Silver sulphate is used as a catalyst and mercury sulphate is used to avoid possible interferences with chloride. Afterward, determination by spectrophotometry at 600 nm is performed. Equipment and instruments are used to determine the various parameters to characterize the wastewater used.

#### *2.2.2 Fats and oils*

Determination of the fats and oils was used in Gravimetric Assay Soxhlet method. This method quantifies substances with similar characteristics, based on its common solubility in appropriate solvent, 213E method [29].

Total suspended solids (TSS) are determined by filtering a known volume of the sample on Whatman (Whatman plc., Maidstone, UK) 4.7 cm GF/C glass fibre filters and then drying it at 103–105°C. The difference in weight of the filter before and after filtration is used to estimate the TSS, 209C method [29].

Volatile suspended solids (VSS): The volatile suspended solids are determined by weight loss after calcination at 550°C, 208E method [29].

#### **2.3 Continuous equipment**

In the current investigation, an activated sludge plant at the laboratory scale is used to conduct biodegradability tests in wastewater with oils and fats. To meet these objectives, experimental work is required, with such parameter information describing the process dynamics regarding the aeration and sedimentation tanks regarding fat and oil content of the wastewater. For this purpose, BIOCONTROL-MARK 2 equipment was used. The details of the equipment are shown in **Figure 1**.

This experimental equipment consists essentially of the following parts:

• Control unit

Composed of the main switch, air cylinder, it is complemented with a flow meter and a flow regulation system. Additionally, the wastewater feed pump, that includes a flow rate regulation system, a timer for intermittent operations, and an ON–OFF switch allowing sludge recycling from the sedimentation tank to the aeration tank.

*Differential Impact of the Prior Mix by Stirring in the Biodegradation of Sunflower Oil DOI: http://dx.doi.org/10.5772/intechopen.100480*

**Figure 1.** *Experimental equipment diagram.*

• Aeration tank

It consists of a transparent Plexiglas® (Vittadini Riferimenti, Milan, Italy) cylinder with a height of 38 cm and a diameter of 20 cm, which has outlets at various heights associated with different volumes (7, 8, 9, and 10 l). There are two separated inlets allowing recirculation of sludge from the top. The influent to be treated is placed at the bottom. In addition, the system has two ceramic diffusers placed in the bottom in a way that they can disperse the air in tiny bubbles [30].

• Sedimentation tank

This consists of a transparent Plexiglas® (Vittadini Riferimenti, Milan, Italy) cylinder, where its lower part is cone-shaped to make sludge sedimentation and thickening easier.

The mixed liquor is fed from the aeration tank, which has an outlet in its upper part. This flow escapes by overflow, when it arrives in the sedimentation tank. The solid phase decantation gives a method to downward flow. Decanted sludge is separated and recirculated at the bottom through the pump towards the aeration tank. Treated water also uses the overflow mechanism to be evacuated to the storage tank.

#### **2.4 Experimental methodology**

a. Feed preparation

The treatment system was initially fed with synthetic wastewater prepared in the laboratory, according to strong urban wastewater typical characteristics of [31]. This wastewater has an approximate BOD of 400 mg/l, with the corresponding

proportions of nitrogen and phosphorus in a relation to BOD: N: P = 100:5:1. Approximately 400 mg of saccharose, 20 mg of phosphate hydrogen of potassium, and 100 mg of ammonium chloride were added per litre of water. Measurements begin when sunflower oil is added, the concentration of this substrate gradually increases. Feed was prepared daily and nitrogen and phosphorus increased according to the organic input coming from fats and oils.

#### b. Operating modes

The synthetic wastewater was poured into a storage pond of approximately 50 l, where the stirring unit has been installed to disperse oil or fat. Through a peristaltic pump, controlled by the control unit, it drives the feed to the aeration tank. Oxygen feed and recirculation flow are controlled by the control unit. Process effluent is collected in a 30-l volume tank, where the samples are taken to be processed. The flow of synthetic wastewater is 25 l/day.

#### **2.5 Mass balance: biodegradability determination**

Experimental method is established in this protocol, which allows to carry out the material balance of fats and oils. From the process diagram, **Figure 2** shows flows and concentrations of inlet and outlet streams in different stages of the activated sludge process are indicated. To carry out material balance and estimate biodegraded oil in the activated sludge process, the oil in the feed tank must be estimated.

This experience works with influents that are biologically treated by active sludge, containing only vegetable oil. This retained fraction is an indicator of the mixing level reached in the feed tank, which corresponds to not emulsified oil, and therefore, it is not part of the influent entering the aeration tank; obviously, the size of this fraction indicates the mixing level of the system quantitatively.

Where:

F0, C0: Flow and concentration oil entering the mixing tank.

F1, C1: Flow and feed oil concentration to the aeration tank.

F2, C2: Flow and concentration of fats and oil leaving the aeration tank = (Flow and concentration of fats and oils entering the secondary settler).

**Figure 2.** *Active sludge plant process diagram.*

*Differential Impact of the Prior Mix by Stirring in the Biodegradation of Sunflower Oil DOI: http://dx.doi.org/10.5772/intechopen.100480*


**Table 2.**

*Mass balance in active sludge for oily influent with prior stirring.*

F3, C3: Flow and concentration of fats and oil from purified effluent.

F4, C4: Flow and fats and oil concentration in recirculation flow.

M1: Oil mass contained in the mixing liquor of the aeration tank.

M2: Oil mass floating in the upper part of sedimentation tank and oil mass at the bottom of sedimentation tank by biomass attached.

*Mass balance and biodegradability determination of fats and oils in bench-scale activated sludge reactor.*

In this experiment made in equipment in **Figure 1**, water and oil were mechanically stirred and mixed in the feed tank. Part of aggregated oil was accumulated in the feed tank, so the oil fraction not going to the aeration tank was known. The influent containing sunflower oil and concentration was gradually increased.

Biodegraded sunflower oil mass was determined from the mass balance equation.

Sunflower oil is part of the influent fed to the treatment system.

Initially, it is assumed the system has no oil.

Results of matter balance are shown in **Table 2**.

From material balance, the mass of biodegraded sunflower oil is obtained, which is part of the influent fed to the system.

For the aeration tank:

$$\frac{\Delta M\_1}{\Delta t} = F\_1 \cdot C\_1 - F\_2 \cdot C\_2 - r\_A \cdot V + F\_4 \cdot C\_4 \tag{1}$$

For the secondary sedimentation tank:

$$\frac{\Delta\mathbf{M}\_2}{\Delta t} = F\_2 \cdot \mathbf{C}\_2 - F\_3 \cdot \mathbf{C}\_3 - F\_4 \cdot \mathbf{C}\_4 \tag{2}$$

It must be noticed that, F6 = 0.

Where *M1* corresponds to the oils and fats in the aerator tank.

M2: Corresponds to oils and fats in the sedimentation tank.

V: Is the volume reactor.

*rA*; Is the vegetable oil biodegradation rate.

The balance for the system as a whole is as follows:

From this expression we have: *rA* � *V*, corresponds to vegetable oil that disappears per unit of time, it means the oil is clearly biodegraded by microorganisms, such that:

$$r\_A \cdot V = F\_1 \cdot C\_1 - F\_3 \cdot C\_3 - \frac{\Delta M\_1}{\Delta t} - \frac{\Delta M\_2}{\Delta t} \tag{3}$$

An important part of the oil floats and therefore does not biodegrade and passes to a secondary sedimentation pond in which it accumulates. For reasons of technical feasibility, the mass balance must be kept integral for some time. Fats and oils accumulated as a result of separation by flotation are measured, allowing to determine the oil biodegradation level in influent.

The equation for integral material balance for a certain period of time is as follows:

$$\mathbf{M}\_1 + \mathbf{M}\_2 = \mathbf{F}\_1 \cdot \mathbf{C}\_1 \cdot \Delta\_t - \mathbf{F}\_3 \cdot \mathbf{C}\_3 \cdot \Delta\_t - r\_A \cdot \mathbf{V} \cdot \Delta\_t \tag{4}$$

Where is *Δt* the time elapsed.

Every day is identified by subscript as shown in the following examples:

Day 1:

$$M\_{11} + M\_{21} = F\_{11} \cdot C\_{11} \cdot \Delta\_t - F\_{31} \cdot C\_{31} \cdot \Delta\_t - r\_A \cdot V \cdot \Delta\_t \tag{5}$$

Day 2:

$$M\_{1i} + M\_{2i} = F\_{1i} \cdot C\_{1i} \cdot \Delta\_t - F\_{3i} \cdot C\_{3i} \cdot \Delta\_t - r\_A \cdot V \cdot \Delta\_t \tag{6}$$

Then, the mass balance for a given number of days of operation is as follows:

$$r\_{\mathbf{A}} \cdot \mathbf{V} \cdot \Delta\_{\mathbf{t}} = F\_{\mathbf{i}\mathbf{i}} \cdot \Delta\_{\mathbf{t}} \cdot \sum (C\_{\mathbf{i}\mathbf{i}}) - F\_{\mathbf{j}\mathbf{i}} \cdot \Delta\_{\mathbf{t}} \cdot \sum (C\_{\mathbf{j}\mathbf{i}}) - \sum (M\_{\mathbf{i}\mathbf{i}} + M\_{\mathbf{i}\mathbf{i}}) \tag{7}$$

Where:

$$M\_1 = \sum M\_{1i} \\ M\_2 = \sum M\_{2i} \tag{8}$$

Then, biodegradability is

$$B = r\_A \cdot V \cdot \frac{\Delta\_t}{F\_{1i}} \cdot \Delta\_t \cdot \sum (\mathbf{C}\_{1i}) \tag{9}$$

From the material balance calculation, the percentage of biodegraded oil of the influent is estimated. The flows and concentrations of the activated sludge process are indicated below.

The feed flow and the concentration of fats and oils (F0, C0) is known, and is set according to the conditions previously defined for the experimental phase.

F1, C1: The flow is determined according to the experimental conditions and regarding the concentration of fats and oils C1, and it is determined by the Soxhlet gravimetric method.

F3, C3: This flow is equal to inflow and, therefore, is given by predetermined conditions of the experiment, and the concentration of fats and oils, C3, is obtained by the Soxhlet method.

M1: This estimates the oil mass accumulated in a given period of time, and it is necessary to remove supernatant fats and oils, dry and weigh them.

M2: The mass of sunflower oil and derivatives accumulated in the secondary sedimentation tank in a determined period of time is determined. The upper or floating phase of the secondary settler is poured into an auxiliary tank, which is oil and its derivatives in humid conditions. Then, the remaining water is evaporated through a heat source and oil mass is obtained by gravimetric. Oil and derivatives accumulated in biomass in secondary settler are added to this mass, which is determined by the Soxhlet method for a 100-ml sample. With the concentration obtained, the oil retained in settled solids is estimated.

*Differential Impact of the Prior Mix by Stirring in the Biodegradation of Sunflower Oil DOI: http://dx.doi.org/10.5772/intechopen.100480*

### **2.6 Incidence of agitation and biodegradation of sunflowers oil**

In this experience, an influent that only has sunflower oil has been fed to the biological treatment system by active sludge, it is the only carbon source available for microorganisms under two sceneries, with and without previous agitation. Different operational parameters are monitored providing information on system behaviour. The oil biodegradation percentage is estimated by mass balance.

## **3. Material balance results: without agitation**

Biodegradability is calculated from the mass balance in the system and concentrations and quantities of oil obtained. In this case, feeding is not subjected to a previous agitation, and this condition is achieved by introducing oil directly to the aeration tank by dripping, and the total volume entered into the system; it is measured every 24 h.

In the present experiment (**Table 3**), influent fed to active sludge system contains only oil and without stirring, and **Table 3** found that oil biodegradation levels range between 28.1 and 42.5%, which is considerable in spite of not being high. This is an interesting result because it opens the viability of biologically degrading substances, such as fats and oils, which evidently presents important comparative advantages when compared with the removal of fats or oils *via* flotation.

The above is explained by the lack of stirring, and it is worth noting that experience with olive oil production wastewater, a culture medium, with a concentration of 5% was used. Effects of agitation speeds were studied on the growth of species Scenedesmus microalgae, in a photobioreactor. A maximum specific growth rate of 0.031 1/h was obtained, using a speed of rotary impeller around 350 rpm, higher than that found in the absence of agitation 0.024 h<sup>1</sup> [32].

On the other hand, the synthesis of bio-lubricant from the effluent of palm oil plant, which is based on enzymatic hydrolysis. Effects of essential parameters were examined, which include stirring speed. The optimal hydrolysis rate (0.1639 mg/s.l) is achieved at 650 rpm, at 40°C, pH 7.0, 20 U ml of loading enzyme [33].

This biodegradation range achieved in this experience, although it is not an optimal result, it shows that oils and fats can be eliminated from influent through biodegradation process; it is also important to note this level of biodegradation can be increased with some modification processes.

The results for the oil removed by biodegradation are due to the fact that a considerable percentage of sunflower oil that floats in the aeration tank reaches the secondary settler, which is due to the very low solubility of substrate in water, which originates a two-phase system. This is increased by the oil tendency to float in water, which is favoured by the flow of air driven from the bottom of the aerobic tank.


**Table 3.** *Matter balance in active sludge for oily influent without previous stirring.*

**Figure 3.** *Oil and grease accumulated in settling tank.*

An increase in emulsification constitutes the vehicle that would generate more favourable conditions for the microbiological attack, since the hydrolysis of oil requires an oil-water interfacial area, which is favoured with the increase in the number of emulsions and with the decrease in their size. One of the causes of the results obtained with respect to biodegradability is the type of hydrodynamics of process reactor, since given the lower oil density in relation to the water and the injection of air from the bottom of the aeration tank, it generates conditions for that the oil floats and, therefore, does not remain in the treatment system during appropriate residence time. There is a part of the oil that is not biodegraded and is separated through flotation (**Figure 3**).

Among the already mentioned characteristics of oils and fats is their low solubility in water and their marked tendency to buoyancy, which is enhanced when the aeration tank is fed with air; as the oil floats, the coalescence process is favoured, reversing the emulsification achieved through aeration by the agitation caused a consequence of air injection into the aerobic pond.

It is noteworthy that the system worked for 50 days and the only carbon source was vegetable oil; with regard to toxicity, some authors [34] claim an inherent toxicity of fatty acids, since they are potential inhibitors of metabolic processes of cells due to their surfactant properties, and for this reason, it is considered they do not remain as such in cells; they are immediately converted into thioesters of their coenzyme A.

As already stated, the low solubility of fats and oils makes it difficult to disperse and distribute this substrate properly in the medium that supports microorganisms in water. In view of the aforesaid, the adequate size of the emulsions is not achieved, so contact surface with the mixing liquor, and therefore with bacterial flocs is limited by this operational factor.

Fats and oils are substances that do not favour the development and growth of bacterial colonies, due to the already explained, but when biomass is acclimatized for a considerable time to the new type of influent, it is found the biomass is adapted.

This is explained from the mutations caused by changes or chemical or physical agents, which change the DNA imparting new characteristics to the cell, thereby

*Differential Impact of the Prior Mix by Stirring in the Biodegradation of Sunflower Oil DOI: http://dx.doi.org/10.5772/intechopen.100480*

**Figure 4.** *Acclimatization of bacteria to oil particles.*

allowing the cell to degrade a xenobiotic substance or facilitate biodegradation. Spontaneous mutations occur in one out of every 1,000,000 cells; however, the DNA molecule is capable of replicating [35]. **Figure 4** shows the environmental condition of the biomass in an oily medium.

The existence of an oily environment for the fat and oil-degrading biomass is an important factor and has to do with the above-mentioned. In kinetic monitoring experiences of the aerobic biodegradable processes of dairy fats and oils, we worked with native biomass from a lagoon that treated dairy water and commercial bioaugmentation inoculum, and the removal percentage was from 78 to 91% and from 82 to 95%, respectively. However, for high substrate concentrations, the native inoculum is more mineralizing than the commercial one [36].

• Oil concentration ratio in influent and effluent

The oil concentration in the effluent is not correlated with oil concentration in influent (**Figure 5**), which is mainly explained by the fact that an important part of the oil is retained in the sedimentation tank, instead of leaving through the effluent, given its buoyancy. There is no evidence that behaviour regarding the elimination of fats and oils in urban active sludge treatment plants is related to the concentration of fats and oils in influents in concentration ranges, which are the discharged influents usually present [37].

As the important part of oil floats is that it does not enter the interior mixed liquor of the active sludge system, this breaks the relationship that should exist between the concentrations of oil at the outlet and in the inlet, unlike other soluble substrates such as saccharose.

Stirring is the driving force that promotes the oil emulsification in the water, which depends on the flow and air pressure in the active sludge system. The emulsified oil will be the most susceptible to biodegrade; it must be considered that the airflow also favours flotation, so both effects are opposed.

**Figure 5.**

*Performance of oil concentration in influent, effluent, and effluent the most accumulated effluent, without previous stirring.*

**Figure 6.**

Biomass constitutes an element of resistance from a hydrodynamic point of view to the flotation of the oil since these are particles that impede the flow of the air and on the other hand are a source of oil consumption, since they use it as a carbon source. The floating of the oil in the aeration tank is reduced according to the biomass concentration since it opposes resistance to the airflow.

**Figure 6** shows that in this experience, removal and biodegradation efficiency do not depend on the mass load, and this indicates the blown air enhances flotation more than emulsification, given the initial system, condition, influencing without previous stirring.

The biodegradation efficiency is low, less than 40%, unlike the elimination that exceeds the average of 85%, which indicates that the flotation phenomenon

prevails, it is worth noting that despite this pessimistic condition of mixture and size of emulsions, biodegradation level over 30% is also achieved.
