Abstract

Oily wastewater is insufficient of nitrogen sources to produce free fatty acid (FFA) for
biodiesel production. To enhance the nitrogen sources for lipase production, the best alternative
way is to produce nitrogen-based fertilizers. This fertilizers will support the process to increase
concentration of nitrogen to convert FFA into biodiesel. The processes in the production of
fertilizers from food waste in this study include fermentation, solid waste fermentation and
composting. Food waste is chose as feedstock as it is rich in nitrogen sources. 15,000 tonnes of
leftover food thrown away each day, including 3,000 tonnes of food that is still good to consume.
The aims of this study are to produce organic fertilizer from nitrogen-composed food wastes, as
well as, supporting biodiesel production using Free Fatty Acid (FFA) from oily wastewater that is
converted by Pseudomonas aeruginosa NR.22. Dirty wastewater is converted into useful clean
water source. Biodiesel basically is the mono-alkyl esters from vegetable oils and animal fat which
can be used as alternative way to replace conventional petroleum diesel fuel which is petrodiesel.

Introduction

Generally, fertilizers are organic or inorganic, natural or synthetic substances that can give
a lot of benefits to plant since it can be used to enhance the plant growth and production. Fertilizers
are also used to enhance the soil’s growing potential because it is able to provide a better growing
condition for plants as compared to natural soil. Plus, fertilizers can provide large amount of
macronutrients such as nitrogen, phosphorous and potassium while natural soil may not contain
sufficient amount of this macronutrients. Fertilizers also provide trace elements such as
magnesium, calcium and copper that are crucial in plant growth (Tan, 2015).

So, this study has shown an alternative way to produce an organic fertilizers from biomass
such as food waste that have been collected. Food waste is food material that is unused, discarded
and not safe to be consumed by human for avoidable reasons at production line (Tan, 2015). Food
wastes consists of lignocellulosic polymers and non-lignocellulosic polymers. All lignocellulosic
as well as non-lignocellulosic materials can be used for energy, food, feed, fertilizer, fuel and
commodity product formation. Figure 1 below shows the biomass raw materials (by Horst W.
Doelle).

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 Figure 1: The biomass raw materials (by Horst W. Doelle).

Rotten food can breakdown to produce methane gas and is harmful to human if not handle
properly. Food waste can cause the emissions of greenhouse gas that possibly increase the
surrounding temperature during its production and disposal. Therefore, we need to convert the
food waste into useful products such as fertilizer and biodiesel. Food wastes can be decomposed
in anaerobic digestion by microorganisms to break down food waste into smaller materials. This
process is carried out inside an enclosed system in the absence of oxygen. Methane gas produced
can be collected and converted into biogas to transport fuels and produce electricity and heat (Tan,
2015).

Hence, we are working on the treatment of oily wastewater from the food waste through
microbial approach by using the strain Pseudomonas aeruginosa NR.22 to convert the oil in the
wastewater into useful free fatty acids with the help of lipase and transformed the dirty wastewater
into useful clean water source (Nik Him & Ahmad Robert, 2017). Initially, composition of nitrogen
is low in the oily wastewater which can cause low production of FFA. Hence, in order to increase
the production of FFA, additional Nitrogen sources needed to react with NR.22 to produce high
production of lipase so that FFA production can be enhanced. Therefore, Nitrogen fertilizer need
to be produced by using the method of fermentation, solid state fermentation and composting after
undergo the pretreatment of food waste (Tan, 2015). As the production of FFA, higher, the
production of biodiesel would be also higher.

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 Biodiesel is known as a fuel composed of monoalkyl ester of long-chain fatty acids derived
from renewable vegetables or animals fats. Vegetables oils and animal fats are made up from
triacyglycerols which there is a long chain of fatty acids bounded chemically. As mentioned earlier,
biodiesel undergo a process named, transesterification reaction in which triacyglycerols is reacts
with a short- chain of monohydrid alcohol by the help of catalyst to form fatty acid alkyl esters at
suitable temperature (Tan, 2015).

Methods

Figure below shows the overview methods used to produce biodiesel from food waste
which supported by nitrogen-rich fertilizer (Nik Him & Ahmad Robert, 2017) (Tan, 2015).

Figure 2 : Overview methods used to produce biodiesel from food waste

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 Food waste is first pretreated which aims to separate the components contain in it, reduce
their sizes and crystallinity, as well as making the oily wastewater and nitrogen-based components
accessible, besides removing the unwanted components before undergo the next process. The
physical pretreatment method is used before the extraction of the oily wastewater and nitrogen-
based compound. The treatment of oily waste water is by using Ps.NR.22 which isolated from a
lake in Selangor. It is a Gram negative-rod shaped bacteria and has been confirmed to carry a 16S
rRNA gene, was isolated to be producing high lipase enzyme (23.44 U/mL) using tributyrin agar.
The strain was maintained at -20℃ in Pseudomonas glycerol agar. Figure 3 shows the Gram-
negative rod species (Nik Him & Ahmad Robert, 2017).

Figure 3: Gram negative rod species (Nik Him & Ahmad Robert, 2017)

Next, 10mL sample of the oily wastewater is added to 100mL of nutrient broth containing
2% (v/v) of olive oil, 0.2% (v/v) potassium dihydrogen phosphate (KH2PO4) and 0.4% v/v
ammonium chloride (NH4Cl) in 250 mL Erlenmeyer flask. The shaking rate was chosen at 150rpm,
35℃, and 24 hours. The 24 hours inoculum preparation was set up using nutrient broth (NB) at
pH = 6.5, shaking rate at 150rpm and 35℃. Figure 4 shows the growth in Erlenmeyer flask (Nik
Him & Ahmad Robert, 2017).

Figure 4: Growth in Erlenmeyer flask

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 The next step is the optical density of the culture broth at 540 nm was measured using
spectrophotometer and about 2.0 mL of the sample from the culture was withdrawn from the flask
and centrifuge at 10,000 rpm for 10 minutes for every 3hours of sampling. After being centrifuged,
the remaining cell then is put into a desiccator at room temperature for overnight. After 24 hours,
the dry cell was weighted. The difference between the final weight and pre-weight were used to
estimate Cell Dry Weight (CDW) (Nik Him & Ahmad Robert, 2017).

The bacteria’s lipase activity was defined as the amount of enzyme required to release one
micromole of fatty acid per minute under the test condition (Nik Him & Ahmad Robert, 2017).

Next, the oily wastewater was sterilized using autoclave at 121℃ for 30 minutes under
15psi of pressure. This was done to prevent growth and other potential intervening biological
agents (Nik Him & Ahmad Robert, 2017).

After that, oil concentration measurement was conducted using the standard method of
gravimetric. The Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography
analysis (GC) was used to detect the functional group that contain in the oily wastewater (Nik Him
& Ahmad Robert, 2017).

As the result from FTIR, we will know that the concentration of nitrogen in wastewater is
low and not enough for lipase production. Therefore, with the help of nitrogen fertilizers produced
from the food waste, this will increase the nitrogen sources for the bacteria to produce lipase which
enable hydrolysis process to take place. Hydrolysis of oil content in wastewater has produces free
fatty acids (FFA), mono- and diglycerides. The hydrolysis has produced six types of unsaturated
FFA namely lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), linoleic acid
(C18:2), oleic acid (C18:1) and caprylic acid (C8:0) (Nik Him & Ahmad Robert, 2017).

Processes in production of organic fertilizers

After undergo the process of pretreatment, the component of Nitrogen, N2 has been
separated from the food waste collected. The processes such as fermentation, solid state
fermentation and composting are involved in the production of organic fertilizers. Fermentation,
solid state fermentation and composting are alternative approaches to manage biological materials.
These processes will definitely convert food wastes of vegetable and fruit into useful materials
such as organic fertilizers for farming (Tan, 2015).

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 Organic fertilizer from organic material of plant and animal under decomposition process
can be in solid or liquid form. In this study, we are producing fertilizers in the form of liquid. This
is because liquid form fertilizer is easier to handle when blending and applying . Next it provides
uniform application and quick acting and sometimes provide an immediate effect. Plus, it also can
be used for both starter and in-season application (Tan, 2015).

Fermentation
Fermentation is an anaerobic process that converts sugar in food waste into acids, alcohol
or gases. During fermentation process, microorganism metabolizes nutrients in food waste and
produce carbon dioxide, methane and acids. Microorganism such as fungi, bacteria and yeast
digest organic compounds in food waste and produce organic acids such as lactic acid, acetic acid
and butyric acid. Microorganism uses simple compounds of food source such as sugar, protein and
starch in fermentation (Tan, 2015).
The compounds with relatively high C: N ratio and high water content are suitable for
fermentation by microorganism. A small amount of water is liberated by microorganism and
fermenting materials during fermentation. The suitable temperature for fermentation is 25°C to
37°C and optimum pH is around 4.0 to 5.5. High temperature can kill the microorganism that used
in fermentation because most of the microorganism is mesophilic which grow at 20°C to 45°C.
Too acidic or alkaline condition is not suitable because food waste is pasteurized and nutrients are
preserved for long time when the pH drops below 4.2 (Tan, 2015).

Solid State Fermentation

Solid state fermentation is fermentation process that involves microorganism that grows on solid
material in the absence of liquid . This process involves inoculation and growth of microorganism
on porous particulate solid substrate that contains low moisture content. The nutrients and water
content that present in solid substrate support the growth of microorganism and cause the
microorganism to produce useful enzyme when grows on solid substrate. The production of solid
state fermentation and the growth of fungal are influenced by temperature. Fungus can grow within
a wide range of 20°C to 55°C. The suitable temperature for the production of organic fertilizers

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from food waste was 25.9°C and fermentation days were 36.7 days. The microorganism that used
in solid state fermentation includes yeast, fungi, bacteria and algae (Tan, 2015).

Composting
Composting is natural process of recycling and decomposing of organic material into humus rich
soil amendment called compost. Microorganism such as bacteria uses nutrients and energy in
organic material and convert hydrocarbons into carbon dioxide and water by oxidation.
Microorganism in compost starts to reproduce and continue decomposition of food waste. Carbon,
hydrogen and oxygen that originally found in solid material are converting into gaseous forms and
release to atmosphere. Higher temperature can enhance microbial growth rate, degradation rate
and enzymatic production in food waste. High degradation rate can release more energy, increase
the temperature of composting and enhance the process of composting. The suitable temperature
for composting food waste is around 50°C to 62°C. The pH for good composting was nearly neutral
in pH is 6.0 to 7.0 (Tan, 2015).

Results and Discussion

Heavily oiled wastewater has been mixed with different nitrogen sources into Erlenmeyer
flask. The effect of the mixture has been shown using 9.1g/L peptone, yeast extract, ammonium
sulphate, ammonium phosphate, urea, ammonium nitrate, ammonium carbonate, nitrogen
compose,nitrogen soil and nitrogen fertilizer. Figure 5 shows the percentage of oil removal and
lipase activity correspond to different nitrogen sources. The graph has proved that the best oil
removal can be achieved by addition of sterile nitrogen compose with rate of oil removal of 82.1%
and lipase activity value is at 18.8 U/mL. Sterile nitrogen composed was chosen due to its cheaper
price compared to other sources. In order to run biochemistry processes, Ps.NR.22 required
additional nitrogen sources to produce enzymes including lipase (Nik Him & Ahmad Robert,
2017).

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Figure 5: Graph of percentage of oil removal and lipase activity correspond to different nitrogen
sources (Nik Him & Ahmad Robert, 2017)

At the optimum conditions (nitrogen compose, 45°C, 200rpm and 11%, v/v cells), the
experiment had been carried out with 92 hours running system before hydrolysis was stopped
which mark a declination of both oil removal and lipase activity. Based on the figure 6A below,
optimum percentage of oil removal (92.8%) is at 36th hours with lipase activity of 29.8 U/mL.
Meanwhile, figure 6B shows comparison between oily wastewater before and after 92 hours
treatment. After treatment, a clear wastewater has been obtained and hydrolysis of the oil content
in the wastewater has produced free fatty acids (FFA), mono- and diglycerides (Nik Him & Ahmad
Robert, 2017).

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Figure 6A shows optimum percentage of oil removal with lipase activity correspond to their time
for treatment while Figure 6B shows comparison between oily wastewater before and after 92
hours treatment (Nik Him & Ahmad Robert, 2017).

Conclusion

In summary, Pseudomonas aeruginosa NR.22 is the best microbes to treat oily wastewater
safely. At the same time, FFA could be extracted from oily wastewater which later can be used in
biodiesel production. Process of fermentation, solid state fermentation and composting has been
applied to produce fertilizer as additional nitrogen sources. The fertilizer created then support the
production of FFA through lipase-enzymatic hydrolysis.

Reference
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Azhdarpoor, A., Mortazavi, B., & Moussavi, G. (2014). Oily wastewaters treatment using
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J. Jara-Samaniego, M. D.-M.-E.-T.-E. (2017). Development of organic fertilizers from food

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Lizardi-Jime´ne, M. A., & Herna´ndez-Martı´nez, R. (2017). Solid state fermentation (SSF):
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Pseudomonas Aeruginosa NR.22 Producing Usable Free Fatty Acids (FFA). International Journal
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