Reviewed Thesis Slops Hydrogel Fertilzier 1.2

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Utilization of Distillery Slops and Gelatin to Produce Slow-Release Gelatin-based

Hydrogel Fertilizer

A Thesis Paper

Presented to the Faculty of the

College of Engineering and Technology

University of St. La Salle

Bacolod City

In Partial Fulfillment of the Requirements

For the Degree of Bachelor of Science

Major in Materials Engineering

Hyacinth C. Gamao

Andrea Marie R. Nardo

Danilo J. Parcon

Engr. Felix Querubin,

Co-Researcher
INTRODUCTION

Background of the Study

Food production has been a challenge to the Philippine Islands owing to both
geographical and meteorological factors. Every Filipino is affected by the economic
inconsistency of farming as a livelihood. The agricultural industry supplies food for
domestic animals as well as meeting people's nutritional needs. Agriculture is one of the
people's principal sources of income in the Philippines, and farmers must maintain high
crop quality in order to provide high-quality fruits and vegetables. Agriculture also
creates jobs opportunities in the country, promotes economic development, and ensures
food security.

Food security is tied to national security since if a nation’s objective is to address


the issues like poverty and global hunger it would very much likely make a nation more
stable to function as a whole. Food is a powerful political commodity, it can be a strategic
weapon of war and a smart investment to foster peace of one’s nation (Hamre & Flowers,
2016).

Food in agriculture is not only dependent on livestock but also in plants. In order
to promote plant growth and crop sustainability by increasing its yield, the most common
way is providing a fertilizer. A fertilizer must carry a sufficient amount of nitrogen,
phosphorus, and potassium (NPK). The amount of NPK varies on the farmer’s ratio or
what type of fertilizer is being used. Nitrogen promotes plant leaves, phosphorus is
responsible for the development of roots, flowers, and fruit, and potassium helps the
overall proper functions of the plant. There are different sources of fertilizers which the
nutrients and the NPK value can vary, fertilizers are made from other organic materials
like plants and animal manures. Food industries can also produce wastes that can be
turned into fertilizer products using anaerobic digestion which breaking down
biodegradable material using bacteria without oxygen (Meyer & Marrs, 2017).

Since the start of the pandemic, there has been an increase on alcohol production
in the Philippines, alcohol distilleries are a good source of liquid fertilizers and since they
produce wastes like slops or stillages from derived from molasses and proven to be an
effective fertilizer since it is a rich source of plant nutrients including potassium,
magnesium, sulfur, and chloride. The distillery slops are also being used by farmers for
quite a while, it contains different types bacteria that is beneficial to the plant, and it is
commonly used as a liquid fertilizer once its acidity is reduced or neutralized.

Liquid fertilizers’ efficacy varies on the properties of the soil it is in and the soil
changes due to climate change. Climate change remains a threat to agriculture, especially
in the Philippines. With the effects of climate change, population growth, and
urbanization, the dangers of economic and food insecurity are projected to increase. The
vulnerability of the Philippines to climate change and agricultural shocks is caused by El
Niño–Southern Oscillation (ENSO), especially because of its high vulnerability to
climate shocks (Sutton et al., 2019). It has been estimated that low levels of and
uncertainty in rainfall limit agricultural production in about three-quarters of the world's
cultivated lands. Crops depend not only on precipitation but also on the ability of the soil
to absorb and store water. Inappropriate agricultural practices like the burning of crop
residues, excessive tillage, eliminating hedges, etc. may destroy soil structure, reduce
organic matter levels, eliminate beneficial soil fauna, and do not favor water infiltration
leading to their negative effects to the natural main causes of soil moisture scarcity, and
then add water stress in crops (Bot & Benites, 2005).

Farmers need to increase their irrigation rate of liquid fertilizers or water to


maintain the moisture in the soil allowing the crops thrive in dry land but during the La
Niña, there is a great chance of fertilizer loss, since La Niña is can be considered as a
counterpart of El Niño, during this time, there can be an increase in the average amount
of rainfalls in most areas of the country and this causes floods, and the nutrients and
fertilizers themselves that were used can be washed away with the runoff water.

Due to the large irrigation rate cause by El Niño, scientists found a way to retain
water and fertilizer in water tressed areas using hydrogels. Hydrogels are crosslinks
polymers that acts as a superabsorbent sponge to hold liquids hundreds of times its
weight, studies also found that plants with hydrogels showed greater results with less
water used in terms of plant yield, and decrease in fertilizer loss rate that could be useful
during La Niña. Some farmers use hydrogel to promote seed germination and keeps the
soil moisturized and absorbs some of the fertilizer which will be locked in the hydrogel.
Though some synthetic hydrogels are toxic to soil and plants, and contributes to pollution
due to their lack of biodegradability.

Due to environmental problems that synthetic hydrogels can cause, natural


hydrogels are needed. Gelatin has been considered a highly promising candidate for use
in hydrogels because of its hydration properties, such as swelling and solubility; gelling
properties, such as texturizing, thickening, and water-binding capacity; and surface
properties include emulsion and foam formation, stabilization, adhesion, cohesion, and
protective properties. Further, its biocompatibility, low toxicity, antimicrobial activity,
and biodegradability makes it suitable for a variety of biomedical applications (Rashid,
2019). Because of the biodegradation and poor mechanical properties of the gelatin,
possible applications of this are limited as a biomaterial. On the other hand, it readily
undergoes chemical crosslinking because of the large number of functional side groups.
Mechanical and thermal stability of gelatin materials are improved by crosslinking of
polymer molecules.

The researchers aim to study the feasibility of the gelatin-based hydrogel with
distillery slops as fertilizer by the hydrogel formation and its characterization, and to
investigate the effects of the gelatin-based hydrogel loaded with distillery slops on plant.
Statement of the Problem

The main objective of the study is to produce a gelatin-based hydrogel fertilizer


from ethanol distillery slops and that can be used as a fertilizer. This study aims to
answer the following questions:

1. Can ethanol distillery slops be concentrated to recover the nitrogen,


phosphorous, potassium, and other minor nutrients useful for plant growth?
2. Can the concentrated distillery slops be used to produce controlled release
hydrogel fertilizer?

3. What will be the properties of the hydrogel fertilizer based on the results of the
following tests?
A. Composition,
B. Hydrogel formation percentages,
C. Swelling,
E. Biodegradability, and
F. Water retention?
4. Is there a difference in plant growth in the following experimental setups:
A. Soil with commercial fertilizer, and
B. Soil with hydrogel fertilizer?

Hypothesis

Below are the null hypothesis of the study:

1. The distillery slops cannot be used to produce gelatin-based hydrogel fertilizer

2. There will be no significant difference in the swelling rate and water retention
of the hydrogel with or without the tannic acid.

3. There will be no significant difference of the fertilizer loss rate in the gelatin-
based hydrogel fortified with distillery slops.

4. There okra with gelatin-based hydrogel fertilizer will have no significant growth
compared to commercial fertilizer.
Scope and Limitations

The study is focusing on utilizing the VMC Distillery’s slop by forming it into a
hydrogel using gelatin and will be used as a fertilizer. The researchers will be testing the
NPK ratio and pH levels of distillery slops. The hydrogels will be characterized using
Fourier Transform Infrared Spectroscopy (FTIR), determining the hydrogel formation,
swelling ratio, biodegradability, and water retention. The release rate of the hydrogel was
calculated to measure the amount of hydrogel released for a particular time. The effect of
the hydrogel fertilizer was tested in terms of stem elongation yield.

The researchers will plant okra seedlings to determine the efficacy of the
produced distillery slop hydrogel fertilizer to know if there is any significant
improvement on the okra’s growth compared to just using a distillery slop fertilizer, okra
was chosen to be tested because it reaches the maturity of 50-65 days and it is not too
long or too short of a time to determine whether the distillery slop hydrogel fertilizer is
more effective or not, and the hydrogel fertilizer contents and materials are applicable
and can be used on the plants okra.

The characterization of hydrogel in terms of hydrogel formation percentages,


swelling, biodegradability, and water retention will be done in school laboratories except
for the characterization by composition using FTIR which will be done in Iloilo. The
hydrogel fertilizer on plants will be done at home. Five samples will be used in each of
the following conditions: Pot with commercial fertilizer and Pot with hydrogel infused
with distillery slops. Samples will be strictly monitored for other uncontrollable variables
such as sunlight and rainfall. This study started on the month of November and is
expected to be completed at the month of June.

Significance of the Study

This research aims to produce a slow-release hydrogel fertilizer that can help the
growth of plants. Furthermore, results of this investigation will be beneficial to the
following:
Farmers. The results of this study would be beneficial to the farmers by helping them
grow their crops where the fertilizer is more applicable. It can also help them lessen
irrigation water consumption, lower the death rate of plants, improve fertilizer retention
in soil and increase plant growth rate.

Distilleries. The main raw material of this study is slops from distillery industry.
Therefore, this study can be beneficial to distilleries in helping them manage their waste
materials that could harm the environment and surroundings. Thereby saving them
money in disposing these waste materials.

Community. The results of this study can give advantage to the community by
mitigating the issues like poverty, and malnutrition, they also play a vital role since they
are considered consumers, they could get affected of the wastes of distilleries if we
couldn’t provide a solution to it.

Environment. This study can be beneficial to the environment by helping in soil erosion
control, environmental cleanups and a substitute of using harmful chemicals as fertilizers
for plants. Also prolongs the fertility of the soil.

Future Researchers. This study will serve as a reference in utilizing distilleries waste
materials that could help in decreasing the probable harmful effects of the wastes. This
will also serve as guide to further develop their research.

Definition of Terms

The following are terms that were used in the study and their conceptual and
operational definitions:

Crosslinker. Crosswise connecting part (such as an atom or group) that connects


parallel chains in a complex chemical molecule (such as a polymer) (Retrieved
from https://www.merriam-webster.com/dictionary/cross-link). In this study, it is
used for improving the properties of the produced gelatin-based hydrogel.

Distilleries. An establishment for distilling, especially for distilling alcoholic


liquors (Retrieved from https://www.thefreedictionary.com/distilleries). In this
study, this alcohol production site is where distillery slops can be sourced.
Distillery Slops. Distillery slop is the waste from alcohol manufacture, as from
breweries or distilleries, which produce highly organic pollutants (Srisatit, 2011),
In this study, this will be used to supply nutrients to be released in the
produced gelatin-based hydrogel fertilizer.

Glutaraldehyde. The colorless, oily liquid glutaraldehyde has a harsh, pungent


odor. Glutaraldehyde is used for cleaning and sterilizing surfaces and equipment
in the industrial, laboratory, agricultural, medical, and some residential settings
(National Center for Biotechnology Information [NCBI], 2021). In this study, this
material will be used as the crosslinker to the gelatin hydrogel to improve the
property of the material.

Hydrogel. Hydrogels can be defined as systems comprising of three-dimensional,


physically or chemically bonded polymer networks entrapping water in
intermolecular space (Ahmed, 2015), In this study, this material is used as a
biodegradable delivery system for the fertilizer made from distillery slops.

Lime. A caustic highly infusible solid that consists of calcium oxide often
together with magnesium oxide, that is obtained by calcining forms of calcium
carbonate, and that is used in building and in agriculture (Retrieved from
https://www.merriam-webster.com/dictionary/lime ) , In this study, this mineral
compound is commonly used for stabilizing the pH levels of the distillery slops.
Review of Related Literature

This section summarizes the relevant literature and studies that the researcher
considered while determining the significance of the current research. It also includes a
summary of the art in order to fully comprehend the research and gain a better
understanding of the study.

Distillery Slops as Fertilizer. The Philippines ranked 13th in the world’s top
sugarcane producers in 2019, supplying 70 billion to the Philippine economy annually and 51%
of sugarcane is planted in Negros making it possible to be recognized for molasses-based alcohol.
In this article it highlights the history of one of the most recognizable alcohol brands in the
Philippines which is Don Papa Rum and one Brand Manager for Asia at bleeding heart rum
company which produces Don Papa rum stated that since their production in this agricultural
product is getting bigger and bigger, there is a continuous and increasing need for ingredients to
supply in creating this product which is the sugarcane. A number of distillery companies are here
in Negros producing thousands of liters of alcohol from molasses as well as a huge amount of
waste water having various characterizations based on the quality of the molasses used (Tan,
2020).

Environmental deterioration, irresponsible endangering of local neighbors, land-grabbing


and displacement of agrarian reform beneficiaries, and a lack of relevant business documentation
and permissions are among the complaints leveled against the distillery. NDB sought the side of
the former employees and of the VMC, one of the complaints from the distillery is on how they
resorted the discharge of slops after the fish kills issues on the rivers in Manapla, VMC
discharges the slops in nearby farms in Manapla, Cadiz, and Victorias. The ammonia from the
slops has also seeped into the artesian wells of residents in the farms, making the water hazardous
to consume and even to wash clothes with, affecting the ecosystem and the health of the people.
Addressing the issues of the distillery, VMC invested in modern structures and technological
processes by installing Anaerobic digester, Automatic Flaring system and made an improvement
to their waste water treatment methods like filtration, aeration and dosing of enzymes to
counteract the odor causing chemical reaction in the digesters, also installing of additional units
of aspirating surface aerators in the Aeration Ponds to assist in eliminating the odor of the waste
which was also complained by the residents affected. Residents of Brgy Purisima have also been
giving positive feedback on the company's efforts in fixing the odor problem. VMC is
now committed to preserving the environment and adding value to the communities in which it
operates (Toga, 2021).

Because ethanol production produces vast amounts of high-strength liquid


effluents with high concentrations of organic matter and nitrogen compounds, low pH,
high temperature, dark brown hue, and high salinity, distilleries are among the most
polluting industries and its alarming that alcohol distilleries are increasing worldwide,
with an increase of ethanol production for about 41 % from 44.3 to 62.6 billion liters
without a proper method in managing the produced wastewater. It is hazardous to the
environment because it pollutes water sources in a variety of ways, for example by
reducing the oxygenation of the water, which is detrimental to aquatic life, it can cause
depletion of dissolved oxygen in water bodies and harms aquatic flora and fauna. In this
review it stated that one method to manage the distillery waste was to use it in
agriculture. Distillery wastewater has potential value in this application, but it also causes
problems and has negative environmental consequences. The use of distillery stillage is
hazardous to plant development and soil qualities at high doses, while it promotes the
sprout, growth, and yield of dry land plants at lower doses owing to its nutritional content
(P, N, K, and Ca). Furthermore, in dry land circumstances, the combination of distillery
stillage and natural organic compounds is acceptable. Elimination of organics from
distillery by-products is becoming increasingly significant in terms of reducing land and
water contamination from an environmental point. This review discusses individual
biological, either aerobic and anaerobic, physico-chemical treatment methods, either
coagulation, electrocoagulation, adsorption, advanced oxidation, and membrane
processes, and thermal, either evaporation and combustion. In this study it also included
the new directions for the improvement of valorization technologies, including the search
for new eutectic solvents for extracting these compounds. Such technologies are essential
for sustainable development, which requires the use of management and valorization
strategies for recovery of valuable compounds with minimal disposal of waste streams.
Distillery slops are rich in organic matter which makes it suitable as a fertilizer but
treatment of slops before disposal is necessary to mitigate the pollution it causes and how
it affects the soil and the bodies of water where it is disposed of (Mikucka & Zielińska,
2020).
The obtained a multinutrient (mixed) fertilizer for acidic soils, such as soddy gley
soils of the forest-meadow zone of North OssetiaAlania is done by mixing the 10-15% of
the total weight of distillery waste with quicklime. The technology of fertilizer
preparation is as follows. Finely grinded quicklime in an amount of 10-15% of the total
weight of distillery waste (100-150 kg per 1 ton) is placed in the container with distillery
waste with moisture content of 85-95%. The moisture rapidly evaporates, resulting in the
decrease of moisture content of the mixture by 28-35% within 20-30 minutes. Quicklime
removes a substantial quantity of moisture from distillery waste, up to 60-70 percent of
the initial humidity.Simultaneously, the inevitable costs of removing moisture from
distillery waste are automatically eliminated. When mixed with distillery waste, the lime
concentration should not exceed 15% of the total weight since the nitrogen compounds in
the waste begin to volatilize with the water vapor, and the protein content, which serves
as a nutritional medium for soil microorganisms, declines, It should be at least 10% of the
entire weight of distillery waste, because otherwise the acidity of the waste is not
appropriately neutralized. Experimental studies indicated that the proportion of lime in
the mixture within the range of 2-9% increased the pH of the medium by only 0.9, i.e. the
medium remained acidic. Increasing it up to 10-15% increased the pH value to 9.8, which
meant creating an explicit alkaline medium required to neutralize the soil acidity. Its
further increase up to 16-20% created an almost maximum alkaline medium, which was
accompanied by a sharp decrease in the content of nitrogen and protein. This study
developed a method of obtaining a multinutrient fertilizer for acidic soils by mixing the
waste of the distillery industry with quicklime in a certain ratio (Kabaloev et al., 2018).
Distillery spent wash is highly acidic and its pH level ranges from 3.8 to 4.4. Which it
can’t be directly applied on soil. Hence, it was neutralized by using 10% of lime slurry to
reach a pH level of 7.0 (Goyal et al., 1996). 10 ± 2 g/L is the amount of sodium
bicarbonate needed when neutralizing the acidic distillery wastewater to 5.6 pH. The
neutralization of the wastewater to pH 7.0 and 7.5 required two and four times the
amounts of sodium bicarbonate required to neutralize its 5.6 pH (Feng at al., 2017).

Magnesium hydroxide in the slurry neutralizes the hydrogen ions (H+) in the acid
wastewater. This can significantly improve the pH value of acid wastewater to reach
industrial wastewater discharge standards. Before and after the treatment, we run tests of
the water quality of the wastewater. Through experiments, first thing is to determine the
relationship between the amount of slurry and pH value of acid wastewater. The pH value
of acid wastewater after treatment can meet industrial wastewater discharge standard (pH
value 6 to 9). Since wastewater is highly acidic, dilute it for 10 times to experiment with.
Under the same conditions, use electrolytic magnesium hydroxide slurry and
commercially available magnesium hydroxide slurry to treat the acid wastewater to run a
comparison test. Add 2 to 7 ml of magnesium hydroxide slurry into 10 diluted acid
wastewater respectively; stir the water in the velocity of 400 r·min-1 for 30min and then
stir the water in the velocity of 500 r·min-1 for 2min; let the water stand for 4h; stir the
water in the velocity of 150 r·min-1 for 2min for complete reaction; then let the water
stand for 3h (Nie & Yi, 2015).

Hydrogel Fertilizer. Superabsorbent polymers are three-dimensional network


materials that can absorb up to 500 percent of their weight in water when in a gel state.
Researchers have recently focused their attention on the utilization of superabsorbent composites
as water and fertilizer carriers. The results observation revealed a reduction in irrigation water
consumption, an improvement in fertilizer retention in soil, a decreased rate of plant mortality,
and a promotion of plant development. In this study Low cost green fertilizer hydrogel packaging
material was prepared based on the waste chicken gelatin using peroxy grafting copolymerization
technique.One kilogram of discharged liquid waste from chicken was obtained during the grilling
method. The liquid was held at a low temperature (0 oC) for 6 hours to solidify, then the top fat
layer was scraped off.The rest was thawed, then filtered through cheesecloth to eliminate any
foreign debris before being evaporated to dryness at room temperature (50 oC, 0.6 mbar). The
fats were removed from the obtained residue with hexane, and the defatted gelatin waste was then
ready for the grafting procedure. Defined pre-weighed extracted waste chicken gelatin (4.0 g) was
dissolved in 40 mL of Millipore water (distilled water with electrical resistance of 18.2 MΩ.cm at
25 o C) and filtered to remove its insoluble salt. The solution was added to a 500 ml 3- neck
double jacket flask equipped with a mechanical stirrer (Cafram BDC 2002, Canada) and the flask
was thermostatic temperature at (80 oC). Then 2-acrylamido-2- methylpropanesulfonic acid (3.0
g) was added to the reactor. After stirring for 10 min, ammonium persulfate (0.20 g of APS in 5
mL of H2O) and methylene bisacrylamide (0.10 g in 5 mL of H2O) were added simultaneously to
the reaction mixture. The temperature was maintained at 80 oC and the reaction mixture was
stirred continuously (300 rpm) for 1 h. At the end of the propagation reaction, the gel product was
poured into ethanol (200 mL) and was dewatered for 12 h. Then the product was cut into small
pieces, washed with 200 mL of ethanol, and filtered. Overnight, the particles were dried in a 50°C
oven. The powdered superabsorbent hydrogel was kept in the absence of moisture, heat, and light
after it was ground (Ibrahim et al., 2015).

It is possible to improve gelatin properties combining it with some different


natural or synthetic polymers; or cross-linking it by enzymatic, chemical or physical
methods. Cross-linking proteins using some compounds (formaldehyde, glyoxal, and
glutaraldehyde) has shown to be effective, however the toxicity of these chemicals has
necessitated additional precautions. Genipin is also an effective less toxic chemical cross-
linker but it has certain drawbacks, such as being expensive and coloring the protein blue
during the cross-linking process. Another approach for protein cross-linking is enzyme
cross-linking, which has several disadvantages such as substrate selectivity,
heterogeneous gels and a lack of control throughout the cross-linking process.
Transglutaminase is commonly used to modify proteins, however it is only commercially
approved for use in food. However, this enzyme has a glutamine residue, which may be a
stumbling block in the food manufacturing process. Physical cross-linking methods such
as high pressure and irradiation are less toxic but they have other disadvantages such as
forming weaker bonds, requiring long periods of cross-linking time, and lack of control
for reaction (Erge & Eren, 2021).

Gelatin-Based Hydrogel Fertilizer. A controlled release gelatin-based fertilizer


by using gelatin microspheres to encapsulate and release the urea was prepared using
glutaraldehyde as a crosslinker using emulsion-cross linking method. The effects of
gelatin/urea ratio, emulsifier, glutaraldehyde concentrations, and cross-linking duration
on urea loading and encapsulation efficiency using response surface approach. The
urea/gelatin ratio was discovered to have a stronger impact on urea loading and
encapsulation efficiency than other parameters. As the amount of glutaraldehyde and the
reaction time increased, the amount of water taken in dropped. Urea release followed a
Fickian trend, showing that release is controlled by diffusion. This research will help with
the design and manufacturing of controlled-release fertilizers (Tang et al., 2017).
To improve fertilizer efficacy and reduce its environmental impacts by producing
controlled release fertilizer (CRF). In preparation, glutaraldehyde was used as a
crosslinker to synthesize biodegradable hydrogels using gelatin and addition of
ammonium nitrate and monoammonium phosphate fertilizers. The hydrogels produced
were characterized by swelling and degradations tests and scanning electron microscope
(SEM) analysis. By increasing the amount of glutaraldehyde/gelatin ratio reduces the
swelling value (Pulat & Yultay, 2016).

Because natural polymers improve material performance while also being


environmentally friendly, they are frequently used as biodegradable matrix in controlled
release technologies. The silk fibroin (SF)/gelatin hydrogel containing chitosan (CS) was
created by solvent casting in this work to reduce the rate of urea release from the
hydrogels. After adding CS to the SF/gelatin, the Fourier transform infrared findings
demonstrated that there were no intermolecular interactions. Furthermore, increasing the
CS concentration in the SF/ gelatin blended hydrogels reduced porosity, affecting
crystallinity, swelling, water solubility, and surface hydrophobicity. The amount of CS
present had a similar effect on the rate of urea release from the hydrogels, with a value of
diffusion exponent characteristics (n) more than 1.0 found using the Korsmeyer-Peppas
model for SF/gelatin/CS hydrogels. According to this, the urea release from the
SF/gelatin hydrogels with CS was a super case II transport type. Furthermore, the
SF/gelatin/CS hydrogels had a lower urea release rate (k) than the SF/gelatin hydrogel
alone, indicating that CS might be employed to extend urea release from SF/gelatin
hydrogels for controlled release applications (Rattanamanee, 2014).

Caffeic acid, a simple plant-derived phenolic molecule, has been used to study
natural crosslinking of gelatin. The creation of a thermo-irreversible gelatin gel at 60
degrees Celsius. Gelatin with changed material characteristics has been obtained by
controlling the crosslinking reaction. To achieve varying degrees of crosslinking, reaction
parameters such as reaction pH, reaction duration, and caffeic acid concentration were
optimized. Small and large deformation rheology were used to investigate the improved
material properties. Gelatin was crosslinked for 20 minutes at 60°C and pH 9 with a 1.5
percent concentration of caffeic acid, resulting in greater melting and setting
temperatures. At higher temperatures, the storage modulus and gel strength of crosslinked
gelatin were also shown to be higher (Kosaraju et al., 2010).

Plant growth and development are harmed by biotic and abiotic stresses such as
nutritional shortages, hormone imbalances, ion toxicity, and osmotic and oxidative
anxiety. The most effective mechanism is the cellular biosynthesis of secondary
metabolites, which include organic compounds that help plants cope with stress by
increasing antioxidant activity, detoxifying toxic ions, regulating nutrient uptake, and
mediating the transport and distribution of various hormones. Caffeic acid is involved in
plant physiology and stress tolerance mechanisms. It is largely used by plants to
synthesize lignin, which thickens cell walls and makes plants resistant to ion toxicity
sodium and heavy metal stress. The synthesis of ferulic acid via the methylation of
caffeic acid mediated by O-methyltransferase also reconciles the uptake of high energy
radiations in mesophyll cells during drought stress. Exogenous administration of caffeic
acid has been found to be the greatest alternative for dealing with salinity, ion toxicity,
drought, and heavy metal stress (Riaz et al., 2018).

Related Studies

Chitosan Hydrogel. The hydrogel was made by dissolving chitosan in a 10 mL


0.1 M (2 percent v/v) acetic acid solution. After that, 1.5 grams of NPK fertilizer was
added to the solution and stirred constantly for about 30 minutes, followed by 0.16 mL
glutaraldehyde solution 25% (0.4 % v/v). The hydrogel was dried using two different
methods: air drying and freeze drying. FTIR and SEM were used to characterize the
hydrogel produced by freeze-drying. Freeze-drying, which produces a larger pore size,
yielded a higher percentage of release than airdrying. Using air drying, the hydrogel was
obtained by heating at 70 °C for 24 hours in the oven. The hydrogel was heated
(Suratman et al., 2020).

A simple freeze-thawing process was used to make the hydrogel. At 60oC,


Mw89,000-98,000 polyvinyl alcohol (Sigma Aldrich) was dissolved in 100 mL distilled
water to form a 10% w/v polymer solution. Chitosan (Mercks) granules were dissolved in
acetic acid and charged into the PVA polymer solution (2, 4, 6, 8, 10 wt percent of PVA).
The PVA-Chi solution was combined with 6 mL glutaraldehyde (Sigma Aldrich) and 10
mL NPK fertilizer solution (Jabatan Pertanian Ayer Hitam, Malaysia) until
homogeneous. The hydrogel solution will be poured into the mold and frozen at -20oC
for 24 hours before being thawed at room temperature for 24 hours (Nayan et al., 2018).

Gelatin Hydrogel. The gelatin-based hydrogel was synthesized by using


glutaraldehyde (GA) as a crosslinker and its characteristics were investigated in terms of
formation, swelling, and degradation. Release of ammonium nitrate (AN) were also
observed. The hydrogels were formed in the glass tube and was cut into 0.5 cm discs. The
gelatin-based hydrogels loaded with AN were tested on tomato cucumber and tomato
seeds, Torf (Gertengold) was the soil used in planting the seeds and each one of 4 pots
was filled with 4L. 3, 6, and 9 hydrogels discs were placed on the pots and the other pot
has none. The depth of the seedlings was 3 cm and the depth of the hydrogel discs were
1.5 cm. The seed germination percentage and stem elongation yields were calculated.
Based on the results, the pot with 9 hydrogel discs were observed to have more
significant growth (Pulat & Saglam, 2018).

Gelatin. Overheating the gelatin could weaken its gelling properties. Additional
gelatin may be needed in salty and acidic ingredients since they tend to soften the gel.
Sugar is hygroscopic, which means it draws water from gelatin molecules, enhancing the
gelling effect. Proteases are enzymes that break down gelatin proteins and boiling could
neutralize the enzymes (Joachim & Schloss, 2020). The collagen can thoroughly dissolve
in 0.1 M acetic acid (Truong et al., 2021).

Drying Distillery Stillages. Thermal treatment of highly polluted distillery


wastewater is thought to be a cost-effective and efficient complement to anaerobic
digestion and oxidation. When the stillage is heated to high temperatures (160–250 °C),
considerable volumes of organic substrate are precipitated as solid precipitates. The
stillage from the distillery can be dried with hot air (180 °C), resulting in a dry powder
with a calorific content of roughly 3200 kcal/kg (Mikucka & Zielińska, 2020).

Glutaraldehyde.

FTIR Analysis. Chemical crosslinking with glutaraldehyde produced poly (vinyl


alcohol) (PVA) hydrogels with varying degrees of hydrolysis (DH) (GA). Fourier
Transform Infrared Spectroscopy (FTIR) and Synchrotron small-angle X-ray scattering
characterisation were used to study the nanostructure of the resultant hydrogels (SAXS).
Swelling ratio assays in various pH solutions were used to conduct in vitro studies. The
absorption bands of the acetal bridges were visible in the infrared spectra of the
crosslinked PVA, which came from the reaction of the GA with the OH groups from
PVA. The crystallinity of the PVA film was also determined using FTIR spectroscopy
based on the relative intensity of the vibration band at 1141 cm -1 (Mansur et al., 2008).

Hydrogel Formation. To determine the hydrogel formation, the dried was


weighed. The samples were then placed in water for 48 hours to remove the unreacted
chemicals and the sample was dried (Pulat & Saglam 2018).
m
𝐻𝐹 (%) = mo 𝑥 100 (1)

The m is the weight of the dried disc sample and mo is the weight of the wet disc
sample.

Swelling. The dried hydrogels were weighed before being soaked in distilled
water for a week at room temperature. The inflated films were weighed after blotting
away the extra water on the surface. After that, each swelled hydrogel was reweighed
after being left at room temperature overnight (Rattanamanee et al., 2014).

The degree of swelling (DS) and water solubility (WS)was calculated using the
equation,

W2 − W1
𝐷𝑆 =
W1

W1 − W3
𝑊𝑆 =
W1

Respectively, the weights of dried hydrogel, water saturated hydrogel, and


swelling hydrogel are W1, W2, and W3

Biodegradability. The biodegradation environment was created using natural soil


from NTU's campus. Okara cellulose hydrogels (510 mm2) were buried at a depth of
around 10 cm below the soil's surface. The typical temperature for incubation is 25
degrees Celsius. After 2 to 30 days of burying, the disintegrated hydrogel fragments were
removed one by one. A balance was used to determine the weight of deteriorated
hydrogel pieces (Cui et al., 2019).

Water Retention. The Agr/GE-co-MA/AA hydrogel was utilized to undertake


water retention studies on two different soil and sand media. In one experiment, 20 g of
soil was mixed with 2 g of Agr/GE-co-MA/AA hydrogel in a plastic container. Then,
with an analytical balance (accuracy: 0.00001 g), 40 mL of tap water was slowly added to
the plastic container and measured (W1). Similarly, the control (soil without Agr/GE-co-
MA/AA) experiment was carried out (W2). The analysis containers were kept at room
temperature and weighed using an analytical balance every day until there was no
discernible weight loss. The weight loss was assessed on a daily basis using an analytical
balance (accuracy: 0.00001 g). Equation was used to calculate the soil's water
evaporation ratio (W percent) (Chaudhary et al., 2020).

W1 − W2
𝑊% = 𝑥 100
40

where W1 is the weight of the plastic container with Agr/GE-co-MA/AA


hydrogel and W2 is the weight of the plastic container without Agr/GE-co-MA/AA
hydrogel, and 40 is the volume of water (mL).

Release Studies. AN (Merck) (53 mg per disc) was added to the gelatin-GA
mixture during crosslinking procedures to produce fertilizer-entrapped hydrogel samples
in this study (Bajpai and Giri, 2002). For 60 hours, the AN release was monitored
spectrophotometrically at a wavelength of 305 nm. In 100 mL of water, AN-entrapped
samples were inserted. To follow the AN release, 0.2 mL aliquots were obtained from the
medium at various periods; a total of 30 aliquots were taken, so the volume could be
deemed constant. The AN release maintained sink conditions at all times (Pulat &
Saglam, 2018). The following equation was used to determine the cumulative release of
AN:

𝑊𝑡
𝑅𝑒𝑙𝑒𝑎𝑠𝑒 (%) = 𝑥 100
𝑊𝑡𝑜𝑡𝑎𝑙
Wt indicates the original total weight of the fertilizer entrapped in the gel system,
and Wtotal reflects the weight of the released fertilizer in the releasing medium at any
time.

Seed Germination. The following formula was used to compute the seed
germination percentage (SG) (Pulat & Saglam, 2018).

Sg
𝑆𝐺 (%) = x 100
S

Where Sg denotes the number of seeds that have germinated and S denotes the
total number of seeds.

Stem Elongation Yields. From the base to the tip of the plant, the height of the
stems was measured with a ruler (Pulat & Saglam, 2018). The following formula was
used to compute stem elongation yields (E percent):

Hs − Hc
𝐸 (%) = 𝑥100
Hc

Hs and Hc are the stem heights of the sample and control, respectively.

Okra pH. Okra can grow in a wide range of soil pH, but it favors soil with a pH
of 6.0 to 6.8. If the pH of the soil is less than 5.8, it should be limed to raise it to 6.0 or
higher (Brandenberger et al., 2019).

Soil. Soils are divided into major categories based on the types of particles
present. The three principal particle types are clay, silt, and sand. The majority of soils
are aggregates with varying amounts of each particle type. Sandy loam soils often have a
high proportion of sand, a minor quantity of silt, and a small to moderate proportion of
organic loam. In sandy loam soils, most common vegetables grow well, especially if the
soil is supplemented with organic matter before planting (Cochran, 2016).

When it comes to planting garden plants, sandy loam soil is ideal. Sandy loam has
a rich, medium-dark color and a light, airy texture. The sandy loam's texture permits
oxygen to reach the plant roots, where it can be put to good use. The sand enables for
proper drainage, while the loam holds moisture for subsequent use by the plants. The
sandy loam's texture permits oxygen to reach the plant roots, where it can be put to good
use. Sandy loam is composed of 50 to 70 percent sand, less than 20% clay, and 10 to 50%
silt or organic debris (Lewis, 2017).

Coarse Sand. Water can collect in pockets in coarse sand, sometimes known as
yellow sand or builder's sand, to feed roots as they search for nutrients in the soil. The
grainy sand also produces pockets of oxygen, allowing exhausted roots that have recently
been transplanted or roots that have recently emerged from well-cared for seeds to
breathe new life. The larger rock material in yellow or coarse sand also aids water
drainage away from roots, preventing them from being submerged overnight or for long
periods of time (Mcgee, 2021).

Kaolin Clay. Kaolin clay has been reported to manage insect pests and disease in
the garden, as well as protect against sunburn or heat stress and possibly improve fruit
color. Kaolin clay insect control is a natural mineral that works by forming a barrier film
by covering leaves and fruit with a white powdery film that sticks to insects and irritates
them, preventing them from feeding on fruit or foliage. Grasshoppers, leafrollers, mites,
thrips, some moth species, psylla, flea beetles, and Japanese beetles can all be deterred by
using Kaolin clay on fruit trees and plants (Grant, 2021).

Loess Soil. About 10% of the earth's surface is covered by loess soil, which is
found primarily in the mid-latitudes. Except for Antarctica, it can be found on every
continent. The primary substrate for the world's most arable soils is loess soil.
Approximately 80% of cereals are said to grow in loess soil. Despite the fact that it forms
the foundation for global food security, loess soil is presently critically endangered and
hence deserves special protection (“Loess Soil,” 2021).
Materials and Methods

This chapter discusses the process of producing gelatin-based hydrogel using


gelatin, glutaraldehyde, lime, and distillery slops.

Research Design

This study will employ the experimental research methods in the production of a
slow-release gelatin-based hydrogel from distillery slops. The independent variables of
this study are the pre-treatment process of the raw materials and …. The dependent
variable is the amount of distillery slops to be optimized in the gelatin hydrogel and…..

Research Procedure

Preparation of
Materials

Concentration of
Slops

Production of
Hydrogel Fertilizer

Characterization

Testing
Figure 1. Production of hydrogel fertilizer

Figure 1 shows the entire hydrogel manufacturing process, from material


collection to manufacture, characterization, testing, and statistical analysis.

Preparation of Materials

The distillery slops will be obtained from the VMC Distillery at the municipality
of Manapla. The slops will be inside a plastic gallon container and it will be used after
received. The slops will be tested for pH, nitrogen, phosphorus, and potassium at Negros
Prawn, Bacolod City.

The gelatin (Ferna) will be used for gelling and to encapsulate the distillery slops.

The quicklime (CaO) will be purchased from Negros Integrated Industries


Corporation, Bacolod City. It will be used to raise and neutralize the pH of the slops.

The acetic acid (Chem Lab CL05.0107.1000), 0.1 M will be purchased at Thermo
Fisher Scientific Inc. and it will be used to dissolve the gelatin to make a solution.

The glutaraldehyde (25% aqueous solution) acid will be purchased online at Sigma-
Aldrich, it will already be in aqueous state once purchased. It will be used as a crosslinker
for the gelatin.

The sandy loam soil will be prepared by mixing 50-70% sand (coarse), less than
20% clay (kaolin), and 10-50% silt (loess) (Lewis, 2017). It will be used test the
biodegradability and water retention of the hydrogel, and it will be used to fill the pots for
planting.

Concentration of Slops

The lime will be slowly added to the distillery slops (Feng at al., 2017). While stirring
the pH will be measured using a pH meter until it will be approximately 6.0 to 6.8
(Brandenberger et al., 2019). The distillery slops and quicklime solution will then be heated
at 180 °C using a convection dryer until the slops turn into a dry powder (Mikucka &
Zielińska, 2020). To maximize the concertation of slops in the hydrogel.
Production of Hydrogel Fertilizer

The hydrogel will be prepared by dissolving gelatin in 10 mL acetic acid solution


0.1 M (2% v/v) in a 100 mL beaker. 1.5 g of concentrated slops will then be added to the
solution under constant stirring for about 30 minutes, followed with the addition of 0.16
mL of glutaraldehyde solution (0.4% v/v) (Suratman et al., 2020). The solution will be
poured in a test tube (20x200 mm) and left for 24 hours at room temperature to crosslink.
The hydrogel rods will be removed from the test tube and will be cut into 0.5 cm long
pieces using a box cutter (Pulat & Saglam, 2018).

Characterization

The hydrogel samples will be analyzed using Fourier Transform Infrared


Spectroscopy (FTIR) to characterize the hydrogel based on its homogeneity to determine
whether the distillery slops are equally distributed inside the hydrogel (Mansur et al.,
2008). The testing will be conducted in UP Miagao, Iloilo.

Air-Drying Method

For the drying the hydrogel discs, it will be heated in an oven 70 °C for 24 hours
(Suratman et al., 2020).

Hydrogel Formation

The hydrogel formation (HF) percentage will be calculated using the formula
below. The dried discs samples will be weighed and placed in water for 48 hours (Pulat &
Saglam, 2018). To determine how much percentage of hydrogel is formed.
m
𝐻𝐹 (%) = mo 𝑥 100 (1)

The m is the weight of the dried disc sample and mo is the weight of the wet disc
sample.

Swelling

The dried hydrogel discs will be weighed before being soaked in distilled water
for a week at room temperature. The inflated discs will be weighed after blotting away
the extra water on the surface (Rattanamanee et al., 2014). To determine the capacity of
the hydrogel to hold liquid.

The degree of swelling (DS) calculated using the equation,

W2−W1
𝐷𝑆 = (2)
W1

Respectively, the weights of dried hydrogel and water saturated hydrogel are W1
and W2

Biodegradability

The biodegradation environment was created using a sandy loam soil. Hydrogel
discs will be buried at a depth of around 10 cm below the soil's surface. The typical
temperature for incubation is 25 degrees Celsius. After 2 to 30 days of burying, the
disintegrated hydrogel fragments will be removed one by one starting on the 2 nd day. A
balance will be used to determine the weight of deteriorated hydrogel pieces (Cui et al.,
2019). This is to anticipate how long will the hydrogel will last before planting.

Water Retention

2 g of hydrogel will be combined with 20 g of loam sandy soil in a plastic container


(4 oz, no brand) in one experiment. Then, with an analytical balance (accuracy: 0.00001
g), 40 mL of tap water will slowly be added to the plastic container and measured (W1). In
a similar manner, the control (soil without hydrogel fertilizer) experiment will be carried
out (W2). The analysis containers will be kept at room temperature and weighed using an
analytical balance every day until there was no discernible weight loss. The weight loss
will be assessed on a daily basis using an analytical balance (accuracy: 0.00001 g)
(Chaudhary et al., 2020). This to measure the ability of the hydrogel to retain water. The
soil's water evaporation ratio (W percent) was estimated using the following equation:

W1−W2
𝑊% = 𝑥 100 (3)
40

Where W1 is the weight of the plastic container with hydrogel fertilizer and W2 is
the weight of the plastic container without hydrogel fertilizer; and 40 is the volume of water
(mL).
Testing

Release of Distillery Slop

For 60 hours, the fertilizer release will be monitored spectrophotometrically at a


wavelength of 305 nm. In 100 mL of water, hydrogel fertilizer disc samples will be
inserted. To follow the fertilizer release, 0.2 mL aliquots will be obtained from the
medium at various periods; a total of 30 aliquots will be taken, so the volume could be
deemed constant. The fertilizer release will maintain sink conditions at all times (Pulat &
Saglam, 2018). The following equation was used to determine the cumulative release of
fertilizer:

𝑊𝑡
𝑅𝑒𝑙𝑒𝑎𝑠𝑒 (%) = 𝑊𝑡𝑜𝑡𝑎𝑙 𝑥 100 (4)

Wt indicates the original total weight of the fertilizer entrapped in the gel system,
and Wtotal reflects the weight of the released fertilizer in the releasing medium at any
time.

Effects of Slow-Release Hydrogel Fortified with Distillery Slops on the Growth of Okra

Following germination and shooting periods, the effect of distillery slop hydrogels
on vegetable growth will be examined based on the study of Pulat & Saglam (2018). Table
1 summarizes the testing circumstances.

Table 1. Growing test procedure


Sample Set-up Pot Seed Number Depth Type of Soil Volume of Watering
of Seed of Soil per pot Schedule
per pot Seeds

1 With Gelatin- 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


based inch Esculentus or Soil per pot
Hydrogel Okra every
Fertilizer discs 50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

2 With Gelatin- 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


based inch Esculentus or Soil per pot
Hydrogel Okra every
Fertilizer discs 50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

3 With Gelatin- 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


based inch Esculentus or Soil per pot
Hydrogel Okra every
Fertilizer discs 50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

4 With Gelatin- 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


based inch Esculentus or Soil per pot
Hydrogel Okra every
Fertilizer discs 50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

5 With Gelatin- 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


based inch Esculentus or Soil per pot
Okra
Hydrogel every
Fertilizer discs 50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

6 With 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


Commercial inch Esculentus or Soil per pot
Fertilizer Okra every
50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

7 With 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


Commercial inch Esculentus or Soil per pot
Fertilizer Okra every
50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

8 With 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


Commercial inch Esculentus or Soil per pot
Fertilizer Okra every
50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

9 With 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL


Commercial inch Esculentus or Soil per pot
Fertilizer Okra every
50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris
10 With 10x10 Abelmoschus 1 5-6 cm Sandy Loam 5 quartz 16.3 mL
Commercial inch Esculentus or Soil per pot
Fertilizer Okra every
50-70 % week
sand, >20%
clay, and 10-
50% silt or
organic
debris

Table 2
Set up for Samples with Gelatin-based Hydrogel Fertilizer

Sample Gelatin Hydrogel Fertilizer Disc per Depth of Gelatin Hydrogel


pot Fertilizer

1 9 1.5 cm

2 9 1.5 cm

3 9 1.5 cm

4 9 1.5 cm

5 9 1.5 cm

Table 3
Set up for Samples with commercial Fertilizer

Sample Commercial Fertilizer Set-up

6 Complete Fertilizer (Triple 14)- Philpos 10 g every 14 days

7 Complete Fertilizer (Triple 14)- Philpos 10 g every 14 days

8 Complete Fertilizer (Triple 14)- Philpos 10 g every 14 days

9 Complete Fertilizer (Triple 14)- Philpos 10 g every 14 days

10 Complete Fertilizer (Triple 14)- Philpos 10 g every 14 days

Two sets of seed germination studies will be conducted. Five Okra seeds that were
healthy and uniform in size will be chosen and put into the soil pots. The seeding pots will
be filled with 9 discs at a depth of 1.5 cm. Commercial fertilizer will be used in the other
five pots. Table 1 shows the watering schedule. During the experiment days, a series of
images will be taken. The following formula will be used to compute the seed germination
percentage (SG).

Sg
𝑆𝐺 (%) = 𝑥 100 (5)
S

Where Sg denotes the number of seeds that have germinated and S denotes the total
number of seeds.

From the base to the tip of the plant, the height of the stems will be measured with
a ruler. The following formula will be used used to compute stem elongation yields (E
percent):

𝐻𝑠−𝐻𝑐
𝐸 (%) = 𝑥 100 (6)
Hc

Hs and Hc are the stem heights of the sample and control, respectively.
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Work Plan

AY 2021-2022
ACTIVITIES

Aug Sept Oct Nov Dec Jan Feb Mar Apr May

2021 2021 2021 2021 2021 2021 2021 2021 2021 2021

Research Proposal and


Revisions

Preparation of raw
materials/procurement
of reagents

Chemical Analysis of
Distillery Slops

Forming of hydrogel
fertilizer

FTIR Analysis

Materials
Characterization

Observation for the


germination of okra
Data Analysis

Budget Requirements:

Materials Amount (Php) Analysis Amount (Php)

Gelatin (5 x 10 g) 92.00

Okra Seeds (11 g) 95.00

Pots (10X10 inch) 170.00

Lime (1 kg) 40.00

Commercial 60.00
Fertilizer per pack

Deionized Water 900.00

Reference 1,800.00

Glutaraldehyde
(100 mL)

Chemical Analysis for 3,800.00


Distillery Slops

Fourier-Transform 4,000.00
Infrared Spectroscopy

Freight 2,500.00

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