PRODUCTION OF ARBUSCULAR MYCORHIZAL

FUNGI AS BIOFERTILIZER

GROUP KK7

NABILA KHAIRANI SABRI

CHONG JIA MIN

NORSYASMIN IZZATI BINTI SAMSUDDIN

NURUL ATIQAH AUNIE BINTI ROSLI

UNIVERSITI KEBANGSAAN MALAYSIA

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DECLARATION

We hereby declare that the work in this project is our own except for quotations and
summaries which have been duly acknowledged.

9 November 2022 NABILA KHAIRANI SABRI

A185243

CHONG JIA MIN

A188216

NORSYASMIN IZZATI BINTI

SAMSUDDIN
A187528

NURUL ATIQAH AUNIE

BINTI ROSLI
A187548
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CHAPTER I

INTRODUCTION

1.1 INTRODUCTION

This chapter will cover the process description and process block diagram that illustrate
the production of Arbuscular Mycorrhizal (AM) as a biofertilizer through pre-treatment
of lignocellulosic biomass, fermentation process, and formulation process. A process
flow diagram (PFD) is designed to give more detailed explanations of the production
conditions and units involved in the process.

1.2 PROCESS DESCRIPTION

1.2.1 Pre-treatment of lignocellulosic biomass

Aiming for a fully sustainable energy and feedstock supply, the sugarcane bagasse
which is the residues from sugarcane production has been chosen as the sustainable
feedstock for the production of Arbuscular Mycorrhizal. The sugarcane bagasse is a
source of cellulosic fiber in which an untreated sugarcane bagasse contains about 47%
cellulose, 31% hemicellulose, 20% lignin, and 2% ash (Mahmud et al. 2021). Hence,
the sugarcane bagasse is chosen as a lignocellulosic biomass to undergo the pre-
treatment process due to its high cellulose content that enables it to undergo an
enzymatic hydrolysis process to produce glucose for fermentation purposes. First, the
sugarcane bagasse is crushed by a crusher (H-101) to produce sugarcane bagasse. The
sugarcane bagasse is then transported by a slurry pump (P-101) to be treated with the
organosolv solution with a composition of 60% ethanol and 40% water in a mixer (M-
101) at 175°C to yield cellulose. Organosolv pretreatment is the most promising
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pretreatment of lignocellulosic biomass due to its high yield of enzymatic hydrolysable

cellulosic pulp. The addition of 0.25% sulphuric acid in the form of dilute mineral acid
could assist ethanol organosolv solution to accelerate the process and reduce
pretreatment time at mild conditions (Vaidya, Alankar A. et al. 2022). Hence, the acid-
catalyzed pretreatment is conducted with 20 mins residence time by maintaining the pH
at 3.5 in the aqueous phase. Lignin and hemicellulose are dissolved in the solution while
the cellulosic pulp is produced in solid form (Montealegre et al. 2017). After acid-
organosolv treatment, the cellulosic pulp is washed with diluted ammonia to remove
the acetylated or formyl functional group on cellulose to avoid decreasing the enzymatic
hydrolysis efficiency of cellulose (Vaidya et al. 2022). The resulting mixture is then
transported by a centrifugal pump (P-102) to the decanter centrifuge (F-101) for solid-
liquid separation at 60°C and 18 bar. The spent liquor is separated and recycled while
the filtered cellulose is transferred by a slurry pump (P-103) to a filter (F-102) to filter
out cellulose from the mixture of dissolved lignin and hemicellulose. The by-product
consists of a mixture of dissolved lignin and hemicellulose can be produced as a
valuable by-product in the glue manufacturing industry and also possesses the potential
to replace petroleum in the future (Juliette Irmer 2017).

Afterward, cellulose is pumped by a centrifugal pump (P-104) for the enzymatic

hydrolysis process in the hydrolysis reactor (R-101) at 50°C, 2 bar, and pH 4.8 (Wang
et al. 2021). During the enzymatic hydrolysis process, cellulase and water are used to
react with cellulose to produce a glucose solution (Lu et al. 2011) as the hydrolysis
reaction equation shown below:

(C6H10O5)n (s) + n H2O (l) = n C6H12O6 (l)

Besides that, the glucose solution is then transferred to the cross-filter membrane
(F-103) via the centrifugal pump (P-105) to separate and purify glucose for the
fermentation process. The water content filtered out will be recycled back to the
hydrolysis reactor (R-101). After filtration, the glucose solution is pumped by a
centrifugal pump (P-106) to the seed fermenter for the fermentation process.
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1.2.2 Fermentation Process

The fermentation process consists of seed fermentation and fermentation process. Two
Paenibacillus Validus bacterial isolates are chosen to culture with Arbuscular
Mycorrhizal fungi in the seed fermenter via the transformed root culture (TRC) method
at 24 ℃ and 2 bar by introducing a medium that stimulates the natural growth
environment of AM fungi (V. Kokkoris et al. 2019). The medium prepared is the
minimal (M) medium previously described by BCcard & Fortin but solidified with 0.4%
(w/v) gellan gum (M. St-Arnaud et al. 1996). Vitamins and 0.8% plant agar are provided
to the seed fermenter for seed germination while the pH value of the medium is kept
between 5.7 to 5.8 with 50% relative humidity (Ho-Plágaro et al. 2018). Paenibacillus
Validus bacterial isolates are under continuous light explosion for 16 hours while 8
hours in the darkness for higher carbon recovery which contribute to a total residential
time of 72 hours (Ho-Plágaro et al. 2018).

After that, fed-batch fermentation is carried out in the fermentor (T-201) at 30℃
and 5 bar to reduce the initial seed concentration required and improve the
performance optimization. Oxygen gas is supplied into the 5L medium during the
fermentation to undergo aerobic fermentation. At the same time, glucose from the
enzymatic hydrolysis reaction and ammonium nitrate solution is fed to the bacteria in a
fermenter. The fermentation is also agitated at 150 rpm with its pH controlled at 5.0 for
roughly 36 hours. 80% of the seed concentration reacted with glucose and ammonium
nitrate to produce AM fungi and carbon dioxide gas. A filter (F-201) is chosen to filter
out the other 20% unreacted seed concentration to be recycled back to the fermenter (T-
201) to fill the fermenter (T-201) with a base number of media to support initial cell
growth with the volume of the fermenter constant at all times. The recycle stream is
able to increase cell concentration while preventing overfed substrate during the process
which contributes to high product yield when AM fungi are positively correlated with
microbial growth.
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1.2.3 Formulation process

The water content of AM fungi is also filtered out in order to produce AM fungi in solid
form for its best performance as a biofertilizer. The wastewater filtered out by the filter
(F-201) is recycled back after being treated with the coagulation method by
manipulating the electrostatic charges of particles suspended in water. The dried AM is
then transported by a slurry pump (P-201) to a mixer (M-301) to mix with
vermicompost and 10% soil with a ratio of 8:1:1 at 50℃ and 1 bar. Finally, the mixture
is produced in solid form and will be stored in the storage at room temperature for a
maximum of 6 months of storage period.

1.3 PROCESS BLOCK DIAGRAM

Production of Arbuscular Mycorrhizal as biofertilizer involves three processes which

are pre-treatment of lignocellulosic biomass, fermentation, and formulation. Figure 2.1
shows the process block diagram of Arbuscular Mycorrhizal biofertilizer production.

Figure 2.1 Process block diagram of Arbuscular Mycorrhizal biofertilizer production

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1.4 PROCESS FLOW DIAGRAM (PFD)

The process flow diagram of production of arbuscular mycorrhizal fungi as biofertilizer.

However, the seed fermentation stage is not included in the diagram.
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