FERMENTATION

❖ INTRODUCTION

• The word Fermentation is derived from Latin word fervere which means
to boil.
• But the conventional definition of Fermentation is to break down of larger
molecules into smaller and simple molecules using microorganisms.
• In Biotechnology, Fermentation means any process by which
microorganisms are grown in large quantities to produce any type of useful
materials.
• In other words fermentation may be define as the process of growing a
culture of organisms in a nutrient media and thereby converting feed into
its desired end product. Its is sometimes described as biochemical reaction
in which microorganisms serve (bacteria or fungi) as biocatalyst.
• This process is carried out in an equipment called as fermentor.

❖ FERMENTOR

• Fermentor can be defined as a vessel in which sterile nutrient media and

pure culture of microorganism are mixed and fermentation process is
carried out under aseptic and optimum condition.
• The fermentor provides sterile environment, an optimum condition that
are Important for microorganism to grow.
• Common features of typical fermenter:
 1.They should be strong enough to withstand the pressure exerted by large
volume of the medium.
2.The materials used for the construction of fermenter should not be
corroded by the fermentation product and it should not yield toxic ion to
the medium.
3.The fermenter should have provision for the control and prevention of
the growth of contaminating microorganisms because industrial
fermentation requires pure culture.
4.If aerobic organisms are used in the process, there should be provision
for rapid incorporation of sterile air into the medium so that the oxygen is
immediately dissolved in the medium and available
to the microorganisms.
5.The Carbon dioxide produced by the microorganisms should be
removed from the medium.
6.Stirring is necessary to mix the organisms with the medium and to make
nutrients and oxygen available to individual microbe.
7.The fermenter should provide provision for the addition of antifoaming
agents intermittently depending on the foaming status of the medium.
8.Thermostatic system should be available to maintain constant
temperature in the fermenter.
9.There should be provision for aseptic withdrawal of culture during
fermentation and also for the aseptic introduction of inoculum at the
starting of the fermentation process.
10. A system should be available for detection of pH of the culture
medium and also for its adjustment.

• Major parts of fermentors :

1. Material used for fermentor
2. Sampling point
3. Impeller
4. pH controller device
5. Temperature controller device
6. Baffels
7. Foam controlling device
 8. Inoculation point
9. Sparger
10. Bottom drainage system

❖ BASIC REQUIREMENT

• Pure culture: Organism, quantity, physiological state

• Culture medium for microorganism growth
• Seed fermentor for initiating inoculum
• Production fermentor: large model equipment – drawing the culture
medium, cell seperation, collection of cell, product purification and
effluent treatment.
❖ Microorganisms used in Fermentation include the commonly used
species are:-

• Bacteria: Acetobacter lacti, Acetobacter woodi, Bacillus subtilis, Bacillus

polymyxa, Clostridium etc.
• Algae: Spirulina maxima, Chlorella sorokiniana etc.
• Fungi: Aspergillus oryzae, Aspergillus niger, Saccharomyces cervisae,
Saccharomyces lipolytica etc.
• Actinomycetes: Streptomyces griseus, Streptomyces noursei etc.

❖ Nutrient requirements : All microorganisms need for their microbial

activity the presence of several nutrients.

• Carbohydrates
• Lipides
• Vitamins and growth factors
• Amino acids
• Nitrogen sources
• Sulfur sources
• Chemical elements and inorganic ions Mineral nutrients required by
microorganisms are species dependent but consists generally of Fe, K, Mg,
Mn. Sometimes S, N, Ca, Co, Cu, P, Zn is required.

❖ Fermentation processes An overall scheme of a fermentation process

can be described as follows:
• Stage 1: inoculum preservation
• Stage 2: inoculum build-up
• Stage 3: fermentor culture

• STAGE 1: INOCULUM PRESERVATION The objective of

preservation is to maintain strains as long as possible without cell division.
The optimal method of preservation must be worked out for each strain.
 The following three techniques are most commonly used:
* Storage at low temperatures (2-6 degrees Celcius)
* Frozen storage (-18, -80 or -196 degrees Celcius)
* Lyophilization Storage at 2-6 degrees Celcius is the least secure, there
is a relatively high risk of contamination and reverse mutation through
frequent transfer.

• STAGE 2: GROWTH OF INOCULUM The preserved culture is

initially revived by growth in a erlenmeyer flask on a biological shaker or
on a solid medium (if spore formation is needed). In order to obtain
sufficient inoculum for small fermentors, a second series of shake cultures
is usually made in more flasks. Out from lyophilized strains the growth of
inoculum takes around 4-10 days, out from frozen cultures the growth of
inoculum takes 4-48 hours for bacteria and 1-7 days for fungi. Finally out
of refrigerated cultures the growth of inoculum takes 4-24 hours for
bacteria and 1-5 days for fungi.

• STAGE 3: FERMENTOR CULTURE The nutrient media for

production must be optimized not only in the ingredients used but also
how the medium is prepared and sterilized, pH value before and after
sterilization.

❖ TYPES OF FERMENTATION PROCESS

The most important types of fermentation are as follows:-

1. Anaerobic Fermentation
2. Aerobic Fermentation
ANAEROBIC FERMENTATION:
• Definition-Anaerobic fermentation is the conversion of complex organic
compounds into simpler ones in the absence of oxygen. It is a method that
cells use to extract energy from carbohydrates when oxygen or other electron
acceptors are not available in the surrounding environment.
• Anaerobic fermentation occurs in the fermentation vessel once the oxygen is
discharged and replaced with N2, CO2, or another by-product of the
fermentation process. Anaerobic fermentation is usually a slower process.
• History-In the mid-1850s, the French chemist Louis Pasteur produced
anaerobiosis by boiling the medium to drive out oxygen and then introducing
inert gas for cultivation. He showed that a microorganism, probably
Clostridium butyricum, was responsible for butyric acid fermentation.
• In anaerobic fermentation, a provision for aeration is usually not needed.
However, initial aeration may be required to support inoculum build-up.
• Once fermentation starts, CO₂ produced during the process ensures sufficient
mixing without external aeration.
• The air in the headspace of the fermenter should be replaced by CO₂, H₂, N₂,
or a suitable mixture, especially for obligate anaerobes like Clostridium.
• Anaerobic fermentation typically liberates CO₂ and H₂ gases, which can be
collected and utilized — for example:
(i)CO₂ can be used for making dry ice or carbonated beverages.
(ii)H₂ can be used for bubbling into freshly inoculated fermenters.
• Recovery of fermentation products generally does not require anaerobic
conditions.
• However, many enzymes from anaerobic organisms are highly O₂-sensitive.
If enzyme recovery is the goal, cells must be harvested under strictly anaerobic
conditions.
• Application- Anaerobic fermentation has been applied to many important
industrial fermentations, such as ethanol production by yeasts, lactic acid
preservation of foods, anaerobic digestion of organic matters in ruminant
cultivation and waste treatment. The most important industrial fermentation is
the anaerobic production of ethanol by S. cerevisiae and other yeasts.

• Glycolysis-
(i) Anaerobic fermentation involve glycolysis as first step in the breakdown of
glucose and other sugar produces molecule of adenosine triphosphate (ATP)
that create energy source for the cell.
(ii) Though this method cell is able to generate nicotinamide adenine dinucleotide
(NAD+) from the reduced form of nicotinamide adenine dinucleotide
hydrogenate (NADH). A molecule necessary for continue glycolysis.
(iii) Anaerobic fermentation relies on enzymes to add a phosphate group to
an individual adenosine diphosphate (ADP) molecule to produce ATP which
mean it is form of substrate level phosphorylation.
(iv) This contracts with the oxidative phosphorylation which use energy
from an established proton gradient to produce ATP.
(v) Anaerobic fermentation have two major types, both restore NAD+ to allow
cell to continue generating through glycolysis- (a) Ethanol Fermentation or
alcohol fermentation and (b) Lactic acid fermentation.

(a)Ethanol Fermentation-
i. The reaction is two step process in which pyruvate is converted to
acetaldehyde and carbon dioxide first by the enzyme pyruvate
decarboxylase.
ii. Ethanol fermentation converts two pyruvate molecule, the products of
glycolysis, to two molecules of ethanol and two molecules of carbon
dioxide.
iii. Yeast and certain bacteria perform ethanol fermentation where pyruvate
is broken into ethanol and carbon dioxide.
iv. Ethanol fermentation use in the production of beer, wine and bread.

(b)Lactic Acid Fermentation-

i. These fermentation method use by animal and certain bacteria, like those in
yogurt.
ii. The fermentation use routinely in mammalian red blood cells and in
skeleton muscles that has an insufficient oxygen supply to allow aerobic
respiration to continue i.e., in muscle used to the point of fatigue. iii. In
muscle lactic acid fermentation must be removed by the blood circulation
and lactate brought to the liver for further metabolism.
iv. The chemical reaction is as follows: Pyruvic acid +NADH Lactic
acid+ NAD+
v. The enzyme used in these reaction is lactate dehydrogenase (LDH). The
reaction is proceed in either direction, but reaction from left to right is
inhibited by acidic condition.
vi. Lactic acid accumulation cause muscle stiffness, fatigue and soreness.
vii. Once lactic acid removed from muscle and circulated to liver, it
reconverted to pyruvic acid and further catabolized for energy.
viii. Types of Lactic Fermentation:
Homo-lactic fermentation- The fermentation in which only lactic acid
produced. There is no any side product formed after reaction.
Hetero- Lactic Fermentation-The fermentation in which lactic acid is
produced along with some by products like gases.

Figure : Alcohol fermentation pathway

 Fig : Lactic acid fermentation.

AEROBIC FERMENTATION:

• Aerobic fermentation occurs in the presence of oxygen. It usually occurs at the

beginning of the fermentation process. Aerobic fermentation is usually a
shorter and more intense process than anaerobic fermentation.
• The main feature of aerobic fermentation is the provision for contant adequate
aeration is essential .
• Oxygen limitation is a major problem in aerobic fermentations because oxygen
has a low solubility in water. Dissolved oxygen (DO) concentration is
generally kept as high as possible by increasing the oxygen transfer rate (OTR).
• High agitation and/or aeration are commonly used to improve oxygen
transfer rate (OTR) by increasing driving force, gas-liquid interfacial area, and
gas bubble residence time.
• Studies have explored the effects of dissolved oxygen (DO) levels and periodic
pressure changes on bioprocess performance.
• Newer strategies include using increased air/O₂ pressure, pressure pulsation,
and oscillating DO tension to enhance OTR and metabolite production.
• Large-scale bioreactors have a reduced surface-to-volume ratio, making
oxygen supply a limiting factor for high cell density and secondary metabolite
production.
• Oxygen solubility in liquids is low, and when DO falls below the critical
concentration, growth rate becomes oxygen-limited.
• Conventional stirred-tank fermenters use high mechanical agitation, gas
flow rates, and bubble dispersion devices to improve oxygen transfer.
• Stirrers break gas bubbles into smaller ones, increasing residence time and
ensuring homogeneity and mixing.
• Low agitation speeds fail to generate enough turbulence for effective gas
dispersion.
• Higher stirrer speeds enhance OTR but may cause shear stress, damaging cells.
• Choosing optimal agitation speed and impeller design balances oxygen
transfer and cell safety.
• ALSA fermenters (Air Lift with Side Arms) are a cost-effective alternative
to stirred tanks by minimizing mechanical agitation.
• In some cases, the amount of air needed per hour is about 60-times the medium
volume. Therefore, bioreactors used for aerobic fermentation have a provision
for adequate supply of sterile air, which is generally sparged into the medium.
• In addition, the fermenters should have befitting device and mechanism for
efficient stirring and mixing of the medium and cells.
• Aerobic fermenters may be either of the
(i) Stirred-tank type in which mechanical motor-driven stirrers are provided.
(ii) Air-lift type in which no mechanical stirrers are used and the agitation is
achieved by the air bubbles generated by the air supply. Stirred-tank
type fermentors (or stirred bioreactor):
• These are usually made of ‘glass’ (i.e., smaller vessels having capacity ranging
between 1-1000 L) or ‘stainless steel’ (i.e., large vessels having capacity
varying between 2000-8000 L).
• In practice, these bioreactors are of closed or batch types, having rather a
definite fixed volumes and are normally agitated with motor- driven stirrer
with loots of variation in design specification, such as: Curved bottom for
more efficient mixing at low speeds water circulated jacket in place of heater
type (electrical) temperature
 control mirrored internal finishes to minimise cell-damage
drastically etc.
Air-lift type fermenters:

• The culture in an air-lift type fermentors are not only subjected to ‘aeration’
but also ‘agitation’ by passing sterilized compered air bubbles introduced
strategically at the bottom of vessel.
• The fermentor has an inner draft tube via which the air bubbls as well as the
aerated medium rise as this help in mixing of culture and aeration
simultaneously.
• Air bubble being lighter lift to the top of the medium and air subsequently gets
released through the outlet.
• In this process, the cells and the medium which eventually lift out of the draft
tube usually moves downwards outside the tube and then recirculated.
• Air-lift type fermentors with a capacity of 2- 90L are invariably available for
large-scale production.
• Fermentors of 2000L are used for specifically for the production of
monoclonal antibodies.
• There are two types of airlift bioreactors –Internal loop type, External loop
type
• Internal-loop airlift bioreactor has a single container with a draft carrying out
the fermentation process.
• External loop airlift bioreactor has an external loop to separate samples or
liquids in different channels carrying out fermentation.
 Fig: Continuous stirred-tank reactors (Fermenter: Equipment design)

Fig: Airlift Bioreacto

Aerobic fermentation can be surface culture or static and submerged.
A. Surface Culture Fermentation/ Solid state fermentation:
i. These are the oldest method of fermentation.
ii. The production of complex flavour extract is fermentation of flavour raw
materials by bacteria and moulds on solid nutrient medium.
iii. In these culture media is simply placed in simple thermostatic boxes on
baking tray like plates to which microorganism are inoculated. After
 incubating the nutrient media for several days, it is extracted with water to
isolated fermented products contained in the media.
iv. Example: production of soy sauce made from rough-ground cereals and soy
beans which are inoculated with special moulds.
v. Vinegar production by acetic acid bacteria grown on the surface of wood
chips.
vi. Production of enzyme secreted by bacteria and moulds into the extracellular
environment (Culture Media)

Fig: SSF bioreactors with continuous agitation and forced aeration. (A) Stirred aired
bioreactor; (B) Gas-solid fluidized bed bioreactor; (C) Rocking drum bioreactor.

B. Submerged Fermentation
i. Submerged fermentation is applicable for the manufacturing of cell products
by propagation of micro-organism and cell cultures in a fluid nutrient media.
ii. The submerged fermenter normally operated in sterile manner.
iii. The whole fermenter consisting of an agitated tank with thermostatic mantle,
a stirrer and several lines for respiratory gases, Ph regulation agents, nutrient
source etc. and has to be autoclaved prior to reaction.
iv. It must be able to withstand sterilization with overheated steam at 121°C.
v. In the aerobe fermentation, the micro-organism have to be supplied with
respiratory gas in the fermenter by intense aeration system.
 vi. Depending on the heat balance of the reaction sometime huge amounts of heat
have to be removed by cooling registers which are built in the fermenter. vii.
With complex measuring and controlling devices the environmental
conditions within the fermenter of ph, temperature, ionic strength and nutrient
concentration are controlled with high accuracy.
viii. Submerged fermentations are mostly operated in batch processes but can also be
run continuously in certain cases (continuous fermentation). Batch
fermentations may last up to 10 days.

Fig: Structure of a modern fermenter used for submerged fermentation.

Microbial Production of Penicillin- Definition, Biosynthesis, Process, Uses

What is Penicillin?

Alexander Fleming in September 1928 accidentally discovered Penicillin. He

found that the fungus, Penicillium notatum prevented the growth of bacteria,
Staphylococcus spp.. Later Clutterbuck and his colleagues in 1932 studied the
nature of Penicillin and found it as an organic acid that dissolves into the
organic solvent at low PH. Chain et al in 1940 cultured fungus and extracted
powdered form of Penicillin. Later, at the time of the second world war,
Penicillin production was done and an adequate amount was produced to treat
wounded people. P. notatum gave poor results so, other species of Penicillium
were tested. As compared, P. chrysogenum NRRL 1951 gave good results
which were induced by UV and other mutagenic chemicals. These selected
strains produced a huge amount of Penicillin and inhibit the growth of the
Oxford strain of Staphylococcus aureus. Czapek-Dox broth was used for the
culture of P. notatum. Later, casein, beef extract were added for the better yield
of penicillin which will be an aid in production. In 1949 chemically produced
mediums like phenylacetic acid ethyl amine etc by maintaining the PH and
addition of buffering agents like Calcium carbonate and also maintain the
temperature.

Microbial Production of Penicillin. Created with BioRender.com

Types of Penicillin
Two types of Penicillin are Penicillin G (Benzyl Penicillin) and Penicillin F.
Penicillin F is also known as Phentenyl Penicillin. Natural Penicillin are
obtained as sodium or potassium salts. These classes of antibiotics are used in
treating both Gram-positive and Gram-negative infections. Penicillin G are a
narrow-spectrum antibiotic. Examples of Penicillins are Ampicillin, Cloxacillin,
Oxacillin, Piperacillin, etc.
Structure of Penicillin

Chemical structure of the Penicillin

core
The structure of Penicillin includes a 4-membered β-lactam ring and thiazolidine
ring. Β-lactam ring contains an amide bond that is broken in an acidic and
alkaline medium and that bond is hydrolyzed by beta-lactamase which is
synthesized by many bacteria. Naturally occurring penicillins have different
structure which is separated by R groups. The basic structure attaches to the N-
acyl group in the substituted amino group. Mainly, Penicillin is categorized as
natural and semisynthetic.
Fig. Industrial fermenter for penicillin production
 Biosynthesis of Penicillin

Figure: Biosynthesis of penicillin G. Image Source: M A Peñalva et al. 1998.

Penicillins yield is done commercially by using P.chrysogenum. Although the
fungus was found earlier in 1928, these biosynthesis processes were concluded
later. Penicillin biosynthesis is described into three main steps; catalytic step,
oxidative, and exchange of different chains.
1. The catalytic step involves an ACV synthetase enzyme that condenses the
lateral chain of cysteine, valine, and alpha aminoadipate into tripeptide ACV.
2. In the second step, tripeptide ACV forms a bicyclic ring by oxidative ring
closure. Isopenicillin N synthase is involved resulting in isopenicillin N which
is a bioactive intermediate in the pathway.
3. The third step involves the exchange of L-aminoadipate. Acyl-CoA synthetase
and Acyl-CoA racemase, a two enzyme system is involved that helps in
converting isopenicillin N into Penicillin N.

Figure: Penicillin biosynthesis. Image Source: Cacycle.

Penicillin Production Process
 Penicillin production is done by fermentation process in a fermenter by agitating
the culture of P.chrysogenum in a suitable condition. The whole process carried
out is aerobic and the method involved is fed-batch.

Figure: Outline diagram of current penicillin production by fed-batch

submerged fermentation. The top panel shows the procedure for inoculum
development. Image Source: David Moore.
This fermentation process is of Penicillin G which involves the following steps:
1. 100ml medium with spores of P.chrysogenum strains is inoculated in
Erlenmeyer flask and is incubated at BOD incubator by placing them on a
rotatory shaker.
2. After 4 days of incubation, the content along with two liters of medium is
transferred into a flask that contains four liters and again incubates for two
days.
3. Then, the content is transferred into a stainless tank containing 500 ml of the
medium that provides suitable conditions for fungal growth.
4. After three days of incubation, the content is used for inoculation and kept in a
fermentor that is well equipped with optimum conditions.
5. The content is filtered after six days of incubation which contains penicillin.
6. The penicillin is extracted into amyl or butyl acetate and is transferred into an
aqueous solution with phosphate buffer.
7. Acidify the extract and again re-extract penicillin into butyl acetate
8. In the solvent extract potassium acetated is added to a crystallization tank to
crystallize as a potassium salt.
9. Crystals were recovered and further sterilization of salt is done.

Application/Uses of Penicillin
 • Used in treating infections caused by both Gram-positive and Gram-
negative bacteria like respiratory tract infections, throat, mouth, gum, and
urine infections and also used in treating bacterial endocarditis.

VITAMIN B2(RIBOFLAVIN)
 A vitamin is a organic compound and a vital nutrient that an organism
requires in limited amounts.
 It has great value in the growth and metabolism of the living cells.

 Vitamins are mainly founds in food, vegetables.

 Humans can produce some vitamins from precursors they consume.

 The vitamin cannot be stored in the body and a constant intake is

required.
 Thirteen vitamins are universally recognized at present.

 Vitamins can be classified as “Fat soluble vitamins” and “water soluble

vitamins”.

VITAMIN B2 -RIBOFLAVIN
• Yellow orange solid compound.
• Water soluble vitamin hence
cannot be stored in our body.
• Chemical structure-
It consist of dimethylisoalloxazin,
ribityl

Production process of Riboflavin-

 In this case study, a batch process using Bacillus subtilis(bacterium),
Ashbya gossypi(fungus),Candida fomate(yeast).
 It is a fed batch fermentation technique. It is operated with 10% inoculum
ratios.
 Upstream processing consists of preparation of medium and associated
continuous counter-current sterilization.
 Feed components are – 70% glucose syrup and sunflower source(carbon
source), peptone,yeast and malt extract(Nitrogen), guanine and
biotin(micronutrients) sulfuric acid and concentrated salt solution at room
temperature.
 It is mixed together and using classical batch conditions for 121˚c,
20minutes.
 Initially medium’s composition does not allow sterilization of all
components, but latter is sterilized by filtration.
 Inoculum is prepared from slants or spores of particular cultures. After
one or two flask stages, the further inoculum is prepared through one or
two small fermentation tanks.
 The fermentation is carried out at 28°C temperature and at pH 6.8 for 4 to
5 days under aerobic conditions. The excess aeration is not suitable for
the growth of cells because it inhibits mycelia production and reduces the
yield.
 An aeration rate at 0.25 to 0.30 volumes of air per volume of medium per
minute is satisfactory for maximum yield.
 Exhaust gases are filtered by a second filter.

 A small fraction of the harvested broth is put into another tank and is used
as inoculum for the next batch.
 After fermentation the broth is harvested into the harvest tank.

 Part of the product crystallizes in the fermenter and also in the harvesting
tank.
Crystallization is completed in the crystallizer by evaporation of some of the
water.
 Afterwards the suspension is stored in tank.

 From the decanter three streams are harvested, two liquid phases and the
cell/crystal suspension. To achieve higher purity, a washing step is used
with a second separation.
 The last step is drying, either using a spray dryer to obtain a powdered
product or applying a spray granulation to obtain granulate. Granulate can
be dosed more precisely.
 The fermentation progresses through four phases.
 (a) First phase: It is the initial rapid growth phase of Ashbya gossypi. In
this phase, glucose is utilized and decreases the pH due to accumulation
of pyruvic acid.
 (b) Second phase: In the phase, sporulation occurs and this phase is called
as production phase. Ammonia in the medium accumulates (deaminase
activity) and increases the pH.
 (c) Third phase: The third phase is characterized by the synthesis of cell-
bound riboflavin in the form of flavin adenine dinucleotide (FAD) and
flavin mononucleotide (FMN). This phase is accompanied by the rapid
increase in catalase activity and subsequently, cytochromes disappear.
 (d) Fourth phase: The free riboflavin is released into the medium due to
autolysis of
the cells.
Fermentation production of Statin.
What are Statins?
• Statins are drugs prescribed to reduce serum cholesterol levels.

• The first statin to be approved by the FDA, lovastatin, is produces as a

result of the fermentation of Aspergillus terreus(isolated from tropical
soil).
• Statins block the conversion of HMG-CoA reductase to malonic acid in
the mevalonate pathway by competitive inhibition. [HMG CoA
(3hydroxy-3-methylglutaryl coenzyme A) catalyst the rate limiting step in
cholesterol biosynthesis.]
• Natural statins, including lovastatin and mevastatin (compactin) are
produced by direct fungal fermentation.
• Semisynthetic statins, simvastatin and pravastatin are synthesised by
stereoselective reaction of natural statins.
• Different fermentation techniques for statin production includes solid state
fermentation (SSF) and submerged fermentation (SMF).

MOA of Statin and Cholesterol biosynthesis

Fermentation production of statin.

 • Statins are produced as a secondary metabolite from a polyketide pathway.
This pathway is regulated by polyketide synthase genes such as LovB,
LovF and LovD that are responsible for the transcription regulation and
production of these secondary metabolites.
• Statins are produced as secondary metabolite during stress of the fungi.
Acetyl CoA acts as a precursor molecule that plays an important role in
bridging the primary metabolism with the secondary metabolism leading
to the production of various secondary metabolites such as terpenes and
polyketides, including statins.
• Lovastatin is commercially produced by fermentation of Aspergillus
terreus and simvastatin is produced by further chemical treatment of
lovastatin, usually involving direct alkylation

Materials and Methods

Microorganism and Substrate Preparation

The fungal strains A. terreus ATCC 74135 and ATCC 20542 were obtained
from the American Type Culture Collection (ATCC) and maintained on potato
dextrose agar (PDA) slants at 32°C. Subculturing was performed biweekly.
Spore suspensions were prepared using sterile 0.1% Tween-80 solution,
adjusted to 10⁷ spores/mL for inoculation.

Rice straw and oil palm fronds were collected locally, ground to pass through a
6-mesh sieve (~3.4 mm), and oven-dried at 60°C for 48 hours.

Solid-State Fermentation (SSF)

Solid-state fermentations were performed in 500 mL Erlenmeyer flasks

containing 20 g of substrate. Moisture was adjusted to 75% with distilled water
or mineral solution (KH₂PO₄, MgSO₄, CaCl₂, FeSO₄, ZnSO₄) before autoclaving
at 121°C for 15 minutes. Post-cooling, 10% (v/w) of the spore suspension was
added. Fermentations were conducted at 32°C for 8–10 days. Sub-experiments
investigated the effects of nitrogen supplementation (urea, ammonium sulfate,
soybean meal), particle size, pH (5–8), inoculum size (5–15%), moisture
content (50–75%), and temperature (25–42°C).
Extraction and Quantification of Lovastatin Following fermentation, cultures
were dried, and 0.5 g samples were extracted with 15 mL methanol, shaken at
220 rpm for 60 minutes. The extracts were filtered (0.2 μm) and analyzed via
high-performance liquid chromatography (HPLC) equipped with an ODS
column (Agilent, 250 × 4.6 mm, 5 μm). The mobile phase consisted of
acetonitrile:water (70:30, v/v) with 0.5% acetic acid at a flow rate of 1 mL/min.
Detection was at 237 nm, differentiating between lactone and β-hydroxyl forms
of lovastatin.

Scanning Electron Microscopy (SEM):Fungal colonization of the substrates

was visualized using a scanning electron microscope (Philips XL30). Samples
were gold-coated and observed at an accelerating voltage of 15–25 kV.