Optimization of Operating Conditions of Increasing HBsAg Protein Expression in FED BATCH Fermentation Process by Changing Pichia Pastoris Culture Medium Conditions and Examining Growth of Yeast Cells by Methanol Testing

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Optimization of Operating Conditions of Increasing

HBsAg Protein Expression in FED BATCH

Fermentation Process by Changing Pichia Pastoris

Culture Medium Conditions and Examining Growth

of Yeast Cells by Methanol Testing

Payam Moradi Zalam Abadi , Alireza Fazlali , Seyed Nezamedin Hosseini , Ehsan Jafarbeigi ¹^,⁴,

Farhad Salimi *

Department of Chemical Engineering, Kermanshah Branch, Islamic Azad University, kermanshah, Iran

Department of Chemical Engineering, Faculty of Engineering, Arak University, Arak, Iran

Production & Research Complex, Pasteur Institute of Iran, Tehran, Iran

Young Researchers and Elite Club, Kermanshah Branch, Islamic Azad University, kermanshah, Iran

Received: 16/12/2019

Accepted: 06/04/2020

Published: 20/05/2020

Abstract

Optimization of the culture medium and induction conditions in the fed-batch fermentor is the commonest and easiest method to

increase the overall productivity of recombinant proteins production. Environmental conditions such as temperature, dissolved oxygen

(DO), PH, and agitation also have a major impact on the expression of recombinant protein. Since the production of the recombinant

protein in pichia pastoris is highly affected by induction conditions, providing induction conditions is one of the most important ways to

increase the productivity rate. AOX1 gene enables recombinant proteins expression at the highest level in methylotrophic yeasts. This gene

is activated by methanol and induced protein expression. Low and high methanol amounts respectively lead to its reduced induction ability

and overproduction of formaldehyde and other toxic substances. However, given that carbon is considered as microorganisms feed,

therefore, the injection of pure methanol in a timely manner in sufficient quantities in specified time intervals, the necessary amounts of

carbon were supplied for feeding them. Also, vitamins such as vitamin A and B were regularly injected to the extent necessary in the

fermentation process. Therefore, this study aimed to investigate the methanol feeding process, an increase in specific growth rate (µ), OD

and dry weight (WW), comparison of the increase in OD and WW. The results showed that the performance of the feeding profile is

improved as much as possible according to the existing facilities.

Keywords: Pichia pastoris; Methanol utilization pathway; Fed-batch fermentation; Recombinant protein expression; Genome annotation

Introduction¹

(

1970) could observe this virus in the blood of those suffering

from this disease. Continuous experiments showed that HBsAg

can stimulate the body's protective system to secrete antibodies.

In 1971, Krugman discovered that the injection of blood infected

with killed HBV can stimulate hepatitis B antibody (5). It was

also proposed that nucleic acid fragments separated by

Bloomberg have the ability to stimulate the immune system

against this virus (6). Blumberg and Millman provided ground

for the development and advancement in making the vaccine, in

which HBsAg can initiate the body's protective response to this

disease. So, they proposed that this vaccine to be prepared from

the antigen in the blood of infected people. The strategy for

vaccine design was first to use the blood infected with the

weakened virus to stimulate the immune system. But, it soon

became apparent that the use of antigen purified from the

infected blood is more effective and safer. Although Fox Chase

Cancer Centre (FCCC) was awarded the patent for this

discovery in 1969, many scientists have carried out extensive

Hepatitis B virus (HBV) is the leading cause of cirrhosis and

liver cancer and HBV infection is now a global problem (1-4).

In a research on various serum proteins in 1963, Bloomberg

accidentally discovered an unknown protein in the blood of an

Australian native. This protein was introduced as Australian

(AU) antigen and it was found that this protein is related to

HBV. In 1968, Prince Okohi and Murakami found that AU

antigen existed in the blood of people infected with hepatitis B

only in the same year. Bloomberg could isolate some amount of

hepatitis B surface antigen (HBsAg) in the blood of an infected

person and continued research revealed that this antigen is the

main symptom for the incidence of this disease. Dane et al.

Corresponding Auther: Farhad Salimi, Department of

Chemical Engineering, Kermanshah Branch, Islamic Azad

University, kermanshah, Iran. Email: f.salimi@iauksh.ac.ir.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

researches on this concept and were able to purify it and enable

its public use. This vaccine provided the immunity rate of more

than 90% without causing any side effects (7). Router et al.

yeasts. Pichia pastoris was first identified as a high-performance

system in producing this antigen. In the first experiment, the

production rate reached to 48 mgHBsAg.L-1 culture 1 (12). At

the industrial scale, a change in the cell structure and use of

alcohol oxidase (AOX) gene promoter reduced the production

rate to 1.3mgHBsAg.L-1 culture. Phillips Co. signed a contract

with Salk Institute Biotechnology / Industrial Associates. Inc.

(1977) proposed making HBV vaccine through recombinant

technology. This production process could provide safe

abundant product. The first system was carried out by bacterial

cells, but experiments yielded no success (8). Before the

development of recombinant technology, the hepatitis B vaccine

was produced by extraction of hepatitis B surface antigen

(SIBIA) for the development of pichia pastoris as

microorganism for the expression of heterologous proteins in

1980. Researchers isolated AOX1 promoter in SIBIA and used it

as the promoter gene in manipulated pichia pastoris. AOX1 can

increase the total extract soluble protein up to 30% and methanol

grows merely in pichia pastoris (13-15). AOX1 enzyme is

responsible for the methanol of the first oxidation reaction in

pichia pastoris.It is synthesized by AOX1 promoter, which has

been widely used in the expression of recombinant proteins in

this microorganism (16-18). This expression system is widely

used for the expression of heterologous proteins (38). The

advantages of pichia pastoris compared to the saccharomyces

cerevisiae yeast include:

1. Pichia pastoris has a very active promoter for methanol

utilization and its oxidation to formaldehyde and hydrogen

peroxide and this activity is carried out by AOX enzyme.

2. The expression of this gene is highly regulated. In other

words, when it uses glucose or ethanol as the carbon source,

AOX is inactive. But when the yeast is cultured in medium

containing methanol, AOX can increase total cell proteins by

3% to 5%. There are 2 AOX genes, including AOX1 and AOX

2. AOX2 plays an inhibitory role for the expression.

(HBsAg), the main ingredient of hepatitis B vaccine, from the

plasma of infected patients (34, 37). Afterwards, Hall developed

this system by yeasts and Valenzuela et al. (1982) (7) managed

to achieve the first purified HBsAg product by recombinant

genetic system using yeasts. Finally, after nine years of research,

the first vaccine was made in 1986 and the US Food and Drug

Administration (FDA) issued the permission for its general use.

After a short while, French scientists were able to make the

same proteins that had the same ability to stimulate the immune

system. In this system, the recombinant plasmid was transferred

into ovary cells of a kind of Chinese mountain hamster mice.

This plasmid was a little different from the yeast plasmid

because it contained surplus of HBV genome. This means that in

addition to protein S gene, it also contained Pre-S2 protein gene.

Although this system was different from the yeast, it contained

same 22nm protein fragments that stimulated the immune

system.

.2 Saccharomyces cerevisiae

The first recombinant HBV vaccines, which can be

produced commercially, were made through genetic

manipulation of the yeasts. Normally after being made in

eukaryotic cells, proteins often enter the Golgi system to

undergo changes in their structure. For this reason, eukaryotic

cells such as yeast are used to produce the recombinant HBV

vaccine. A variety of yeasts that are used in the production of

recombinant HBV vaccine include: saccharomyces cerevisiae

and pichia pastoris. There were certain limitations associated

with the use of saccharomyces cerevisiae to produce HBV

vaccine, because its total protein efficiency (TPE) is 1% to 5%.

Also, placing a transgene in the yeast causes tensions in host

cells, and the produced foreign protein is considered toxic for

saccharomyces cerevisiae in many cases. Currently, P. pastoris

has attracted major attention in the healthcare industry for

efficient largescale production of recombinant products (34-36).

The first plasmid, in which hepatitis B antigen is produced under

the control of alcohol dehydrogenase gene promoter (ADH1),

was transformed in saccharomyces cerevisiae by Valenzuela et

al. This system has yielded an expression rate of about 10-25

μgHBsAg.L-1 culture(8). Later, the expression of this gene

reached to about 50 μgHBsAg.L-1 culture in saccharomyces

under the control of 3-phosphoglycerate kinase gene promoter

3. The use of methanol for this yeast leads to creation of a

special characteristics compared to other yeasts because

saccharomyces cerevisiae and most of other yeasts lack this

ability. Also, this yeast's ability to grow in the methanol can be

used to prevent contamination of the fermentation medium

during fermentation. Considering reasons mentioned above,

today, pichia pastoris has become a more suitable candidate for

use in the genetic engineering. To achieve an optimal strategy to

achieve the highest amount of recombinant protein production,

factors such as temperature, PH, specific growth rate of yeast

physiology, culture conditions and feed composition percentage

have been investigated.. Factors such as temperature have been

investigated frequently. Pichia pastoris fermentation process is

carried out in three ways: 1) Glycerol batch phase

2)Transformation phase (from development to production) 3)

Methanol induction phase and the induction phase was

investigated among the three above-mentioned phases.

1.3 Glycerol batch phase

The purpose of this phase is to obtain the maximum amount

of yeast in the shortest time. The highest µ in glycerol (1/h) has

been reported to be 0.18(19) that is much higher in comparison

with that of methanol (1 / h) (0.14) (20). Usually µ is much less

than this amount in methanol phase that is due to the production

of protein and negative impact of its metabolic burden on the

growth. This phase begins with glycerol concentration of 40g / L

independently of the type of pichia pastoris. Glycerol with

higher concentration acts as an inhibitor. Brierley et al. proposed

6% as the maximum amount for glycerol concentration (21).

Scientists measured the ethanol amount as about 2% in glycerol

concentration of above 7%. This ethanol concentration is toxic

to the yeast. The dry biomass obtained from this amount of

substrate is approximately 0.5. The amount of dry weight cells

(PGK) (9). Miyanohara et al. achieved expression rate of 2.8

μgHBsAg.L-1 culture under the control of acid phosphatase

gene promoter (10). Under the control of glyceraldehyde-3-

phosphate gene promoter (GPD), S gene expression rate reached

μgHBsAg.L-1 culture and about 20 culture mgHBsAg.L- 1 (6-

fold increase) in saccharomyces cerevisiae using the batch

fermentor and the batch-fed fermentor, respectively (11). The

fact that the cell can secrete the protein out of the cells is very

important. Chen et al. made a tremendous effort on this issue,

but it was useless (9). Due to problems associated with the

saccharomyces cerevisia use, researchers used methylotrophic

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

obtained at the end of this phase is 100g / L. The end of this

phase is characterized by a sudden increase in DO, which is due

to the end of substrate and lack of oxygen consumption.

by stopping the feeding process. The most important indirect

control devices include Gas chromatography (GC), High

Performance Liquid Chromatography (HPLC) and enzymatic

reactants. In this method, we can measure the concentration of

other substances and even protein. In case of limited methanol

concentration, DO response is rapid and methanol feed can be

increased. This indirect measurement should be continued until

the DO amount reaches the desired minimum level. Then

.4Transfer phase

The biomass amount needed to be increased until achieving

high amount of cell mass. This is one of the objectives of this

phase to the extent that the presence of glycerol does not prevent

methanol from entering the induction phase. Some researchers

have added glycerol with a fixed amount to the culture medium

and some others have added glycerol exponentially. In this

method, the dry weight of cells can be increased even to more

than 120g/L. This phase is usually completed with a gradual

increase in methanol level. In this method, the cells become

ready to enter the induction phase. Different methanol feeding

programs have been reported. (a) Gradual increase in methanol

in proportion to the DO amount (20). (b) Maintaining the

methanol concentration of about 4g / L (22). (c) Inducing 1hour

hunger before entering the induction phase to ensure that all the

available glycerol is consumed (23). (d) Adopting glycerol

reduction program along with fixed glycerol feed rates (24).

methanol feeding rate is maintained at

a constant rate.

Sometimes a feedback control loop is used to maintain DO level.

But it is practically unusable because the sampling and testing

take a long time. Therefore, direct methods are usually used.

1.6 Fermentation

Fermentation is a process that is carried out by using energy

released from the oxidation of organic materials such as

carbohydrates. In industry, fermentation refers to so-called

chemical reactions, the catalyst of which is microorganisms,

whether aerobic or anaerobic process.

1.7 Fermentor

Fermentor is a device, in which fermentation operation is

carried out. Fermentor used in this research, was Vessel single

jacket model fermentor with Winpact brand. Fig 1 shows a view

of the fermentor and other parts of it.

.5 Methanol induction phase

Pichia pastoris starts producing the recombinant protein

under the control of AOX promoter in this phase. Specific

growth rate (µ) has a major impact on the amount of protein

expression, thus feeding must carried out in such way that we

achieve the maximum expression rate. It is very important to

control the concentration level because its high levels and cause

toxicity and its low levels prevent protein expression. Induction

with methanol is a very important phase, because AOX activity

is low at the beginning and the methanol accumulates easily. At

this stage, the oxygen consumption rate is limited with rapid

increase in AOX activity. Pichia pastoris strains are sensitive to

high methanol concentrations remained in the medium and

sudden methanol accumulation decreases AOX activity and

leads to cell death. Methanol is controlled using direct or

indirect measurements. Methanol direct measurement in this

process is an important tool in optimizing feeding strategies. A

methanol sensor with silicone tube is commercially available in

the market for the direct measurement of methanol (25). Gas

analyzer is a unique method that is based on the measurement of

oxygen and carbon dioxide leaving the fermentor. In this

method, other output gases such as ammonium may create a

nuisance, but it can be solved with a modification(26). This

device can be used to control methanol concentration feedback

and in methanol and temperature-limited fermentation processes.

When the necessary equipment is not available for direct

control, another strategy is used to adjust the methanol

concentration that is called DO spike method. In this feeding

method, methanol concentration is adjusted in such way that the

methanol steams are prevented from being exited, which is

detected through a sudden increase in DO (27). This method has

its own defects, too. If an inhibitor stops the process, DO level

rises, as a result of which the injected methanol level increases,

which in turn leads to methanol accumulation and cell death.

Some direct methods are based on the indirect method. For

example, in sequential injection analysis, seven samples are

taken and analyzed per hour. But the disadvantages of this

method include an increase in the likelihood of contamination

and yield loss due to continuous sampling (28). Methanol

accumulation control is also feasible using indirect method, i.e.

1.8 Conventional methanol feeding methods in batch culture

1.8.1 Pichia pastoris yeast

Methylotrophic yeast pichia pastoris is a suitable host for

production of recombinant proteins. One advantage of this host

is, in fact, having a very effective regulation promoter called

AOX. Pichia pastoris uses methanol as a source of carbon and

energy. Methanol is also considered as an inducer in this system.

AOX enzyme is a catalase that decomposes hydrogen peroxide

to water and oxygen. Some of the formaldehyde produced by

AOX leave the peroxisome and is oxidized mainly to carbon

dioxide and formate. This reaction occurs with cytoplasmic

dehydrogenase that provides the energy source for cell growth

on methanol. High cell density culture with pichia pastoris,

AOX promoter enables achieving production of large quantities

of recombinant protein. However, due to the accumulation of

formaldehyde and hydrogen peroxide in the cell through

methanol metabolism, methanol concentration in the fermentor

is effective both on cell growth and level of protein secreted by

cells. Therefore, proper adjustment of methanol concentration in

pichia pastoris fermentation is necessary not only to maintain the

induction of genes under the control of the AOX promoter, but

also to prevent the accumulation of excessive methanol

concentration in the medium sites in the centrifuge.

1.8.2 Methanol consumption cycle

Methylotrophic yeasts belong to four species: Hansenula,

Pichia, Candida, tropicalis, all of which have methanol

consumption cycle. The main reactions in this cycle are shown

in Fig. 2. To grow in methanol, the yeast is adapted by induction

of AOX gene promoter, dihydroxyacetone synthase (DAS) and

many different enzymes that can participate in this metabolism

(29). In these species, AOX can produce more than 35% of the

cellular protein, while this gene is inactive in glucose and

glycerol or ethanol. AOX oxidizes methanol in the first step and

then converts it to formaldehyde and hydrogen peroxide.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

Figure 1: A view of the used fermentor

This reaction is performed in an organ, called peroxisome to

prevent the toxicity of hydrogen peroxide in the reaction (30).

Peroxisomal catalase breaks down hydrogen peroxide to water

and oxygen. Formaldehyde enters the cytosol from peroxisome,

where it forms a complex with the regenerated glutathione.

Then, it is oxidized and carbon dioxide is produced by the

dehydrogenation reaction in two stages. Formaldehyde-GSH

complex is converted to formate by formaldehyde

dehydrogenase enzyme and formate is oxidized and is converted

to carbon dioxide by formate dehydrogenase enzyme. Oxidation

in this cycle not only produces energy in the form of NADH, but

also plays a very important role in eliminating formaldehyde

toxicity in the methylotrophic yeast (31). Formaldehyde can then

be re converted to methanol by formaldehyde reductase enzyme.

Formaldehyde reductase and alcohol oxidase enzymes create a

cycle that controls the cellular amount of formaldehyde and

NADH. The residual formaldehyde reacts with xylose -5-

phosphate (Xu5P) through transketolase reaction and produces

two products of Dihydroxytryptamine acetone (DHA) and

glyceraldehyde - 3-phosphate (GAP). This reaction is carried out

by Dihydroxyacetone synthase enzyme. Dihydroxyacetone

synthase enters the cytosol and is phosphorylated by

Dihydroxyacetone kinase enzyme. It is later converted to

fructose-1, 6-bisphosphate (FBP) after reacting with

glyceraldehyde-3-phosphate in the aldolase reaction and is

finally converted to fructose 6-phosphate (F6P) by 8-phosphate

phosphatase. F6P enters the pentose phosphate pathway and

reproduces xylose phosphate. During each of three cycles, one

glyceraldehyde - 3 - phosphate molecule is produced and the

biomass is formed by the standard gluconeogenesis reaction.

Fig. 3 shows a simple metabolism shape in methylotrophic

yeasts (14). Tri-carboxylic acid cycle (TCA) plays an important

role in energy supply for the growth of methylotrophic yeasts.

Materials and method

Required materials and equipment have been listed in Tables

and 2. Fig. 4 shows the algorithm of how to implement the

project.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

Figure 2: Pichia pastoris metabolism cycle in methanol. 1, alcohol oxidase; 2, catalase; 3, formaldehyde dehydrogenase; 4, formate dehydrogenase, 5,

dihydroxyacetone synthase; 6, dihydroxyacetone kinase; 7, fructose 1,6-biphosphate aldolase; 8, fructose 1,6-bisphosphatase

Figure 3: Pichia pastoris simplified metabolism cycle in methanol (14)

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

.1 Used materials

In this study deionized water (hardness of less than 1

9. Beginning the fermentation process and continuing it until

inhibition of cell growth.

microsiemens), extract of microbiological yeast, agarose,

sodium chloride, calcium chloride dehydrate, methanol, crystal

violet, EDTA, glycerol, di-sodium hydrogen phosphate

anhydrous, agarose, diethyl barbituric acid, diethylbarbituric

acid sodium, caprylic acid, sodium acetate dehydrate, copper

sulfate heptahydrate, ammonium sulfate, manganese sulfate

heptahydrate, zinc sulfate monohydrate, iron sulfate

heptahydrate, potassium chloride, potassium hydrogen

phosphate, potassium iodide, acrylamide, bis acrylamide,

ammonium sulfate, bromophenol blue, acetic acid monohydrate,

standard molecular weight marker, and pichia pastoris yeast

have been used.

10. Harvest

11. Biomass centrifugation and collection

Table 2: Required nutrients and design of medium

Vitamin A & B

Di-Potassium hydrogen phosphate

Biotin

Myo-inositol

Calcium Pantothenate

Pyridoxol Hydrocholoride

Thiamine Hydrocholoride

Nicotinic Acid

Concentration

4 g/L

0.8 g/L

8 g/L

0.8 g/L

0.2 g/L

W.F.I

.2 Needed equipment

In this study Eppendorph centrifuge model 5804, peristaltic

.4 Determining cell dry weights

pump model VERDERLAB (2 pcs), Nikone optical microscope,

glass beads with a diameter of 0.5 micron, pH meter model

Mettler Toledo, Eppendorph spectrophotometer, IKA horizontal

stirrer, IKA horizontal shaker, Eppendorph 37 ° C incubator,

Eppendorph 30 ° C incubator, Emersan 2 to 8 ° C refrigerator,

REVCO -20 ° C freezer, IKA Vortex, microbial hood, elisa

reader, glass utensils, magnet, pence, syringes, dowel, millipore

filter paper Wharman GB002, WinPact Glass 10 liters

fermentor, and oxygen capsule have been employed.

First, four clean 15 ml falcons, two of which are named A

and B are prepared. The falcons are later weighed using a

sensitive scale. The yeast mixture is taken to the extent

necessary and 5 ml of the mixture is added to each of A and B

falcon. Then they will be placed in two opposite Mixtures are

later centrifuged at 22 °C for 15 minutes at 3000 rpm. The

supernatant is poured into another falcon and kept into the

freezer so that it is used later to measure methanol. After being

dried, the falcons are weighed again. If we multiply the primary

and secondary weight difference by 200, the dry weight will be

obtained.

Table 1: Required nutrients and design of medium

Medium

NH So

MgSO

HPO

CaCl

Concentration

.5 Expression under the control AOX1 gene promoter

Heterologous proteins are often produced in the P. pastoris

(

)

20.31 g/L

6.92 g/L

,7H₂O

yeast under the control of very effective AOX1 promoter. This

promoter controls the expression of the AOX1 enzyme. For the

first time, Ellis et al. (1985) were able to isolate AOX1 gene. P.

pastoris genome consists of two alcohol oxidase genes (AOX1,

AOX2). AOX1 has the greatest responsibility in regulating

alcohol oxidase activity in cells. This gene produces high levels

of AOX1 enzyme in cells (more than 30%); therefore, this

promoter can cause expression of recombinant proteins higher

than 12 g / L (32). Considering the ability their methanol

consumption rates, yeasts are divided into three sub-groups:

16.77 g/L

0.46 g/L

8.8 g/L

1 g/L

,2H

%85

Trace element

Glycerol

W.F.I

40 g/L

2 lit

.3 Working method

. Preparation of 4.5 lit complex medium (including different

Positive methanol consumption (Mut ): Both AOX1 and

types of proteins, lipids and carbohydrates)

AOX2 genes are present and have good efficiency and yeast

grows well in methanol.

. Pouring the medium into the fermentor vessel.

. Blocking all fermentor container openings by cotton, foil and

Slow consumption of methanol (Mut ): AOX1 gene is

clamp.

. Autoclaving the fermentor container along with its contents

for 30min.

. Connecting feed, air, sensors, and temperature and pH probes

damaged and AOX2 gene only works, but the resulting slows

methanol consumption depends on the poor transcriptional

properties of AOX2.

Negative methanol consumption (Mut ): Both AOX1 and

to the fermentor container after autoclaving under complete

sterile conditions.

AOX2 genes are inactive, methanol cannot be metabolized, but

its presence is necessary for induction of recombinant proteins

expression. The maximum specific growth rates in methanol are

. Setting feed, temperature, rotational speed of the blades and

pH parameters by fermentor monitor.

. Inoculating 0.5 lit of inoculated culture medium by siphon

and peristaltic pump in sterile conditions

. Injection of Vitamin A, Vitamin B, Trace Element

-1 -1 -1 + s

.14h , 0.04h and 0.0 H , respectively for Mut , Mut and

Mut species of P. pastoris yeasts (33). Figures 5, 6, 7, 8 and 9

respectively show methanol feeding process, an increase in µ,

OD, dry weight (ww), comparison of an increase in OD and ww.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

Revision

Yes

Evaluation

ofselected

parameter

Testing in

lab scale

Data analysis

Parameter

selection

(theory)

Effective

Next analysis

Ineffective

Testing in large

scale

Yes

Validation tests

Completion

Figure 4: Algorithm of how to implement the project

.6 Fed-batch culture (the induction phase)

Fed-batch culture is a method of choice to achieve high cell

pastoris yeast, methanol is considered as an inductor for the

expression of recombinant protein and energy source for cells.

Accumulation of methanol in the medium is considered as a

growth and production inhibitor. On the other hand, its

deficiency reduces the growth rate and increases the process

time. Each of limited methanol feed rate, fixed-rate and

exponential feeding, feeding strategy based on the growth rate,

and etc. attempts to improve the production of proteins. The

exponentially methanol feeding strategy was used in Pasteur

Institute in such a way that the specific growth rate was almost

at about fixed rate of 0.016. The medium conditions of the

process were almost unchanged and only the amount of input

methanol was changed. Medium pH was controlled at 4.5 and

density. Implementing fed batch fermentation process, despite

its numerous advantages, due to problems such as restriction or

inhibition of raw materials (especially carbon source) required

for yeast growth, high-speed production of CO , heat, excessive

need for oxygen and limited capacity to transfer oxygen, a sharp

increase in viscosity and reduced mixing efficiency, plasmid

instability and reduced yield in case of prolonged process,

product degradation by intracellular proteases, accessory

metabolite accumulation to the inhibition level, has always been

associated with challenges. Considering its ease of

implementation and possibility of changing the growth rate of

the cell, the exponential feeding method is a method that is more

commonly used for the implementation of high cell density

culture of various microorganisms. Research has shown that the

specific productivity in high cell density culture is usually less

than the "fed-batch culture that is probably due to the fact that

most fed-batch processes are implemented under low specific

growth rate and the limited substrate level conditions. At this

stage of high cell density culture (induction phase) of Pichia

since CO gas is produced in this process, the medium pH

decreases and ammonia is used to control it. In addition to

regulating pH, ammonia is also considered as the nitrogen

source. The needed oxygen was first supplied by the compressed

air and Due to the impossibility of increasing the pressure in the

fermentor and atmospheric nature of the process, oxygen

deficiency was evident. Therefore, pure oxygen capsules were

used at this stage. Agitation plays a fundamental role in mass

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

transfer and increasing oxygen time both in terms of creating

dispersion and breaking apart the oxygen bubbles. At the

beginning of the process, the agitation was set at 250 rpm and

finally was set at 600 rpm due to the increased viscosity of the

solution. Medium temperature was controlled unchanged at 30

at the end of fermentation for different batches before

optimization.

600

200

800

°C. Temperature control system was comprised of a heat

exchanger and a pump that circulates the water in the pipe inside

the fermentor.

Results and discussion

As can be seen in Fig.5, Since the yeast is inadaptable with

the change in type of carbon source (carbon source was changed

to glycerol source) in the early hours of its growth, Methanol is

injected slowly into the fermentor and the increasing trend of

methanol consumption rises with a more gentle slope. In this

phase, the yeast needs time to metabolize the methanol, and then

methanol consumption begins at higher rate after the yeast is

adapted to the medium change. DO suddenly undergo a sharp

drop at this time that indicates a high consumption of methanol

and increased fermentation speed.

2000

4000

6000

8000

Time (min)

Figure 5: Methanol consumption rate during fermentation

As shown in Fig.6, the growth rate in the early hours has a

negative value due to the yeast inadaptability. But the growth

rate was increased quickly and then maintained at a constant

level. Higher specific growth rate was observed in the

fermentation process until 80th hour and its level was later

decreased because of the increased concentration of the

fermentation and higher level of toxic substances or oxygen

deficiency. Fig. 7 shows an increase in dry weight of the cells

compared with fermentation time. This increase reflects the

successful growth of the cells. In the final fermentation hours, in

addition to the lack of ascending growth in the diagram, a

decrease is observed in the dry weight as the fermentation time

passes, which indicates the end of the logarithmic growth phase

and entering the stationary phase and eventually the completion

of fermentation process. In Fig. 8, according to samples taken

from the fermentor during the fermentation process, the number

of cells was measured using turbidimetry. The obtained data

indicate an increase in the number of cells in the logarithmic

phase of growth. Considering the direct relationship between the

bacteria growth and OD, the OD level was increased and with

beginning of the stationary or death stage, the OD graph

undergoes reduction and recession in line with the bacteria

growth. Fig. 9 shows a comparison of the two parameters of OD

and ww after in terms of fermentation time and we see that these

parameters have a direct relationship with each other.

.025

.015

0.005

-10 10 30 50 70 90 110 130 150

0.005

0.015

0.025

Fermentation hour (hr)

Figure 6: Trend of specific growth rate increase (μ)

Conclusion

In this research, considering the transfer of technology from

the production sector (production and research complex of

Pasteur Institute of Iran), to achieve the amount of biomass

produced in elapsed specified time, numerous experiments were

carried out and their results have been reported in Table 1. As is

evident, much time has been spent in some of the experiments

and amount of the produced biomass has been increased. As

presented in Table 3, the average ratio of methanol to biomass

amount is 1.14, i.e. 89% and 11% of methanol has been

converted to biomass and energy, respectively. It should be

noted that methanol purity grade is 79%. Fig. 10 shows the

results obtained from the different consumption methanol rates

100

120

Time (hr)

Figure 7: Relationship between the dry weight and fermentation time

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

Table 3: Methanol consumption rate and biomass specifications for different batches at the end of fermentation

Methanol

consumption (L)

Time (h)

w.w (g/l)

Biomass (kg)

Methanol consumption/biomass (L/Kg)

292

270

349

280

350

296

290

310

1.54

1.50

1.74

1.57

1.77

1.67

1.66

1.81

2.03

2.15

2.28

2.32

2.40

2.5

1.13

1.04

1.18

1.04

1.24

1.07

1.17

1.96

2.61

2.73

(

avoidance of guest authorship), dual submission, manipulation

of figures, competing interests and compliance with policies on

research ethics. Authors adhere to publication requirements that

submitted work is original and has not been published elsewhere

in any language.

Competing interests

The authors declare that there is no conflict of interest that

would prejudice the impartiality of this scientific work.

Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analyses and manuscript writing.

100

150

Time (hr)

Figure 8: Relationship between OD and fermentation time

600

400

200

130

135

140

145

150

155

Time (hr)

Figure 10: Pre-optimization methanol consumption rate diagram for

different batches

Wet Weight (g/l)

References

Gerlich WH. Medical Virology of Hepatitis B: how it began and

where we are now. Virology Journal. 2013;10(1):239.

120

2. Jing M, Wang J, Zhu S, Ao F, Wang L, Han T, et al. Development

of a more efficient hepatitis B virus vaccine by targeting hepatitis B

virus preS to dendritic cells. Vaccine. 2016;34(4):516-22.

Time (hr)

Kosinska AD, Zhang E, Johrden L, Liu J, Seiz PL, Zhang X, et al.

Combination of DNA prime–adenovirus boost immunization with

entecavir elicits sustained control of chronic hepatitis B in the

woodchuck model. PLoS pathogens. 2013;9(6):e1003391.

Tsai S, Chen P-J, Lai M, Yang P, Sung J, Huang J-H, et al. Acute

exacerbations of chronic type B hepatitis are accompanied by

increased T cell responses to hepatitis B core and e antigens.

Implications for hepatitis B e antigen seroconversion. The Journal of

clinical investigation. 1992;89(1):87-96.

Figure 9: Comparison of dry weight with OD

Acknowledgement

This research was supported by Pasteur Institute of Iran

IPI), Tehran, Iran.

(

Ethical issue

Authors are aware of, and comply with, best practice in

publication ethics specifically with regard to authorship

Blumberg BS. Hepatitis B virus, the vaccine, and the control of

primary cancer of the liver. Proceedings of the National Academy of

Sciences. 1997;94(14):7121-5.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 2, Pages: 708-717

Szmuness W, Stevens CE, Zang EA, Harley EJ, Kellner A. A

controlled clinical trial of the efficacy of the hepatitis B vaccine

21. Curvers S, Brixius P, Klauser T, Thömmes J, Weuster‐Botz D,

Takors R, et al. Human Chymotrypsinogen B Production with

Pichiapastoris by Integrated Development of Fermentation and

Downstream Processing. Part 1. Fermentation. Biotechnology

progress. 2001;17(3):495-502.

22. Chen Y, Krol J, Cino J, Freedman D, White C, Komives E.

Continuous production of thrombomodulin from a Pichia pastoris

fermentation. Journal of Chemical Technology & Biotechnology.

1996;67(2):143-8.

23. Cos O, Serrano A, Montesinos JL, Ferrer P, Cregg JM, Valero F.

Combined effect of the methanol utilization (Mut) phenotype and

gene dosage on recombinant protein production in Pichia pastoris

fed-batch cultures. Journal of Biotechnology. 2005;116(4):321-35.

24. Surribas A, Cos O, Montesinos J, Valero F. On-line monitoring of

the methanol concentration in Pichia pastoris cultures producing an

heterologous lipase by sequential injection analysis. Biotechnology

letters. 2003;25(21):1795-800.

25. Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Tomomitsu

K. High level secretion of recombinant human serum albumin by

fed-batch fermentation of the methylotrophic yeast, Pichia pastoris,

based on optimal methanol feeding strategy. Journal of Bioscience

and Bioengineering. 2000;90(3):280-8.

26. Lim H-K, Choi S-J, Kim K-Y, Jung K-H. Dissolved-oxygen-stat

controlling two variables for methanol induction of rGuamerin in

Pichia pastoris and its application to repeated fed-batch. Applied

microbiology and biotechnology. 2003;62(4):342-8.

27. Jennings VM. Review of Selected Adjuvants Used in Antibody

Production. ILAR Journal. 1995;37(3):119-25.

28. Ramon R, Feliu J, Cos O, Montesinos J, Berthet F, Valero F.

Improving the monitoring of methanol concentration during high

cell density fermentation of Pichia pastoris. Biotechnology letters.

2004;26(18):1447-52.

(Heptavax B): a final report. Hepatology. 1981;1(5):377-85.

Valenzuela P, Medina A, Rutter WJ, Ammerer G, Hall BD.

Synthesis and assembly of hepatitis B virus surface antigen particles

in yeast. Nature. 1982;298(5872):347-50.

Hitzeman RA, Chen CY, Hagie FE, Patzer EJ, Liu CC, Estell DA, et

al. Expression of hepatitis B virus surface antigen in yeast. Nucleic

acids research. 1983;11(9):2745-63.

Miyanohara A, Toh-e A, Nozaki C, Hamada F, Ohtomo N,

Matsubara K. Expression of hepatitis B surface antigen gene in

yeast. Proceedings of the National Academy of Sciences.

983;80(1):1-5.

0. Hsieh JH, Shih KY, Kung HF, Shiang M, Lee LY, Meng MH, et al.

Controlled fed‐batch fermentation of recombinant Saccharomyces

cerevisiae to produce hepatitis B surface antigen. Biotechnology and

bioengineering. 1988;32(3):334-40.

1. Cregg JM, Tschopp JF, Stillman C, Siegel R, Akong M, Craig WS,

et al. High–Level Expression and Efficient Assembly of Hepatitis B

Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris.

Bio/Technology. 1987;5(5):479-85.

2. Ogata K, Nishikawa H, Ohsugi M. A yeast capable of utilizing

methanol. Agricultural and biological chemistry. 1969;33(10):1519-

3. Couderc R, Baratti J. Oxidation of Methanol by the Yeast, Pichia

pastoris. Purification and Properties of the Alcohol Oxidase.

Agricultural and Biological Chemistry. 1980;44(10):2279-89.

4. Hartner FS, Glieder A. Regulation of methanol utilisation pathway

genes in yeasts. Microbial cell factories. 2006;5(1):39.

5. Macauley‐Patrick S, Fazenda ML, McNeil B, Harvey LM.

Heterologous protein production using the Pichia pastoris

expression system. Yeast. 2005;22(4):249-70.

6. Cayetano-Cruz M, Pérez de los Santos AI, García-Huante Y,

Santiago-Hernández A, Pavón-Orozco P, López y López VE, et al.

High level expression of a recombinant xylanase by Pichia pastoris

cultured in a bioreactor with methanol as the sole carbon source:

Purification and biochemical characterization of the enzyme.

Biochemical Engineering Journal. 2016;112:161-9.

29. Egli T, Van Dijken J, Veenhuis M, Harder W, Fiechter A. Methanol

metabolism in yeasts: regulation of the synthesis of catabolic

enzymes. Archives of microbiology. 1980;124(2-3):115-21.

30. Lee B, Yurimoto H, Sakai Y, Kato N. Physiological role of the

glutathione-dependent formaldehyde dehydrogenase in the

methylotrophic

yeast

Candida

boidiniib.

Microbiology.

7. Kim S, Warburton S, Boldogh I, Svensson C, Pon L, d’Anjou M, et

2002;148(9):2697-704.

al. Regulation of alcohol oxidase

peroxisome biogenesis in different fermentation processes in Pichia

pastoris. Journal of Biotechnology. 2013;166(4):174-81.

(AOX1) promoter and

31. Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA.

Functional characterization of the two alcohol oxidase genes from

the yeast Pichia pastoris. Molecular and Cellular Biology.

1989;9(3):1316-23.

32. Stratton J, Chiruvolu V, Meagher M. High Cell-Density

Fermentation. In: Higgins DR, Cregg JM, editors. Pichia Protocols.

Totowa, NJ: Humana Press; 1998. p. 107-20.

33. Jahic M, Wallberg F, Bollok M, Garcia P, Enfors S-O. Temperature

limited fed-batch technique for control of proteolysis in Pichia

pastoris bioreactor cultures. Microbial Cell Factories. 2003;2(1):6.

8. Vogl T, Glieder A. Regulation of Pichia pastoris promoters and its

consequences for protein production. New Biotechnology.

013;30(4):385-404.

9. Brierley R, Bussineau C, Kosson R, Melton A, Siegel R.

Fermentation development of recombinant Pichia pastoris

expressing the heterologous gene: bovine lysozyme. Annals of the

New York Academy of Sciences. 1990;589(1):350-62.

0. Brierley RA, Davis GR, Holtz GC. Production of insulin-like

growth factor-1 in methylotrophic yeast cells. Google Patents; 1994.