Recent Progress of Commercially Available Biosensors in China and Their Applications in Fermentation Processes

December 2014

ScienceDirect

Vol. 21 No. 4 73-85

Journal of Northeast Agricultural University (English Edition)

Available online at www.sciencedirect.com

Recent Progress of Commercially Available Biosensors in China and Their Applications in Fermentation Processes Chen Yan1, 2, Feng Dong1, Bi Chun-yuan1, Zhu Si-rong1, and Shi Jian-guo1* 1

Biology Institute of Shandong Academy of Sciences, Key Laboratory for Biosensors of Shandong Province, Jinan 250014, China

2

Institute of Oceanology, Chinese Academy of Science, Qingdao 266071, Shandong, China

Abstract: Biosensors, which are the products of the biotechnology industry, are among the key projects of the 7th, 8th, and 9th Fiveyear Plans of China Science & Technology Developing Programs, respectively, and they play an important role in developing and reforming traditional biotechnology. SBA series biosensor analyzer, as the only one commercial biosensor in China, has attracted lots of attention in the process of information gathering and measurement for biological industry with the development of technology and society. In this paper, we presented an overview of the most important contributions dealing with the monitoring of the biochemical analytes in fermentation processes using SBA series biosensor analyzers in China. Future trends of the biosensor analyzer in China were also mentioned in the last section. Key words: fermentation industry, biosensor analyzer, process monitoring, biochemical analytes, off-line, on-line CLC number: F426

Document code: A

Article ID: 1006-8104(2014)-04-0073-13

Biosensors can have several advantages over tradi-

Introduction

tional analytical techniques when they come to process

In fermentation industries, process safety and product

view, a biosensor is a "compact analytical device

quality are two important issues of interest. Process

sensitive 'recognition' element integrated or associated

monitoring is a widely adopted tool for process safety

with a physio-chemical transducer" (Turner, 2000).

and quality enhancement. An optimized process leads

Biosensors can be used to monitor the presence of

to streamlined performance, reduction in running and

product, either biomass, enzyme, or antibody, or of

material costs and improvements in quality con-trol.

a by-product of the process as an indirect measure

The effective monitoring of the fermentation pro-

of process conditions, which could make monitoring

cess is necessary to develop, optimize and maintain

and control in the fermentation industry accurate and

biological reactors at the maximum efficiency (Kumar,

reproducible. Since the first biosensor, an enzyme-

2011). However, due to the complex nature of fer-

based glucose sensor, was developed by Clark

mentation process, the use of the traditional analytical

and Lyons at the New York Academy of Sciences

techniques is often hindered by the need for expensive

Symposium in 1962, the biosensor field has grown

instrumentation, experienced operators and complex

enormously (Clark and Lyons, 1962). Most notable

sample purification and separation before analyses.

of the commercially available biosensors today is

monitoring and control. From a general point of the

Received 28 September 2014 Supported by the Postdoctoral Innovation Fund of Shandong Province (201303032); the Independent Innovation Projects of Shandong Province (2012CX20505); the National 863 High Technology Project of the Ministry of Science and Technology of China (2012AA021201) Chen Yan (1983-), Ph. D, research assistant, engaged in the research of biosensor. E-mail: [email protected] * Corresponding author. Shi Jian-guo, Ph. D, professor, engaged in the research of biosensor. E-mail: [email protected] http: //publish.neau.edu.cn

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Journal of Northeast Agricultural University (English Edition)

probably the range of the biosensors sold by the

Vol. 21 No. 4 2014

discussed.

Yellow Springs Instrument Company (YSI), and their glucose biosensor was successfully launched commercially in 1975. This was the first of many biosensor-based laboratory analyzers to be built by

Development of Serial SBA Biosensor Analyzers

companies around the world. YSI followed this in

One of the major driving force for the development

1982 with a whole blood lactate analyzer. Since then,

of biosensors is fermentation monitoring and pro-

these products have become the gold standard for

cess control. In China, there are more than 6 000

clinical diagnostic work in hospitals and laboratories.

companies working on traditional fermentation

Nowadays, several kinds of YSI life sciences products,

or the modern biotechnology, being involved in

YSI 1500, YSI 2300, YSI 2700, YSI 2900, YSI 5300,

different industries, such as food, pharmacy, light

YSI 7100, and YSI 8500 biochemistry analyzer, have

manufacturing, environmental protection, and energy.

been found around the globe in research institutions,

Process monitoring during fermentation is widely

hospitals and clinics, athletic training facilities, and

applicable in the field of phyaramaceutical, food

biopharmaceutical companies. These products offer

and beverages and wastewater treatment. Since, the

scientists, technicians, physicians, and clinicians

ability to process monitor has direct relevance in

precise, consistent data for life.

improving performance, quality, productivity, and

　Biosensor in China started relatively late. The first

yield of the process, much attention has been focused

biosensor system applied in China, the Model 23-L

on improving process monitoring tools. Biosensor may

Lactate Analyzer developed by the YSI Corporation,

be a suitable tool for fermentation monitoring, due

was introduced in the late 1980s. However, its high

to their attractive features including relatively simple

price limited its application in China. From then on,

instrumentation, excellent selectivity, low prices,

lots of the researchers did researches on the biosensor

minimal sample preparation and easy automation

system (Jin et al., 1993; Sun et al., 1993; Wang and

of the measurements. Application of biosensor can

Deng, 1993; Hu and Leng, 1995; Hu, 1996; Liu et al.,

provide insight into some of biochemical parameters

1996; Kang et al., 1997; Liu et al., 1997; Liu et al.,

and facilitate decision making. During the past

1997; Qian et al., 1997; Ruan et al., 1997). The

30 years, research and development institutions,

transition from laboratory achievement to the practical

universities, and various industrial sectors have

use of the biosensors is sometimes a complicated

developed biosensors, in which Biology Institute of

process. Although there are lots of laboratory

Shandong Academy of Sciences is the only institute

achievements in China, few of them have been

specializing in researches and applications of the

transformed into practical applications. So far only

industrial biosensors in China. The work in serial SBA

SBA-serial biosensors are widely used in fermentation

biosensor began in 1983, and now more than 10 kinds

process in China.

of biosensors have been developed for measurement

　So, this review mainly focused on the development

for substrates, products and intermediate metabolites.

of SBA biosensor analyzers and their applications in

Comparing to the foreign similar products (YSI-serial

fermentation including production of the starch sugar,

products, Nova Biomedical products), SBA-serial

ion exchange process, optimization of fermentation

biosensor analyzers have advantages such as small

processing, quality control of fermentation products,

volume, simple and convenient operation, low cost, in-

and effective use of biomass waste. In addition, the

time and cheap service.

recent studies on the on-line measurement and control

　Nowadays, several kinds of commercial SBA bio-

system for the fermentation industries were also

sensors are available, which are able to detect bio-

E-mail: [email protected]

Chen Yan et al. Recent Progress of Commercially Available Biosensors in China and Their Applications in Fermentation Processes

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chemical parameters such as glucose, lactate, gluta-

products: glutamate, glucose, or lactate. It has been

mate, lysine, ethanol, starch, dextrin, saccharose,

used to control fermentation, saccharification, and

lactose, saccharifying enzyme, pyruvic acid, and

recovery in more than 90% of the glutamate factories

hypoxanthine. These biosensor analyzers have been

in China, each of which produces more than 10 000

listed in Fig. 1 (Feng, 2003).

tons of glutamate products.

　 Model SBA-30 lactate analyzer, a mono-elect-

　Model SBA-60 on-line quadraelectrode biosensor

rode biosensor, is widely used in physical train-

analyzer system, authenticated by specialists in 1996,

ing and its consumers number more than 140 organi-

can be fitted out simultaneously with four kinds of

zations.

biosensors. It is controlled by a computer, performs

　Model SBA-50 monoelectrode biosensor is a new

controlled dilution of samples, and can be connected

type of SBA-30, produced in 1995. It is more auto-

to the fermenter or the biochemical reaction vessel to

mated compared with SBA-30 and is used to detect

detect a substance on-line.

lactate and also other substances as well, such as

　Model SBA-70 automatic analyzer based on FIA

glucose, by replacing the immobilized-enzyme

and auto-sampling system, can analyze glucose,

membrane and setting multiple parameters.

L-lactate, pyruvic acid, hypoxanthine, and ethanol. It

　Model SBA-40 analyzer, a bielectrode analyzer,

is especially suitable for the measurement of glucose

simultaneously analyzes any two of the following

and lactate simultaneously in whole blood or plasma.

SBA-40 analyzer

SBA-60 on-line analyzer

SBA-50 analyzer

SBA-70 analyzer

SBA-90 analyzer

Fig. 1　Prototypes of analyzers that have been developed

　 Model SBA-90 analyzer, terelectrode analyzer,

status of the system is obtained from the computer

simultaneously analyzes three products: glutamate,

display, including data collection, and parameter

glucose, and lactate. The system operated in interac-

setting. We can also change the status of the system

tive mode with Chinese character menu, and more

and display and print the previous records.

than 3 200 measurement results can be stored in a data

　 At present, these analyzers are widely used in

collector for the future printout or computation. The

enterprises in China, occupying more than 90% of http: //publish.neau.edu.cn

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Vol. 21 No. 4 2014

Journal of Northeast Agricultural University (English Edition)

the market in China. Among them, Model SBA-40

for process monitoring and control of nutrients and

analyzers are used the most widely in biological

byproducts, SBA-serial biosensors with fast, accurate

industry, and now at present there are more than 4 000

results and low cost occupy China market dominant

users.

position continuously. They hold more than 90% market share in China. The main vertical markets for

SBA Biosensor Analyzers Market

SBA biosensors include research laboratories, clinical

China has been a great biotechnological country in the

environmental monitoring and industry control. Fig. 2

world. In China, the demanded amount of biosensor

shows the percent of SBA biosensor users in 2013.

is large and the market of it is vast. The biosensors

From Fig. 2, we can see the applications of the bio-

market is categorized as a growth market with appli-

sensor analyzer for research laboratories and industry

cations increasing with the development of each new

control dominate the market, accounting for 85%,

biosensor. Biosensor developments have resulted in

followed by food analysis and physical fitness train-

standardized equipment, standardized biomolecules,

ing, accounting for 8% and 3%, respectively. Clinical

and standardized test processes for use in drug dis-

detection and environment monitoring only account

covery, biodefense, environmental monitoring, pro-

for 3% and others account for 1%. The market of the

cess industries, and the development of an artificial

biosensor is stabile, and research laboratories and

nose for security applications, such as narcotics and

industry control continue to dominate the market by

explosive material detection (Scheller et al., 1991).

2020 and beyond. In biosensors for fermentation pro-

Although YSI is the market leader in most countries

cess applications, we expect a gradual shift from lab

in the world in providing biochemistry analyzers

tests to on-line biosensors to ensure real-time analysis.

Clinical detection 1% Physical fitness training 3%

Environment monitoring 2%

Food analyses 8%

detection, physical fitness training, food analysis,

Others 1%

Research laboratories 43%

Industry control 42%

Fig. 2　Percent of SBA biosensor users in China in 2013

　 The sale volume of SBA biosensor analyzers in

been sold from 2006 to 2010, currently, there are more

the last 24 years is shown in Fig. 3. From 1996 to

than 5 000 users, including academic institutions,

2000, there were about 400 biosensor analyzers,

enterprises, and other domains. Preliminary analyses

almost triple the number of the biosensor analyzer that

indicated that the number of biosensor analyzers was

appeared from 2000 to 2005. Because the explosion

steadily increasing. By 2015, the total number of

of biotechnology over 2 700 biosensor analyzers had

biosensor analyzers is expected to reach 7 000 or so.

E-mail: [email protected]

Chen Yan et al. Recent Progress of Commercially Available Biosensors in China and Their Applications in Fermentation Processes

3 000

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Biosensor analyzer

Sale volume

2 500 2 000 1 500 1 000 500 0

1991-1995

1996-2000

2001-2005

2006-2010

2011-2015

Years

Fig. 3　Sale volume of biosensor analyzers in last 24 years

biosensor has many advantages and has attracted a

Applications of Single Biosensor Analyzers in Fermentation

lot of attention in saccharification. In recent years,

SBA biosensor analyzers, as one of the products of

glucose in the glucose solution on a dry substrate is

the biotechnology industry, play an important role in

defined as DX value. DX value is always lower than

developing and reforming traditional biotechnology

DE value, and the specific ratio of DX to DE can be

in China. Benefit of the application of the biosensors

used to judge the final quality of the glucose solution,

in biological industry has been obvious. For example,

in fact, which is equal to the reducing sugar value. The

annual total productivity of amino acids in China

concept has been utilized fairly well in some factories

is three million tons, which values about 40 billion

equipped with the glucose biosensor. Zhoukou MSG

Yuan. Biosensors are used in the process optimization,

Factory has successfully used glucose biosensors

fed-batch control and down-stream process, resulting

to control production in the saccharification and

in a 10%-15% increase of the productivity, which

fermentation workshop, and they have developed the

accounts for about four billion Yuan increase value

technique of increasing the quality of glucose solution

with exclusive of the environmental benefit. In the

and the fermentation level.

following, a number of the applications of the bio-

　When fermentation factories use the bienzymatic

sensor analyzer in relation to bioprocesses are over-

method to produce glucose, the amount of gluco-

viewed. These examples clearly illustrate the potential

amylase added must be correctly controlled. So the

of the biosensor analyzer in fermentation process.

enzyme activity must be tested to properly control

the glucose biosensor used in glucose production has made significant progress. The percentage of the

the saccharification process. After utilization in Wuxi Application in production of starch sugar

Xingda Bioengineering Co., the largest enzyme-

The determination and control of the glucose

producng factory in China, the evaluation is as

production in saccharification is very important. For

follows: SBA-50 biosensor analyzer needs only

several decades, saccharification has been controlled

3 min to determine the glucoamylase level, the regular

by the traditional Fehiing's method. However, this

iodometric method needs 60 min for the same task.

method is used to titrate the content of reducing sugar and its result cannot provide fully accurate information

Application in ion exchange process

on the change of the glucose content. The glucose

In ion exchange retrieval, detection of the change of http: //publish.neau.edu.cn

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Journal of Northeast Agricultural University (English Edition)

Vol. 21 No. 4 2014

biochemical composition is the key to increase the

is produced when the substrate, glucose, is presented

total product extraction rate. For example, in 1995, the

above certain concentrations. Accurate measurement

extraction rate of the glutamate in 10 MSG enterprises

of glucose concentration in E. coli fermentation is

in China ranged from 91.76% to 83.90% (according

of great importance for achieving higher product

to a report in the Bulletin of Fermentational Science

concentration. Pan et al. (2011) studied the effects of

and Technology in February, 1996). This means that

carbon source feeding, nitrogen source feeding and

the extraction rate of the glutamate in ion exchange

feeding with pH control to obtain the optimal culture

was very low, about 43.9%-55.9%. Much was

conditions. After culture 24 h under the optimal

wasted and lost. By using the glutamate biosensor,

conditions, the cell dry weight concentration of E. coli

our group has conducted simulated experiments on

increased by 104.7%. It is of great significance for re-

the ion exchange retrieval of an isoelectric liquor

ducing the production cost of the preparing L-phenyla-

supernatant of the glutamate. According to this simu-

lanine by enzyme method, enhancing the production

lated experiment, the retrieval rate in the test is about

efficiency of L-phenylalanine. Chai and Zhang (2011)

90%. We increased the retrieval rate by more than 30%

applied SBA-40 analyzer to determine the optimal

and reduced the loss caused by ineffective circulation

glucose for the growth of the strain LG-65 that has

in the ion-exchange process of the glutamate. The

evoked considerable interest as a large-scale L-gluta-

expenses of acid, alkali and environmental pollution

mine producer. Qin et al. (2011) used SBA biosensor

are also reduced. Hao et al. (2008) used SBA bio-

analyzer to determine L-lactic acid and reducing

sensor analyzer to evaluate the adsorption capacity of

sugar and optimization experiments to investigate the

ion-exchange resins for propionic acid, glucose and

high L-lactic acid production strain of Lactobacillus

amino acids in vitamin (B12 (VB12) fermentation broth.

casei. Under the control of the biosensor, the strain

Results indicated that propionic acid was removed

can accumulate L-lactic acid 188 g • L-1, sugar conver-

from the fermentation broth and the concentration of

sion efficiency more than 90% and the fermentation

VB12 increased from 9.1

mg • L

-1

to 13.1

mg • L

-1

, an

increase of 44%.

period less than 44 h. Jining MSG Factory using lactate biosensor to determine the lactate content in fermentation, established that the optimal lactate

Application in optimization of fermentation

content in the glutamate fermentation process was

processing

0-0.2%. At that point, the turnout of glucose-glutamate

Optimization of the operational procedures in

was the highest. Without proper operation, the

bioprocessing either can concern state variables that

highest content of the lactate in fermentation liquid

should be kept under control at a predetermined set-

was 4.0%, and the glutamate content was only 5.0%,

point or should follow the course of a preset trajectory,

which caused a great loss of the production. Under

or discrete sequential actions. An optimized process

the control of a lactate biosensor, the production rate

leads to streamlined performance, reduction in running

of the glutamate increases by 0.5%, thus increasing

and material costs and improvements in quality

glutamate output by 6.0%.

control. Monitoring biomass or substrates or the

　 Mathematical modeling of the bioprocess data

inducer can provide insight into the feeding strategies

has been discussed in biotechnology for the past 30

for realizing better efficiency from the reactors.

years. They could be applied for the calculation of the

Most applications of SBA analyzer have concerned

optimal feeding profiles and other control strategies. In

optimization of the composition of the growth and

general, related parameters (such as concentration of

production culture media. Many examples can be

substrate and product, temperature, pH, and dissolved

found for production of the stains. Escherichia coli

oxygen) in fermentation are critical for establishing the

E-mail: [email protected]

Chen Yan et al. Recent Progress of Commercially Available Biosensors in China and Their Applications in Fermentation Processes

·79·

mathematical model. The number of model parameters

metabolites and thus optimize the biomass yield.

required, and the ease by which their values may be

Biosensor monitoring in fermentation is increasingly

obtained, will influence the utility of mathematical

preferred as it affects better control of substrate and

models. Lack of suitable methods for parameter

product levels. In China, SBA biosensor analyzers,

determination has proved to be a significant obstacle

as the most popular commercial biosensor, have

for establishing the optimal model. The development

been developed to monitor the levels of substrate

of new and more robust analytical techniques will

and product in fed-batch fermentation. For example,

lead to improved model, and deliver more precise

SBA-40C biosensor analyzer was used for monitoring

estimate of important parameters, such as the growth

the fed-batch feeding of the glucose and results showed

rate and product formation rates. SBA series analyzers

that the optimal initial glucose concentration was

have injected a flow of fresh vitality into model

20 g • L-1 and the fed-batch feeding strategy of adding

establishment. For example, He et al. (2010) reported

22, 24, 24, 24, 24, 22 g • L-1 glucose at intervals of

batch fermentation kinetic model of the glutathione

3 h from 12 h to 27 h of fermentation resulted in the

producing by Saccharomyces cerevisiae. They

best effect (Pan et al., 2010). Sang et al. (2011) used

monitored biomass, glutathione and glucose concentra-

SBA biosensor for determination of residual sugar in

tion to draw the profile of the bacth fermentation

cephalosporin C (CPC) fermentation to control carbon

and establish the dynamic model of the cell growth,

source concentration and DO at adequate levels. Using

product formation and substrate consumption,

this strategy, final CPC concentration and CPC yield

respectively. Liang et al. (2013) used SBA biosensor

reach 35.77 g • L-1 and 13.3%, respectively. The major

analyzer to determine the residual glutamic acid

by-product, de-acetoxycephalosporin (DAOC) and

and sugar content. With MATLAB software, they

DAOC/CPC were only 0.178 g • L-1 and 0.5%. Likewise

proposed the kinetic models for γ-polyglutamic acid

strategies on feeding depending upon the process

and substrate consumption. The results showed that

states and key process variables can be adopted by

the models could provide a reasonable description for

monitoring.

the batch fermentation. Zuo et al. (2013) used YPDF basic medium to simulate sugarcane juice to conduct

Application in quality control of fermentation

ethanol fermentation at different sugar concentrations.

products

Parameters of the fructose and glucose consumption

Fermentation processes are often sensitive to even

process and ethanol production process were deter-

slight changes of the conditions that may result in

mined for curve fitting. Data suggested that lower

unacceptable end-product quality. One of the most

sugar concentration could result in a lower discrepancy

promising ways of the developing rapid and relatively

in fructose and glucose utilization.

inexpensive methods for quality control in fermen-

　Fermentation processes are commonly operated in

tation processes is the use of the biosensor systems. No

fed-batch mode in order to prevent the accumulation

matter which approach of the target product preparation

of toxic substrates or products thus allowing the

is determined, one indispensable step should be taken

achievement of higher product concentrations. Fed-

into account for final isolation and purification of

batch fermentation is superior to conventional

this product. This helps in reducing production of

fermentation when controlling concentrations of

undesirable by-products like ammonia and lactate.

substrates affect the yield or productivity of the

Moreover, effective monitoring coupled with appro-

desired products. In industrial fed-batch cultivations,

priate control strategy will facilitate increase of the pro-

it is often necessary to control substrate concentrations

duction of desired product with high quality. Glucose,

at a low level to prevent the production of overflow

as the simplest form of sugar and the foundation of all http: //publish.neau.edu.cn

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Journal of Northeast Agricultural University (English Edition)

Vol. 21 No. 4 2014

the carbohydrates, is one of the main components in

steeping is the first and the most important step in

fermentation broth. Therefore, determination of such

the milling process, and is also a capital-intensive

analytes (glucose, lactic acid, ethanol and glycerol)

and time-consuming step because it involves grain

in the process of isolation and purification is very

soaking in weak solution of sulphurous acid at

important. Using SBA biosensor analyzer to detect

subgelatinisation temperatures (50-55℃) for 30-55 h.

such analytes content in fermentation broth has the

Reduced steep time decreases energy cost, increases

advantages of rapid response and high precision. For

plant capacity, and reduces the capital cost involved

example, Sun et al. (2009) used glucose to product

in constructing new wet-milling plants. A great

2-keto-D-gluconic acid and applied membrane

number of the investigations have been approached

separation technology to extract fermentation product.

to find a steeping method that may decrease the time

In order to increase productivity and quality of the

of this step. In order to solve the crucial problems of

product, glucose concentration was monitored by

environmental pollution from high concentration of

SBA series biosensor analyzer in this process. In this

sulfite and the long soaking cycle, our group explored

case, the conversion rate of the glucose into 2-keto-D-

a new technology to improve the soaking process.

gluconic acid was as high as 90%. Further monitoring

We employed biosensor analyzer to determine the

enables generating early warning signals based on

contents of the reducing sugar, glucose and lactic acid

observed process deviations and devise appropriate

in corn steep water and presented their variation rule.

control strategies. With these insights, it is possible to

Under the optimum conditions, the soaking cycle was

achieve high productivity e.g. by monitoring glucose

shortened by 42 h, and the amount of SO2 was reduced

and glutamine and maintaining the balance of these

by 0.1% (Feng et al., 2011).

carbon and nitrogen sources, cell cultures can be run

　 Corn pericarp, a waste of industrial corn starch

at optimal conditions. Wu et al. (2010) applied SBA

production, is mainly composed of carbohydrats

biosensor analyzer for sensing residual sugar and

including cellulose, hemicellucose, and residual starch.

studied the complexation extraction of succinic acid

However, it used to be discarded or utilized as animal

in simulated fermentation broth. Liquid ammonia in

feed. Application of hydrolyzed corn pericarp in

glutamate fermentation was a nitrogen source and pH

biology ferment will supply a kind of cheap biomass

conditioner and was added in feeding. In fermentation,

material for industrial fermentation. Determination

ammonium ion might interfere with the determination

of total sugar content and glucose content is very

and produce low data quality. Xing et al. (2011) pro-

important for process optimization of corn pericarp

posed a pH feedback-controlled glucose feeding

hydrolysis. The biosensor detection method has

method to simplify the feeding method for glutamate

less error and offers simpler operation, thus rapid

fermentation. The linear relationship between the

detection of sugar can be achieved. For example,

consumption amounts of ammonia and glucose was

by using SBA biosensor analyzers to determine the

used as the ratio of the amount of ammonia and

glucose and xylose, Zhao et al. (2007) investigated

glucose in the feeding broth. Compared with the con-

the technologies in enzymatic hydrolysis of corncob,

stant feeding method, the concentration of ammonia

ethanol production from cellulosic hydrolysate,

was controlled and the quality and productivity of

simultaneous saccharification and fermentation using

glutamate was enhanced.

a thermotolerant yeast strain and a recombinant yeast. By this technique, the simultaneous conversion of

Application in effective use of biomass waste

cellulose and hemicellulose to ethanol was realized,

Wet-milling is the common process used to produce

which was very meaningful in decreasing the ethanol

corn starch with high quality and yield. Kernel

production cost and increasing the productivity of the

E-mail: [email protected]

Chen Yan et al. Recent Progress of Commercially Available Biosensors in China and Their Applications in Fermentation Processes

ethanol fermentation.

·81·

in order to facilitate rapid optimization and also to control a bioprocess. So, it is crucial to provide solu-

Application of On-line Measurement and Control System in Industrial Fermentation

tions to on-line sensing and optimization technique

The fermentative process is a complicated dynamic

specificity, simplicity and quick response, has also

process with multiple key variables, most of which

attracted increasing scientific interest in on-line moni-

are difficult to be measured in real time. Furthermore,

toring of the fermentation process (Kumar et al.,

the response to changes in the process environment

2001; Akin et al., 2011; Kumar, 2011; Moeller et al.,

is less reproducible making the tasks of the process

2011; Bäcker et al., 2013; Mentana et al., 2013).

development, optimization and scale up difficult. Off-

Although the literature on various biosensors is

line measurement though is useful, causes delay in

extensive, examples of their use for on-line monitoring

process control and hence is generally used for the

are rather limited. So far only Nova Bioprofile Flex

development of mathematical models and process

and YSI 7100 MBS Multiparameter Bioanalytical

development. On-line monitoring of the critical

System are widely used for on-line monitoring of the

metabolites including substrate and product is desirable

bioprocesses around the world (Fig. 4).

Nova Bioprofile Chemistry and cell analyzers

for bio-process to break down the bottleneck on the productivity of the fermentation. 　 In recent years, biosensor, due to its inherent

YSI 7100 MBS Multiparameter Bioanalytical System

Fig. 4　Prototypes of on-line analyzing system that have been fully commercialized

　With a well-established industry in fermentation,

biosensor analyzer. Though biosensor can be appli-

on-line monitoring of the fermentation processs

ed for measurement for substrates, products and

has also gained wide attention in China. Recently,

intermediate metabolites, their use in bioreactors is

many efforts have been focused on the design and

scare owing to drift or fouling. In order to solve

development of on-line analyzing system (Yang et al.,

the problem of the contamination caused by a bio-

2003; Chen and Jin, 2011). Gao et al. (2012) developed

sensor, we drained the sample from the fermenter

the glucose on-line detection system for fermenta-

and diluted it to the concentration range that could

tion process. Glucose concentration was measured

be detected by the biosensor. Nowadays, we have

on-line in the coli fermentation process with the

successfully developed SBA-60 on-line measurement

detection system, and the results exhibited the same

and control system of the fermentation (Feng et al.,

variation trend as the commercial off-line SBA-40E

1998) (Fig. 5). This system consists of the three http: //publish.neau.edu.cn

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Journal of Northeast Agricultural University (English Edition)

Vol. 21 No. 4 2014

parts: (1) biosensors; (2) auto-samplers, which is

reactors with different biosensing configurations.

used for sampling purposes and is installed outside

Weifang Shengtai Pharmaceutical Co., Ltd., is the first

the reactor; (3) a signal transmission lines and

to apply this on-line measurement and control system

computing devices to implement the feed-back

in large-scale fermentation. They used a large-scale

control. Compared with Nova Bioprofile Flex and

fermenter together with SBA-60 on-line analyzing

YSI 7100 MBS, SBA-60 on-line analyzing system

system and pH automatic analyzer to keep oceanic

possesses strong anti-interference ability, which can

red yeast fermentation in an optimal condition. In

be used directly in industrial environments. The

the fermentation process, the amount of the glucose

application of SBA-60 on-line analyzing system is

decreased continuously and amount of the oceanic

discussed below:

red yeast increased continuously. The dynamic

　Simulating production with a small-scale fermenter

change of biochemical substances in oceanic red yeast

parallels to a large scale fermenter to resolve produc-

fermentation process are shown in Fig. 7. Under the

tion problems and explores new technological condi-

control of this on-line system, the cost of red yeast

tions. We used a small-scale fermenter together with

was reduced, and the profits increased. Calculated on

SBA-60 on-line analyzing system and pH automatic

eight tons fermentation tank, the production capacity

analyzer to simulate a fermentation test. The total

of red yeast hyperchromic feed would increase

initial 6 000 mL of the fermentation liquor was put

by 120 kg. Calculated on the prices of 100/kg,

into the small-scale fermenter, the target product in

increased sales income would be 12 000 Yuan. They

fermentation was glutamate and the by-product was

also applied this on-line system in Saccharomyces

lactate. In the fermentation process, the amount of

boulardii fermentation process. Calculated on eight

glucose decreased continuously and 1 075 mL of 30%

tons fermentation tank, the production capacity of

glucose was added in the middle of the fermentation.

Saccharomyces boulardii feed would increase by 80

At the end of the fermentation, 4 190 mL remained.

kg. Calculated on the prices of 400/kg, increased sales

The concentration of the glutamate, glucose, and

income would be 32 000 Yuan. Applying this on-line

lactate were 9.26%, 0.38% and 0.085%, respectively.

system in yeast fermentation process optimization

The fermentation data are shown in Fig. 6.

has increased the production and solved the pollution

　SBA-60 on-line analyzing system can be integrated

problems of by-product, which has considerable

to laboratory fermentors as well as industrial bio-

economic and social benefits.

Fig. 5　On-line measurement and control system for industrial fermentation process E-mail: [email protected]

Chen Yan et al. Recent Progress of Commercially Available Biosensors in China and Their Applications in Fermentation Processes

derivative components, chemical additives added to

16 14

the fermentor, and gases such as oxygen and other

Glucose   

12

 

 

metabolic gases, is a very complex soup or solution. 

10

Content (%)

·83·



8



  

 

6



4





 

 

 









   

  

   

0 0

5

10







YSI's newest multiparameter bioanalytical system



offers simultaneous multiparameter measurement





          

more likely are we to understand our bioprocesses.





2

The more (useful) measurements we can make the

Glutamic acid





for enhanced productivity. For example, YSI 7100



        

Lactic acid

15

20

25

30

35

MBS offers multiparameter measurement of up to six distinct parameters at one time, but SBA-serial

40

Time (h)

Fig. 6　Dynamic change of biochemical substances in glutamic acid fermentation process

biosensors offer no more than four biochemistry parameters. In order to dominate Chinese industry biosensor market for a long time, we have sought to exploit multi-biosensors which can measure many

OD

120

of the biosensors, chemical sensors and physical







         

 



    

Lactate (%)







   









pH

80



 

     





Glucose (%)

4

metabolites simultaneously. Second, realizing online measurement and control of bioprocess by integration

Ethanol (%)

0

                                 

–10

0

10

20 30 Time (h)

40

Value

Value



12 8

160



DO



16

40

0 50

Fig. 7　Dynamic change of biochemical substances in oceanic red yeast fermentation

sensors. The complex dynamics of bioprocesses require automated tools for having a greater insight into the process in realtime. Indentification of phase states in brocesses and predicting the direction of the biosynthesis require knowledge of biochemical as well as physical and chemical variables. At present, we just realized the integration of SBA-60 biosensor with pH automatic analyzer. So the work need to be focused for integration of the biosensors, chemical sensors and

　At present, on-line operation and control of fer-

physical sensors for bioprocess on-line measurement.

mentation is challenging. SBA-60 on-line analyzing

Third, constructing intelligent bioprocess operation

system is continuously improved and the connection

model by combination of the biosensors, IT and the

of a large-scale fermentation to biosensors still needs

internet of the things. With the intelligent bioprocess

to be studied.

operation model, process development could be enhanced, quality control and safety of the bioreactor

Conclusions and Prospects

production be improved and process control be realized more efficiently.

China biological industry is growing fast and has been put into the national strategic emerging

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