Measuring the
fermentation broth of
NADH photoelectric sensor
量測發酵液之NADH光電感測器
1
Presenter: Shih-Chieh Chao
Adviser: Dr. Hung-Chi Yang
Chairman: Dr. Hung-Chi Yang
Date:05.08.2013
Outline
 Background
 Introduction
 Purpose
 Methods & Materials
 Experiment
 Future works
 Reference
2
Background
 Fluorescent luminous phenomenon is a light cooling. When
some kind of substance at room temperature by a certain
wavelength of the incident light (usually ultraviolet or Xray) radiation, absorb light energy into excited states and
decay and issued by the outgoing light.
 Human tissue containing many fluorescent substances,
including collagen, NADH, FAD, rhodopsin, will be
excited by UV fluorescence.
3
Background
 Human tissue in the cancer process, will produce a change
in the structure, such as: the cells become less dense
connective tissue collagen tissue to reduce; metabolism of
cancer cells than normal cells, so that the increased NADH;
caused by increased blood flow absorbed rhodopsin rise.
Therefore, the fluorescence spectrum of the cancer tissue
and normal tissue will be different, and performance in the
fluorescence spectral peak position and strength of the ebb
and flow.
4
Introduction
 NADH(煙醯胺腺嘌呤二核苷酸) NADH is a widespread
variety of animals, plants and humans in living cells,
natural substances , and is a very important compound, was
not discovered until 1905, usually with many enzymes in
the body co-participate in the reaction, usually called
"coenzyme“.
5
Introduction
 Cell growth and energy metabolism in the cells are required
to NADH, food into energy process requires NADH
participate, and in the cell to produce energy in the process,
NADH plays the important role of the portable electronic,
in addition to NADH is also a very important the
antioxidants can protect cells from damage of the harmful
substances.
6
Introduction
 NADH Excitation light of 340 ± 20nm.
7
Introduction
8
UV-A
UV-B
UV-C
Wavelength
400～315nm
315～280nm
280～200nm
Nociceptive
weak
medium
strong
Introduction
 The most important function of the Fermenter, is to make
the microorganisms in the growth conditions it needs to
reach the desired proliferation.
 Therefore the design and production of the fermentation
tank you need to have a lot of considerations.
9
Introduction
 Need to maintain long-aseptic operation.
 Microbial metabolism of aeration and agitation, but can not






10
give microbial mechanical shock stirring intensity.
Need to configure the device temperature system Imperial.
Need to be able to control the pH.
Need to be able to sample.
Must be appropriate size.
Internally to maintain a smooth inner surface, not have
welded junction.
By fermentation of a variety of different, you need to install
the necessary equipment incidental.
Introduction
11
Purpose
 This study focused on :
 The sensor can be fully inserted fermenter
 Continuous and real-time performance measurement
12
Purpose
PC
Detector
MCU Control
450 nm
LED
340 nm
13
Broth
+
fungus
Purpose
14
Methods & Materials
 Solidwork
15
Methods & Materials
 Type 304—the most common grade; the classic 18/8 stainless steel.
Outside of the US it is commonly known as "A2 stainless steel”.
 Type 316—the second most common grade (after 304); for food and
surgical stainless steel uses; alloy addition of molybdenum prevents
specific forms of corrosion. It is also known as marine grade stainless
steel due to its increased resistance to chloride corrosion compared to
type 304. 316 is often used for building nuclear reprocessing plants.
16
Methods & Materials
17
Methods & Materials
LED
Detector
18
Methods & Materials
19
Methods & Materials
20
Methods & Materials
20mm
44mm
12mm
10mm
10mm
8mm
20mm
40mm
44mm
40mm
21
Methods & Materials
22
Methods & Materials
23
Methods & Materials
26mm
30mm
18mm
14mm
14mm
26mm
30mm
24
26mm
Methods & Materials
 Excitation light ： NSHU551B
25
Methods & Materials
 Kupo：UV Transmission
stut
%T
100
90
80
70
60
50
40
30
20
10
0
300
26
400
500
600
nm
Methods & Materials
 NADH emit light of 450±20nm， so the selection of the
received light 320-730nm.
 Hamamatsu：S1133
spectral response range
27
320~730nm
Methods & Materials
 Kupo：C11715
stut
%T
100
90
80
70
60
50
40
30
20
10
0
300
28
400
500
600
nm
Methods & Materials
 Kupo：UV Blocking
stut
%T
100
90
80
70
60
50
40
30
20
10
0
300
29
400
500
600
nm
Methods & Materials
 UV Blocking+C11715
stut
%T
100
stut
90
%T
100
80
90
80
70
70
60
60
50
50
40
40
30
30
20
20
10
0
10
30
300
400
500
0
300
400
500
600
600
nm
nm
Experiment
31
Experiment
32
Experiment
33
Experiment
34
Experiment
 Fluorescein, 90+% L13251
 Formula: ：C20H12O5
 Molecular weight :332.32
 Orange powder
35
Experiment
 Deployment method, beginning first with a 1-to-1 water
diluted to 47.5% alcohol with 95% alcohol(AOH)
36
5mL AOH(47.5%)
5mL fluoresein(1mM)
0.5mM
5ml AOH(47.5%)
5mL fluoresein(0.5mM)
0.25mM
5ml AOH(47.5%)
5mL fluoresein(0.25mM)
0.125mM
5ml AOH(47.5%)
5mL fluoresein(0.125mM)
0.0625mM
Experiment
37
Experiment
38
Experiment
 Sigma N8129 ：
 Reduced coenzyme
 Yellowish white powder
 Stored at -20 ℃
 Excitation light = 340 nm。
 Emit light = 460 nm。
39
Experiment
 Fluorescence Spectrometer F-7000
40
Future works
 Circuit design
 30F4011
 Labview
41
Reference
[1] Y-C Liu, F-S Wang, W-C Lee, “ On-line monitoring and controlling system for fermentation processes. “ Biochemical
Engineering Journal 7 (2001) 17–25.
[2] Monici M. (2005). "Cell and tissue autofluorescence research and diagnostic
applications".BiotechnolAnnu.Rev.11:227–56.doi:10.1016/S1387-2656(05)11007-2. PMID 16216779.
[3] Georgakoudi I, Jacobson BC, Müller MG, Sheets EE, Badizadegan K, Carr-Locke DL, Crum CP, Boone CW, Dasari
RR, Van Dam J, Feld MS (2002-02-01). "NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of
epithelial precancerous changes". Cancer Res. 62 (3): 682–687. PMID 11830520
[4] Windholz, Martha (1983). The Merck Index: an encyclopedia of chemicals, drugs, and biologicals (10th ed.). Rahway
NJ, US: Merck. p. 909. ISBN 911910271 Check |isbn= value (help).
[5] Bakker BM, Overkamp KM, van Maris AJ, et al. (2001). "Stoichiometry and
compartmentation of NADH
metabolism in Saccharomyces cerevisiae". FEMS Microbiol. Rev. 25 (1): 15–37.
[6] Nicholls DG; Ferguson SJ (2002). Bioenergetics 3 (1st ed.). Academic Press. ISBN 0-12-518121-3.
[7] Sistare FD, Haynes RC (15 October 1985). "The interaction between the cytosolic pyridine nucleotide redox potential
and gluconeogenesis from lactate/pyruvate in isolated rat hepatocytes. Implications for investigations of hormone
action". J. Biol. Chem. 260 (23): 12748–53. PMID 4044607.
[8] Freitag A, Bock E (1990). "Energy conservation in Nitrobacter". FEMS MicrobiologyLetters 66 (1–3): 157–62.
doi:10.1111/j.1574-6968.1990.tb03989.x.
[9] Starkenburg SR, Chain PS, Sayavedra-Soto LA, et al. (2006). "Genome Sequence of the Chemolithoautotrophic NitriteOxidizing Bacterium Nitrobacter winogradskyi Nb-255". Appl. Environ. Microbiol. 72 (3): 2050–63.
doi:10.1128/AEM.72.3.2050-2063.2006. PMC 1393235. PMID 16517654.
[10] Dawson, R. Ben (1985). Data for biochemical research (3rd ed.). Oxford: Clarendon Press. p. 122. ISBN 0-19855358-7.
[11] 曾百由 編著 “數位訊號控制器原理與應用”，洪有圖書開發股份有限公司，九十三年十一月
[12] 庭田企業有限公司，網址：http://www.dichroic.com.tw/cn/index.htm。
[13] 日亞化學株式會社，網址：http://www.led-shop.com.tw/index.htm。
42
The End
43