基礎生物技術通論

Environmental
Biotechnology
環保生物技術

黃啟裕 , Ph.D.

東海大學 環境科學工程系
環境微生物實驗室

http://140.128.122.97
 Objectives of the Class
Environmental Biotechnology not just
bioremediation!
Practical understanding of biotechnology as
it relates to environmental science
Develop a critical eye to understand,
evaluate and use biotechnology

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 Reference books
Barnum, S. R., 2005. Chap. 5: Microbial
biotechnology, in “Biotechnology”. Thomson
Brooks/Cole, U.S.A.
Rittmann, B. E and McCarty, P.L. 2001.
Environmental Biotechnology: principles and
applications. McGraw Hill, U.S.A.
鍾竺均、陳偉。環境生物技術， in “ 生物技
術概論” ， pp106~138 。新文京出版社。
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Journal Examples

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Review Journal Examples

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Trade Journal - Magazine

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生物技術 (Biotechnology)

利用生物所得到對人類有益之產品與新
技術、新製程。 using living organisms or
their products for commercial purposes
生物：動、植物及微生物。
應用於農業、醫藥、工業、食品與環境
保護
環境生物技術 (Environmental
Biotechnology)
• 歐洲生物技術協會 (European Federation of
Biotechnology) 之定義，「環境生物技術」是指生
物化學、微生物學及工程技術相結合之整合性科
學。主要目的是利用微生物、動物或植物應用於
農業、環境及工業上，以發展永續事業。
• Using human developed biological processes or
products to impact the environment, in a positive way.
• Public awareness of environmental problem issues:
 Chernobyl, Bhopal, Exxon Valdez, Greenhouse
gases

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 Why Biotechnology?
 Biotechnology approaches are replacing /
augmenting physical/chemical technology due to:
Higher specificity
Lower temperature, pressure
Less energy
Less waste products
Less harmful end products

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 Environmental Biotechnology
• Application of all components of biological
science (mostly Microbiology and
Biochemistry) and engineering science
(especially Chemical and Environmental
engineering) to environmental problems.

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What “Basic Skills” are Required?
1. Basic Understanding of Microbiology
2. Basic Understanding of Biochemistry
3. Quantitative understanding of microbial growth and
metabolism
4. Quantitative understanding of biological reactions and
reactors (Rates vs. Yields ； that is, HOW FAST a process
works and HOW EFFICIENTLY it works)
Ability to make relevant design calculations (e.g. reactor
size, inflow rate, hydraulic retention time etc)
Ability to combine 1-4 above to quantitatively understand
existing and new biological metabolisms and processes
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 What is Microbiology?
Micro - too small to be seen with the naked
eye
Bio - life
ology - study of
The science of microorganisms (very small,
unicellular organisms)

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 微生物 (Microorganism)
細胞構造簡單、形體微小 (nm ~ m) 、肉眼看不見
之生物  光學或電子顯微鏡觀察
無組織分化之單細胞或多細胞生物  原生物界
種類：原生動物 (Protozoa) 、黴菌 (Fungi) 、
藻類 (Algae) 、細菌 (Bacteria) 、病毒
(Virus)
依細胞結構：
真核生物 (eucaryotes): Protozoa 、 Fungi 、
Algae
原核生物 (procaryotes): Bacteria
其他 : Virus

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 Bacteria Algae

Virus

Protozoa Fungi

http://news.bbc.co.uk/2/hi/health/3419537.stm 16
 原核細胞 vs. 真核細胞

http://www.microscopy.fsu.edu/cells/procaryotes/images/ http://micro.magnet.fsu.edu/cells/animals/images/animalcell.jpg
procaryote.jpg
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 微生物分類地位
Haeckel (1866) Whittaker (1969)
三界分類法 五界分類法

 動物界 (Animalia)  動物界 (Animalia)

 植物界 (Plantae)  植物界 (Plantae)

 原生生物界 (Protista)  無核生物界 (Monera)

真菌 (fungi) 細菌 (bacteria) ，藍綠細菌
藻類 (algae) (cyanlbacteria)

原生動物 (protozoa)  原生生物界 (Protista)

細菌 (bacteria) 藻類 (algae) ， 原生動物 (protozoa)

 真菌界 (Fungi)
酵母 (yeast) ， 黴菌 (mold)
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 Whittaker’s 5 kingdoms
真菌界
吸收
動物界
植物界
消
光 化
合

原生生物界 ( 真核
單細胞 )
藻類、原生動物

無核生物界 ; 原核生物界
細菌、藍綠菌

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Carl Woese (1990), 分子生物分類法
Based on small ribosomal ribonucleic acid gene (16S rRNA
gene; 16S rDNA)
 3 Domains ( 域 , 超界 superkingdom)
1. Archaea 太古細菌類 : archaebacteria
2. Bacteria 真細菌類 : eubacteria
3. Eukarya 真核生物類

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Impact of Microorganisms on Humans

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 Microorganisms, Energy, and the
Environment
The role of microbes in biofuels production
• e.g., methane, ethanol, hydrogen

The role of microbes in cleaning up pollutants

(bioremediation 生物復育 )

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環保生技應用領域
Biological treatment of pollution 污染處理、去
除 : Removal or reduction of pollution from
current processes

Bioremediation of existed polluted area 汙染場

址復育 Cleanup of Superfund sites, oil spills

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 環保生物技術應用領
域
Environmental Monitoring 環境監測 : Indicator,
Ames test, Microtox, Biosensors, ELISA
(Enzyme-linked immunosorbent assay), PCR
Prevention of pollution 汙染防制 (clean
technology, Green technology) microbial removal
of S compounds from coal, fungal pretreatment of
logs before pulp and paper production,
biodegradable plastic, biofuels
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Mechanisms of Biological Treatment

• Use the microorganisms to degrade the

pollutants (Biodegradation)
• Biodegradation 生物降解 : a general term
referring to the microbially mediated
decomposition of compounds (natural or
synthetic)
Reduction or removal of current wastes
Biodegradation
The natural process in which microorganisms
(bacteria, fungi) are able to completely or
partially break down organic compounds to CO 2
+ H2O or other simple organic molecules that are
inert or are readily metabolized by other
organisms.
Mineralization 礦化作用
The degradation process is carried to the
extreme, in which organic compounds are
biodegraded to inorganic compounds.

有機化合物經微生物作用下，轉化為無機
化合物過程的總稱。
Biodegradation of Problem Environmental
Contaminants

Pesticides
Hydrocarbons
BTEX, MTBE
Poly-Aromatic Hydrocarbons (PAH’s)
Chlorinated Solvents
Halogenated Aliphatic Hydrocarbons
Polychlorinated Biphenyls (PCB)
Explosives

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Biological Waste Treatment
• One of the earliest (late 19th ~ early 20th century)
acceptable engineering concepts for wastewater
treatment was Dilution by dumping wastes into a
large body of water (lake, ocean) or a fast-moving
receiving stream (stream, river)
 No longer a tenable philosophy today.
• Today, biological/chemical treatment of waste
effluents before discharge
 Control of waste before dumping.
WASTE TREATMET HAS TWO
PURPOSES
1. To lower the organic load
lower the BOD in order to decrease the effect on the
dissolved oxygen (DO).
BOD 生化需氧量 = Biochemical Oxygen Demand
The amount of oxygen (mg/L) required by bacteria
in order to oxidize decomposable organic matter
under aerobic conditions.
2. To kill off pathogens
cholera ( 霍亂 ), typhoid ( 傷寒 ),
paratyphoid ( 副傷寒 ), dysentery ( 痢
疾 ), hepatitis ( 肝炎 ), liver flukes ( 肝
吸蟲 ), polio ( 脊髓灰質炎 ),
enterovirus ( 腸病毒 ) diseases.
The best-known application of
environmental biotechnology to date is
biological wastewater treatment
(domestic and industrial wastewater)

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Biological wastewater treatments
a. 好氧 (Aerobic process): activated
sludge, oxidation pond, rotary
biological contactor (RBC)
b. 厭氧 (Anaerobic process): anaerobic
digestion, denitrification.
Aerobic vs. Anaerobic wastewater treatment
Aerobic processes: much more efficient than anaerobic
processes for energy conversion of starting substrates into cell
material
 faster reaction rate
 ADVANTAGE of aerobic processes
- O2 as the terminal e- acceptor, the standard free energy
change for the oxidation of glucose completely to CO2 and
H2O is – 686 Kcal/mole, 45% of which is conserve in the
form of ATP.
-  Can produce more cells (vs. anaerobic one)
 larger biomass
 higher volume of sludge
 DISADVANTAGE of aerobic processes
Anaerobic processes: obtain very little of this available substrate
(glucose) energy for growth.
 lower growth rate
 smaller biomass
 less amount of sludge produced
 ADVANTAGE 1 of anaerobic processes
- Most of the energy (ca 90%) remains in the fermentation
products (volatile fatty acids, acetate, methanol, ethanol, etc.)
these fermentation products are may be converted entirely to
CH4and CO2, which release 4-5% more of the substrate energy.
85% of original energy is still bound up in the endproducts
CH4!  ADVANTAGE 2
 DISADVANTAGEs of anaerobic processes
(1) long retention time
(2) fragility of the system
(3) odors (very bad smelling) due to VFAs, H2S
Biological
wastewater
treatment:
activated-
sludge process

- an aerobic, cell-
suspended process

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Activated sludge 活性污泥

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Activated sludge
under microscope
Sludge drying bed
Land application of dried sludge
Anoxic Sludge Digester
台北八里污水處理廠蛋型消化槽 ( 六座 ): 造價 51 億元
民國八十三年完工，從未驗收發揮功能
固定化生物膜 (Biofilm)

生物濃度高
污泥量少
抗毒性高
利於生長緩慢之微生物生長
Trickling Filter 滴濾池 , Rotary Biological
Contactor (RBC) ), Fluidized bed reactor
 Biofilm
A layer of microbes attached and proliferated on the
surface of an object.

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 Trickling Filter
An aerobic, attached-growth wastewater treatment processes
 Rotary Biological Contactor

An aerobic, attached-growth wastewater treatment processes

primary
sedimentation clarifier
inflow outlet

sludge return

primary sludge
Gujer 2000

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 Bioremediation
The process of cleaning up contaminated sites (soil,
beach…) using microorganisms to remove or degrade
toxic wastes or other pollutants (crude oil, refined
petroleum products, heavy metals…..)
Ex situ vs. in situ

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 EXXON
VALDEZ
oil spill
(1989)

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 Exxon Valdez tanker accident

The microbes were not added to the site, but were native
bacteria. A nutrient solution was sprayed onto the oil to
encourage the growth of oil-degrading microbes. This was
one of the most successful bioremediation tried to clean up
the oil 65
 Heavy metals

Removal of toxic heavy metals from water or

soil relies on adsorption 吸附 of metals to
microbial cells or plants (Phytoremediation
植生復育 ) or transformation of toxic metal
wastes by microroganisms.

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 微生物分析技術
Culture-dependent techniques: cultivation approach
 ENTEROTUBE II
 API 20E system
 BIOLOG microstation system
Nonculture-dependent techniques: molecular approach
- Restriction-enzyme fingerprint
- Denaturing gradient gel electrophoresis (DGGE)
- Temperature gradient gel electrophoresis (TGGE)
- Fluorescent in-situ hybridization (FISH)
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優缺點：
Culture-dependent techniques
選擇性 (Biased results)
耗時
可得到純化分離之特殊菌種，適合基礎研究
Nonculture-dependent techniques
分解細胞
DNA 粹取 ( 不需分離培養 )
PCR 過程
探針穿透細胞壁
誤差較小，時效較佳 ( 族群組成及特性研究 )
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Isolation of Pure Culture

The “Great Plate Count” Anomaly

環境樣品 Culturability (%)

海水 0.001 – 0.1
淡水 0.25

土壤 0.3
活性污泥 1 - 15
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 Environmental
Samples
Cells
isolated
Direct observation
using fluorescent
probes Isolated DNA

Colonies fixed to a Specific genes

membrane, lysed and amplified by PCR
DNA denatured

Probed using dot blot

DNA analyses using

The DNA hybridized with probes labeled RFLP or DGGE
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with P32, biotin or fluorescent dyes
Biological removal of nitrogenous compounds
from wastewater

含氮化合物對環境之影響
水體優養化 ( 臨界濃度 : 0.3 ppm)
高濃度之含氮化物會對人體及水體生態造
成危害
亞硝酸鹽易使六個月以下嬰兒得到藍嬰症
Eutrophication results in
Algal blooms 藻華現象
Fish kills
Taste & odor problems
Clogged filters
Natural, but accelerated
by humans
Need to lessen nutrients
availability
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Nitrogen Removal from wastewater

氮之型態 處理方式
Organic-N 活性碳吸附。生物合成轉換為較易處理
之氮型態。

NH3-N 離子交換法。氣提法。折點加氯法。生
物脫氮法 (Nitrification)

NO3-N 離子交換法。生物脫氮 (Denitrification)

 除氮方法中以生物處理法之效率最高，
應用也最廣 。

優點：
反應效率高、系統穩定、操作容易、成本低
。
缺點：
容易受到環境 (pH 、溫度、其他有毒物質
) 的影響。
 脫氮基本原理簡介
N2(g)
大氣
N2O(g) Nitrogen fixation

NO3-(aq) Nitrite reduction

Nitrate reduction
(denitrification)
NH4 +(aq)
(denitrification)

Nitrite oxidation Ammonia oxidation

(nitrification) NO2 (aq)
-
(nitrification)

水體 Anaerobic ammonification
 Ammonia-Oxidizing Bacteria
Nitrification Nitrosomonas,
硝化反應 Nitrosococcus, Nitrosovibrio,
Nitrosospira, Nitrosolobus.

 3 AOB 
NH  O 2   NO 2  2H  H 2 O
4
-

2
 1 NOB
NO 2  O 2   NO3-

Nitrite-Oxidizing
Bacteria Nitrobacter,
Nitrococcus
 Denitrification
NO3-

NO2- NO2-
Plant,
microorganisms
NO microorganisms

N2O

NH3 N2
Assimilatory nitrate reduction
Dissimilatory nitrate reduction
(Denitrification)
 NH3(g)
KH
NH2-groups of
Ammonification Ka
protein (organic N) NH3(aq) NH4+(aq)
2 [H] + O2
AMO
H2 O
HAO
NO2 (aq)
-
NH2OH(aq)
2 [H] + O2 2 H2 O [H] + O2 (hydroxylamine)
H2 O
NO3-(aq) nar nir S or nir K
NO2 (aq)
-
NO (g)
2 [H] H2 O [H] + H+ H2 O
nosZ nor
N2 (g) N2O (g)
 Real-time PCR for monitoring
ammonia oxidizing bacteria
NH3(aq)
amoA
H2 O

NO2-(aq) NH2OH(aq)
2 H2 O [H] + O2 (hydroxylamine)

Standard AOB strain: Nitrosomonas europeae C-31

Ammonia monooxygenase gene partial fragment: amo A
利用氨氮氧化酵素基因﹙ amoA﹚ 偵測
氨氮氧化菌群
Ammonia monooxygenase A (amoA) primers for PCR
(nucleotide position based on Nitrosomonas europeae C-31)

amoA-1F (targeting position 332 to 349):

5’-GGG GTT TCT ACT GGT GGT-3’

﹙Appl. Environ. Microbiol., 1997, 63(12), 4704 - 4712﹚

 a b c

amoA 基因 PCR 放大後產物 (size = 491 bp) 分布

a. 標準菌 Nitrosomonas europeae C-31 ATCC
25978; b. 及 c 環境樣本。
Fluorescent In Situ Hybridization (FISH)
Target molecule is 16S rRNA

Properties of rRNA
 Composed of nucleotides (G-C, A-T/U)
3’-CGCCGAGTTACGT-5’
 Regions of variable sequence conservation
GCGGCUCAAUGCA
5’
 Multiple copies/cell, related to growth rate

Develop probe complementary to target sequence

3’
 Compare sequences of target and nontarget organisms
 Find region in which sequence is unique for target organisms

Label probe with a measurable marker (e.g., fluorescent molecule)

Denitrifiers
(Green)

Nitrifiers
(Red)
Environmental Monitoring
Detection of pathogenic microorganisms in various
environments
Measurement of changes in species within the
environment (Biomarkers) upon exposure to pollution
Biological material to estimate the toxicity of a
particular chemical in isolation (Daphnia test,
Microtox, Ames test)
 Biosensor 生物感測器
“A chemical sensor is a device that transforms chemcial
information, ranging from the concentration of a specific
sample component to total composition analysis, into an
analytically useful signal”
“Biosensor - a subgroup of chemical sensors where
biological host molecules, such as natural or artificial
antibodies, enzymes or receptors or their hybrids, are
equivalent to synthetic ligands and are integrated into the
chemical recognition process.

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 Enzymes
Antibodies Biological material
Organelles
Whole cells
Conductimetric
Optical
Transducer Oxygen electrode
pH electrode
Photodiodes
Thermistor

Data processing

The basic components of a biosensor

生物技術在環境保護之成功例子
生物殺蟲劑取代原有危害性化學殺蟲劑
環境污染物生物處理技術
替代性生質能源開發 ( 生質柴油，酒精，
甲烷 )
污染場址生物復育技術 Bioremediation
改良現有廢水生物處理技術
開發生物或生化性產品偵測環境中污染物
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 舊問題需要新工具
環境系統中微生物族群之監測
• 廢污水處理程序
• 上水道及下水道
• 海灘及泳池
• 特殊區域環境
• 基因工程微生物
• 水華藻毒
• 生物復育