Beruflich Dokumente
Kultur Dokumente
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
2
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。
3
Journal Examples
4
Review Journal Examples
5
Trade Journal - Magazine
6
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
8
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
9
Environmental Biotechnology
• Application of all components of biological
science (mostly Microbiology and
Biochemistry) and engineering science
(especially Chemical and Environmental
engineering) to environmental problems.
11
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
12
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)
13
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
14
15
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
17
18
微生物分類地位
Haeckel (1866) Whittaker (1969)
三界分類法 五界分類法
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
20
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
21
22
Impact of Microorganisms on Humans
23
Microorganisms, Energy, and the
Environment
The role of microbes in biofuels production
• e.g., methane, ethanol, hydrogen
24
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
25
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
26
Mechanisms of Biological Treatment
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
30
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)
34
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
38
Activated sludge 活性污泥
39
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.
56
Trickling Filter
An aerobic, attached-growth wastewater treatment processes
Rotary Biological Contactor
primary
sedimentation clarifier
inflow outlet
sludge return
primary sludge
Gujer 2000
61
62
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
63
EXXON
VALDEZ
oil spill
(1989)
64
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
66
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)
67
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 )
68
Isolation of Pure Culture
海水 0.001 – 0.1
淡水 0.25
土壤 0.3
活性污泥 1 - 15
69
Environmental
Samples
Cells
isolated
Direct observation
using fluorescent
probes Isolated DNA
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
72
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)
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
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)
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
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.
86
Enzymes
Antibodies Biological material
Organelles
Whole cells
Conductimetric
Optical
Transducer Oxygen electrode
pH electrode
Photodiodes
Thermistor
Data processing