Methods to study microorganisms

in the environment

PART 1: Cultivation-based techniques (Chapter 10)

- how to identify and enumerate microorganisms?
PART 2: (Eco)physiological methods (Chapter 11)
- how to measure microbial activities (functions)?
PART 3: Tracking specific microorganisms (Chapter 13)
- how to track presence/activity of specific microorganisms?
PART 4: Nucleic acid based techniques (Chapter 13)
- how to identify and enumerate microorganisms?

1
 Lecture 4
Soil and Water Pollution – Experimental Assessment
Kristian K. Brandt

Methods to study microbial communities in

polluted environments

PART 1: Cultivation-based techniques (Chapter 10)

Enrichment and isolation of microorganisms
Viable counts and MPN enumeration of bacteria
The ‘great plate count anomaly’ and the uncultured majority
New approaches for enhanced or targeted cultivation of bacteria
污染环境中微生物群落的研究方法 第 1 部分：基于培养的技术（第 10 章）

微生物的富集和分离 细菌的活菌计数和 MPN 计数 “大平板计数异常”和未培养的大多数细菌

2
 Enrichment cultures
“The use of certain growth media to favor the growth
of a particular type of microorganism over others,
enriching a sample for the microorganisms of
interest”.
– Can prove the presence of an organism in a habitat.
– Cannot prove an organism does not inhabit an
environment.
– The ability to isolate an organism from an environment
says nothing about its ecological significance.
富集培养“使用某些生长培养基有利于特定类型微生物的生长，从而丰富感兴趣微生物的样本”。

– 可以证明栖息地中存在生物体。

– 无法证明生物体不栖息在环境中。
3
– 将有机体从环境中分离出来的能力并不能说明其生态意义。
 Enrichment
Enrichment bias (cultivation bias)
– Microorganisms cultured in the lab are
frequently only minor components of the
microbial ecosystem
• Reason: the nutrients available in the lab culture
are typically much higher than in nature (i.e.
selection for fast-growing copiotrophs rather
than slow-growing oligotrophs)
• Practical solution: dilution of inoculum (dilution-
to-extinction) to eliminate rapidly growing, but
rare microorganisms during the enrichment step
4
富集偏差（培养偏差）——实验室培养的微生物通常只是微生物生态系统的次要组成部分 原因：实验室培养物中可用的营养素通常比自然界中的要高

选择快速生长的富养生物而不是缓慢生长的寡养生物） 实用的解决方案：稀释接种物（稀释至灭绝）以在富集步骤中消除快速生长但稀有的微生物
 通过在琼脂培养基上铺板进行分离

Isolation by plating on agar media

Can also be used to enumerate viable bacteria counted as
colony-forming units (CFUs)

Spread or pour plating

5
用于快速生长的细菌的选择，但如果选择长时间后才出现的菌落，也可能获得生长较慢的细菌

Isolation
Visible colonies derived from colony-forming units (CFU)
Selection for fast-growing
bacteria, but slower-growing
bacteria may also be obtained if
picking colonies appearing only
after a long time

6
纯培养物包含单一种类的微生物——可以通过划线平板、琼脂摇动或液体稀释

获得琼脂稀释管是在熔融琼脂中稀释的混合培养物

Isolation
——可用于纯化厌氧生物

Pure cultures contain a single

kind of microorganism
– Can be obtained by streak
plate, agar shake, or liquid
dilution

Agar dilution tubes are mixed

cultures diluted in molten
agar
– Useful for purifying
anaerobic organisms

7
© 2012 Pearson Education, Inc.
通过最可能数 (MPN) 方法对活细菌或活性细菌进行计数

Enumeration of viable or active bacteria

by most-probable-number (MPN) method

Serial 10  dilutions of inocula in a medium

(dilution-to-extinction)

Used to estimate number of microorganisms

in food, wastewater, and other samples

Growth is scored (+ or -) in each tube

Calculate MPN ± confidence interval

Often combined with use of 14C-pollutants to

enumerate pollutant-mineralizing
microorganisms (ecophysiological detection
method; not cultivation)
8
 The ‘great plate count
anomaly’ and the
uncultured majority
– Typically ≤1% of cells
can be cultured
– 2004: Several phyla
have no cultured
representatives (marked
in red color)

9
Keller and Zengler (2004) Nature Reviews Microbiology
 The ‘great plate count
anomaly’ and the
uncultured majority (2016)
– We get better at
cultivating bacteria, but
we also continue to
discover new phyla by
molecular techniques
– Several phyla still have no
cultured representatives
(marked with red dots)

10
Hug et al. (2016) Nature Microbiology
没有文化的大多数——如何解释这种现象？

细菌的生理状态 – 活细胞 – 活细胞但不可培养 – 休眠细胞 – 死细胞 方法学解释 – 由于培养技术不当，即使是活细胞也无法培养

The uncultured majority

- how to explain the phenomenon?

• Physiological states in bacteria

– Viable cells
– Viable, but unculturable cells
– Dormant cells
– Dead cells

• Methodological explanation
– Even viable cells cannot be cultured due to
inappropriate cultivation techniques

11
 培养未开化者的新方法

New approaches for cultivating the

uncultured

• Mimic natural low-nutrient conditions

• Improved use of conventional techniques
– Microbiologist skills and patience
• Improved instrumentation
– Flow cytometry and cell sorting
• Guided cultivation by parallel molecular analysis
of enrichment cultures
– Parallel molecular community analysis
模拟自然低营养条件 改进传统技术的使用——微生物学家的技能和耐心 改进

仪器——流式细胞术和细胞分选 通过富集培养物的平行分子分析引导培养——平行分子群落分析 12
 微生物的可培养性

Culturability of microorganisms

• Culturability
– Operational definition: capable of growth on
specific medium
– Theoretical (ideal) definition: capable of growth in
laboratory media
– Not possible to grow all bacteria on one medium
– Growth in the lab should mimic natural conditions of
the microbes to be isolated
可培养性 – 操作定义：能够在特定培养基上生长 – 理论（理想）定义：能够在实验室培养基中生长

– 不可能在一种培养基上生长所有细菌 – 实验室中的生长应该模拟待分离微生物的自然条件

13
 Culturability of microorganisms
(semantics matters)

• Common misconceptions
– ‘>99 % of all cells in the environment are unculturable’
– ‘Unculturable species of bacteria’

• In stead we should talk about

– ‘uncultured bacteria’
– ‘as-yet-uncultured bacteria’
语义很重要） 常见的误解 – “环境中所有细胞中 >99% 是不可培养的” – “不可培养的细菌种类”

相反，我们应该谈论 – “未培养的细菌” – “尚未培养的细菌”

14
 Part 1: Take home messages
• The ability to isolate an organism from an environment
– can prove the presence of an organism in a habitat.
– cannot prove that an organism is NOT present.
– says little about its ecological significance.
• Cultivation bias (cultivation conditions are often
unrealistic)
• The uncultured majority can be studied by
– improved use of existing cultivation-based techniques
– novel improved cultivation-based techniques
– nucleic acid based techniques (Lecture 5)
将生物体与环境隔离开来的能力——可以证明栖息地中存在生物体。

– 不能证明有机体不存在。

– 很少提及其生态意义。 15

培养偏差（培养条件通常不切实际） 未培养的大多数人可以通过以下方式进行研究 – 改进现有基于培养的技术的使用

– 新的改进的基于培养的技术 – 基于核酸的技术（第 5 讲）
Lecture 4
Soil and Water Pollution – Experimental Assessment
Kristian K. Brandt

Methods to study microbial communities in

polluted environments

PART 2: (Eco)physiological methods (Chapter 11)

Microbial functions
生态）生理学方法（第 11 章）微生物功能

17
 基础（实际）和底物诱导呼吸 (SIR) 实际和潜在的微生物活动

Basal (actual) and substrate-induced respiration (SIR)

Actual and potential microbial activity

Respiration rate (CO2-C g-1 h-1)

Actual rate: In situ activity of heterotrophs

Potential rate: Reflects biomass of non-dormant heterotrophs
True in situ rates must take microbial C assimilation into account

Growth

Time

Addition of glucose
18
Measuring microbial activities performed by
specific functional groups in nature

测量自然界中特定官能团执行的微生物活动

示踪剂方法 微电极

• Tracer methods

• Microelectrodes

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 Sulfate reduction Photosynthesis
Radioisotopes

incorporation
Formalin-killed control
Light

Lactate or H2S
H 2S

• High sensitivity and specificity can be

2
14CO
achieved with radioisotopes Killed
Lactate Dark
– Proper killed cell controls must be used
Time Time
放射性同位素 放射性同位素可实现高灵敏度和特异性——必须使用适当的死细胞对照

Sulfate reduction 14C-Glucose respiration

evolution
H2 present
H2 35S

2
14CO
Killed H2 absent

Killed

Time Time
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© 2012 Pearson Education, Inc.
微电极 – 可以高空间分辨率测量多种化合物 p H、O2、CO2、H2S、NO2-、NO3- 等。

– 电极被小心地插入栖息地 每 10–100 洀洀 行一次测量 微生物活动可以通过对水饱和系统（例如沉积物或水饱和土壤团聚体）中的化学梯度

Microelectrodes
• Microelectrodes
– Can measure a wide range of compounds at
high spatial resolution
• pH, O2, CO2, H2S, NO2-, NO3- etc.
– Electrodes are carefully inserted into the
habitat
• Measurements taken every 10–100m
• Microbial activities can be inferred by modeling of
chemical gradients in water-saturated systems (e.g. in
sediments or water-saturated soil aggregates)
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 Gold Glass Platinum

5 m

Membranes Glass

50–100 m NO3 N 2O 2e 1 N2O  H2O N2  2 OH

Bacteria Cathode Nutrient solution

22
© 2012 Pearson Education, Inc.
 Oxygen (O2) concentration (M)
0 100 200 300

Seawater
Depth in sediment (mm)

0
Commercial spin-off
O2 NO3
from microbial ecology
Oxic sediment research established in
1998 in Aarhus,
5 Denmark
(www.unisense.com)

Four daughter
Anoxic sediment companies established
in 2003, 2013, 2015
10
and 2018

0 4 8 12
23
Nitrate (NO3) concentration (M)
© 2012 Pearson Education, Inc.
单细胞分辨率下的细菌活性（左侧）

Bacterial activity at single-cell resolution

(left side)

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Nybroe et al., from Modern soil microbiology 2007.
 Fluorescence microscopy can be used to enumerate active
cells (CTC staining) in environmental samples
荧光显微镜可用于计算环境样品中的活性细胞（CTC 染色）

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Bartosch et al., (2003). J. Microbiol. Methods
流式细胞术也可用于基于荧光染色对液体样本中的活性细胞进行计数（数秒内可计数数千个细胞）

Flow cytometry can also be used to enumerate active cells

in liquid samples based on fluorescence staining
(thousands of cells can be counted in a few seconds)

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KKB2

Measurement of in situ
microbial growth rates
Often uncoupling between growth and activity
in nature (i.e. non-growing microorganisms
may respire or otherwise be active)
原位微生物生长速率的测量通常在自然界中生长和活动之间解耦（即非生长微生物可能呼吸或以其他方式活跃）

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Slide 27

KKB2 Kristian Koefoed Brandt; 10-09-2012

纯培养物中的微生物生长周期——它与环境有关吗？ - 我们如何测量环境中微生物的生长？ - 细菌数量通常非常稳定，并不代表原位生长率

The microbial growth cycle in pure cultures

- Is it relevant in the environment?
- How can we measure microbial growth in the environment?
- Bacterial numbers are often quite stable and not indicative of in situ
growth rates

Growth phases
Lag Exponential Stationary Death
10 1.0

Optical density (OD)

0.75
organisms/ml
Log10 viable

9 Turbidity
(optical density) 0.50

Viable count
8
0.25

6
0.1
Time
28
© 2012 Pearson Education, Inc.
 通过 [3H] 胸苷掺入测定细菌生长

Bacterial growth determination by

[3H]thymidine incorporation
Cellular
membrane

Synthesis of radiolabelled DNA

containing incorporated [3H]
from [3H]thymidine

Ribosomes
**
DNA **
*
**

[3H]thymidine
incorporation (1-3
hour incubation)
DNA synthesis
Cell division 29
Bacterial growth
 Bacterial growth determination by [3H]leucine
Cellular incorporation
membrane
Synthesis of radiolabelled
proteins containing
incorporated 3H-leucine

Ribosomes
**
DNA **
*
**

3H-leucine incorporation
(1-3 hour incubation)
Protein synthesis
Biomass production
Bacterial growth 30
胸苷和亮氨酸掺入测定

The thymidine and leucine incorporation assays

1. Pre-incubation Killed control (TCA)

1-2 h after addition of [3H]thymidine or

2. Incubation [3H]leucine

TCA (ice cold), centrifugation

3. Stop

4. Remove Collect waste

5. Measure
Add scintillation liquid

31
 Fungal growth determination by [14C]acetate
incorporation in ergosterol
乙酸盐用作真菌中麦角甾醇生物合成的前体，但不用于细菌（真菌生物标志物）

Acetate is used as a
precursor for the
biosynthesis of ergosterol
in fungi, but not in bacteria
(fungal biomarker)

14C-acetateincorporation in ergosterol (4 hour incubation) followed

by ergosterol extraction and measurement of radioactivity
Cell membrane synthesis
Biomass production
Fungal growth 32
 将微生物功能与特定生物联系起来 谁在做什么或“谁吃了香蕉”？将在第 5 讲中介绍

Linking microbial functions to

specific organisms

Who is doing what or “who ate the

banana”?
To be covered in Lecture 5

33
区分实际和潜在过程速率很重要 可以使用化学和同位素示踪剂技术研究污染物生物降解，但需要严格的实验控制

可以在单细胞分辨率下研究微生物活动 我试图提供可用的代表性快照方法，但也可以使用许多其他方法 多相方法（即平行或组合使用多种

Part 2: Take home messages

• Important to distinguish between actual and potential

process rates
• Pollutant biodegradation can be studied using both chemical
and isotope tracer techniques, but requires strict experimental
controls
• Microbial activities can be studied at single-cell resolution
• I have attempted to provide a representative snapshot of
available methods, but many other approaches can be used
• Polyphasic approach (i.e. parallel or combined use of
multiple methods) is recommended for in-depth studies of
biodegradation
34
Lecture 4
Soil and Water Pollution – Experimental Assessment
Kristian K. Brandt

Methods to study microbial communities in

polluted environments

PART 3: Tracking the presence or activity of specific

microorganisms in complex environments (Chapter 13)
Marker and reporter gene technology
Whole-cell bacterial biosensors (bioreporters)
第 3 部分：跟踪复杂环境中特定微生物的存在或活动（第 13 章） 标记和报告基因技术 全细胞细菌生物传感器（生物报告器）

35
全细胞细菌生物报告器可以作为污染物的活体传感装置

Whole-cell bacterial bioreporters can work

as living sensing devices for pollutants
“开灯”生物传感器 – 由特定化学物质或一般应激反应途径诱导的启动子 – 在亚毒性浓度下信号增加

“关灯”生物传感器 – 启动子持续活跃 – 报告对任何化合物的毒性 – 信号减弱在有毒浓度

The whole-cell bioreporter concept

(bioreporter = biosensor)
• ‘Lights on’ biosensor
– Promoter induced by specific
chemicals or by induction of
general stress response
pathways
– Increased signal at sub-toxic
concentrations

• ‘Lights off’ biosensor

– Promoter is active
continuously
– Reports toxicity to any
compound(s)
– Reduced signal at toxic
concentrations
 Reporter genes encoding
luciferases (e.g. luxAB)

FMNH2 + RCHO + O2 FMN + RCOOH + H2O + light

• luxAB encodes the functional luciferase enzyme

• luxCDABE also encodes the enzymes needed
for biosynthesis of the aldehyde substrate
 Reporter genes encoding fluorescent proteins
and use of sensor plasmids

The green-fluorescent protein (GFP) of Suite of fluorescent proteins are

the hydromedusa Aequorea victoria now available
www.nobelprize.org/nobel_prizes/chemistry/laureates/
2008/shimomura_lecture.pdf

Most bacterial bioreporters are

made by in vitro construction of
recombinant plasmids followed
by plasmid transfer to host cell
(transformation)
大多数细菌生物报告基因是通过体外构建重组质粒，然后将质粒转移到宿主细胞（转化）而制成的
 Choice of reporter gene(s) for environmental
application of bioreporters
Advantages of luxAB genes relative to gfp
• Lower detection limits for “lights on” biosensors
• Shorter response time
• Higher signal-to-noise ratio (e.g. no autofluorescence)
• No external light source is required
• Allows for construction of “lights off” toxicity biosensors

Disadvantages of luxAB genes relative to gfp

• Single-cell resolution not possible
• Decanal substrate must be added which limits real-time
detection capabilities
– Solution: luxCDABE, but can be a big energy burden for the cell
优势 “开灯”生物传感器的检测限较低 响应时间较短 信噪比较高（例如无自发荧光） 无需外部光源 允许构建“关灯”毒性生

gfp 的缺点 无法实现单细胞分辨率 必须添加癸醛底物，这限制了实时检测能力 – 解决方案：luxCDABE，但对细胞来说可能是一个很大的能量负担

Pseudomonas fluorescens gfp-tagged
cells colonizing straw

- use of gfp as a marker gene monitoring the

presence of the gfp-tagged bacterium 41
 Part 3: Take home messages
• Marker genes (e.g. gfp) make it possible to monitor
specific microorganisms in the environment (e.g.
leaching to ground water of a specific bacterium)
• Reporter genes (e.g. gfp or lux genes) make it possible
to report on environmental conditions sensed by specific
microorganisms (i.e. whole-cell bioreporters)
• Whole-cell bioreporters represent an elegant way to
study pollutant bioavailability processes in the
environment, but should preferably be complemented by
chemical analysis
标记基因（例如 gfp）可以监测环境中的特定微生物（例如

特定细菌渗入地下水） 报告基因（例如 gfp 或 lux 基因）使报告特定微生物感知的环境条件成为可能（即全细胞生物报告基因）

42
全细胞生物报告基因代表了一种研究污染物的优雅方法环境中的生物利用度过程，但最好辅以化学分析