MIC 221

SOIL AND ENVIRONMENTAL MICROBIOLOGY

DR. NONTONGANA & MR NTOZONKE
COURSE OUTLINE

 Introduction to Environmental Microbiology

 Methods used in Environmental Microbiology for Identification and Enumeration of Microorganisms
 Soil Microbiology
 Biogeochemical Role of Soil Microorganisms
INTRODUCTION TO ENVIRONMENTAL MICROBIOLOGY

 ENVIRONMENTAL MICROBIOLOGY - The study of microorganisms and their activities in the environment
for the purpose of understanding their role in maintenance of the biosphere and their potential use in remediating
sites that have been disturbed.

 MICROBIAL ECOLOGY - Microbial Ecology is the study of interactions between populations of

microorganisms and between microbial populations and organisms in other tropic levels

 APPLIED MICROBIOLOGY - The exploitation of microorganisms, their activities, and their products for the
benefit of society, e.g. wine making, beer making, vinegar, etc.
‘ENVIRONMENTAL MICROBIOLOGY AS A DISCIPLINE’

 Rachael Carson published “Silent Spring” bringing to the public’s attention the poor condition of the
environment

 Emergence of new waterborne and foodborne pathogens that posed human health threats
 Posed a threat to both human and animal health
 As a result waste disposal practices, both surface water and groundwater supplies are frequently contaminated with
organic and inorganic chemicals.

 New technologies emerged to study microbes in their natural environment rather than as pure cultures in
the laboratory
 The discovery of the structure of DNA in 1953 by James Watson and Francis Crick
 ”THE BOOK THAT BIRTHED
MODERN ENVIRONMENTALISM”

- Born and lived in the Northeast United States from 1907-1964

- Rachel Carson started her career as a Marine Biologist. Being such a

strong conservationist, she began writing full time in the mid-1950's.
• The main theme of Silent Spring is the destruction of the delicate balance of nature by the wholesale use of
insecticides. 
• Rachel Carson carefully explains what the balance of nature is.
• She describes the balance of nature of the soil, of the earth's waters, and of the organisms of the earth

Polluted our streams

Damaged bird and animal populations

Caused severe medical problems for humans.

Instrumental in advancing the global environmental movement

 The first outbreak of a waterborne disease to be scientifically
documented in modern Western society occurred in London,
England, in 1854.

• Waterborne diseases, such as cholera and typhoid, are the diseases that are transmitted through drinking water.

• Disease can be spread while bathing, washing or drinking water, or by eating food exposed to contaminated water.

• Microorganisms causing diseases that characteristically are waterborne prominently include protozoa and bacteria. 

• Foodborne diseases are illnesses that result from ingestion
of contaminated food.

• More than 250 different foodborne hazards have been

recognized including infectious bacteria, viruses and
parasites, and noninfectious chemicals and toxins.

• Many of these agents commonly cause diarrhea and

vomiting, but there is no single clinical syndrome for all 
foodborne diseases.

• The sources of these agents range from being an inherent

constituent of the food to inadvertent (or intentional)
addition during food production, processing, or
preparation.
HISTORICAL PERSPECTIVE

 Initial scientific focus of the field environmental microbiology - water quality and the fate of pathogens.
 Treatment - Dramatic decrease in the incidence of typhoid fever and cholera.

 Pathogens in our food supply are second area of immense concern.

 Import of vegetable and fruits
 Consumer demand for a wide variety and high quality of imported produce
 Increased danger of importing a pathogen with the produce.

 Illustrate the types of questions that are addressed by environmental microbiologists.

MICROBES THAT HAVE HAD A SIGNIFICANT
Agent Mode of IMPACT ON HUMAN HEALTH.
Disease/Symptoms
transmission
Rotovirus Waterborne Diarrhea
Legionella Waterborne Legionnaire’s disease
Escherichia coli 0157, H7 Foodborne Enterohemorrhagic
fever, kidney failure
Waterborne
Hepatitis E virus Waterborne Hepatitis
Cryptosporidium Waterborne Diarrhea
Foodborne
Calicivirus Waterborne Diarrhea
Foodborne
Helicobacter pylori Waterborne Stomach ulcers, cancer
Foodborne
Cyclospora Waterborne Diarrhea
Foodborne
 CHEMICAL CONTAMINANTS THAT ROUTINELY FOUND IN SOIL AND
GROUNDWATER

Chemical class Frequency of occurrence Status of bioremediation

technologies
Fuel oil Very frequent Established
PAH polycyclic aromatic hydrocarbons Common Emerging
Creosate Infrequent Emerging
Alcohols, ketones, esters Common Established
Ethers Common Emerging
Chlorinated organics Very frequent Emerging
PCBs Polychlorinated biphenyls Infrequent Emerging
Nitroaromatics Common Emerging
Metals Common Possible
MONITORING APPROACHES

 Molecular methods...
 No one approach is free of bias
 Best strategy is to use several independent approaches and compare results
LISTERIOSIS OUTBREAK

 South Africa - Jan 2017 to March 2018

 978 cases confirmed by NICD
 59% Gauteng
 12% Western Cape
 7% Kwa-Zulu Natal
 22% Other provinces
 183 Mortalities
LISTERIOSIS

 Bacterial infection
 Foodborne
 Illness include sever sepsis, meningitis or encephalitis, sometimes resulting in lifelong harm or even death
BREAKTHROUGH

 Jan 2018
 9 children aged 5 from a pre-school in Soweto were hospitalized.
 Environmental Health practitioners intervened
 Source was discovered
SOURCE

 Whole genome sequencing was performed on isolates from patients

 91% strains belonged to Listeria monocytogenes Sequence Type 6 (ST6).
 Same isolate identified in source implicated in Soweto
 Same strain found in the processing environment of the manufacturer
http://www.free-powerpoint-templates-design.com
CHAPTER 2: METHODS USED IN ENVIRONMENTAL
MICROBIOLOGY FOR IDENTIFICATION AND
ENUMERATION OF MICROORGANISMS.

• LIGHT MICROSCOPY AND CULTIVATION

• POLYMERASE CHANI REACTION
• 16S RRNA GENE APPROACH
• 16S RRNA GENE-BASED FINGERPRINTING TECHNIQUES
• DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) AND TEMPERATURE GRADIENT GEL
ELCTROPHOESIS (TGGE)
• HYBRIDIZATION TECHNIQUES USING RRNA-TARGETED PROBES
• FLUORESCENT IN SITU HYBRIDIZATION
• MICROARRAY TECHNOLOGY
 LIGHT MICROSCOPY AND CULTIVATION
 Light microscope
 Identification made using Gram-staining
 Morphological characteristics
 not able to distinguish between the different
bacterial domains
 polymorphism of certain filaments is
known,
 complicates morphology-based
identification.
 LIGHT MICROSCOPY AND CULTIVATION

 Cultivation/Culturing and isolation

 Isolation: Obtaining a pure culture by separating one species of microbe from a mixture of other species
 Cultivation: is a method of multiplying microorganisms by letting them reproduce

 Most probable number (MPN) technique

 Total plate counts
 PLATE COUNT
 a method used to estimate the concentration of viable microorganisms in a sample by means of replicate liquid
broth growth in ten-fold dilutions.
POLYMERASE CHANI REACTION

PCR employs oligonucleotide

primers and thermostable
DNA polymerase to amplify
target DNA sequences by
temperature-controlled cycles
which enable strand
separation, primer annealing
and primer extension.
 16S RRNA GENE APPROACH

 16S rRNA gene sequence analysis is a standard method in bacterial taxonomy and identification, and is based on the
detection of sequence differences (polymorphisms) in the hypervariable regions of the 16S rRNA gene which is present in
all bacteria.
 Current 16S rRNA gene databases contain more than 20 000 entries and thereby provide a high resolution framework for
the assignment of those sequences obtained in 16S rRNA gene libraries from environmental diversity surveys.
 The approach consists of:
 DNA extractions
 Amplification of the 16S rRNA gene using primers targeting regions conserved in the bacterial genome
 Cloning
 Sequencing.

 The obtained sequences are analyzed together with adequate reference sequences to deduce their phylogenetic affiliation.
16S RRNA GENE APPROACH

 It is however important to realize that even if the complete diversity of an environmental 16S rRNA
gene library is harvested
 The obtained species inventory might not represent the naturally occurring diversity.
 This failure might be caused by: 
 Inefficient DNA extraction
 Inadequate coverage by the selected PCR primers.
 Kinetic and stochastic biases introduced by the PCR amplification
 Cloning bias.
 Different numbers of copies of rRNA genes.
16S RRNA GENE-BASED FINGERPRINTING TECHNIQUES
 16S rRNA-based fingerprinting techniques supplement the 16S rRNA approach.
 The common principle of these techniques is to separate PCR products of the same length but different sequence
to visualize the diversity within the amplificon by banding pattern.
 The most frequently applied fingerprinting techniques are
 Denaturing gradient gel electrophoresis (DGGE)
 Temperature gradient gel electrophoresis (TGGE).

 Advantages
 A high sample number can be processed in a relatively short time in order to gain an overview on the diversity of
the complex microbial community in wastewater treatment plants.
 Bands of interest can be excised, cloned and sequenced for subsequent identification.
DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE)

 The principle is to separate DNA strands, based on the ratio of CG and AT base pairs.
 During the process, the DNA is exposed to a gradient of denaturant at high temperatures within a polyacrylamide
gel.
 As the DNA sample progresses through the gel, from low to high denaturant concentration, it begins to melt at
varying points.
 The theory goes that the higher GC content makes it harder to melt while samples with lower GC content tend to
melt more rapidly.
TEMPERATURE GRADIENT GEL ELCTROPHOESIS (TGGE)
 HYBRIDIZATION TECHNIQUES USING RRNA-TARGETED
PROBES

 Hybridization methods represent standard techniques in molecular biology.

 In general, they are used to detect particular sequences (target) within a complex mixture of DNA or RNA molecules.
 DNA or RNA are usually transferred and immobilized to nitrocellulose or, more commonly, to nylon membranes.
 Complementary single-stranded probes are labeled radioactively or non-radioactively.
 When hybridized to the filter, probes bind to their complementary target sequence via hydrogen bonds.
 Un-hybridized probe is then washed away, and specifically-bound probe is detected by autoradiography or color
reaction.
 FLUORESCENT IN SITU HYBRIDIZATION

 Fluorescence in situ hybridization is a molecular cytogenetic technique that uses fluorescent probes that bind to
only those parts of a nucleic acid sequence with a high degree of sequence complementarity.
MICROARRAY TECHNOLOGY
 Microarray technology is a novel tool in molecular biology, capable of quantitating hundreds or thousands of
gene transcripts from a given cell or tissue sample simultaneously
 SOIL MICROBIOLOGY

 Soil microbiology is the study of organisms that live in the soil.

 Metabolic activities
 Their role in the energy flow
 Cycling of nutrients associated with primary production.

 Additionally, the discipline is concerned with the environmental impacts, both favourable and unfavourable, of soil
organisms and the process they mediate.

 The co-disciplines were initially concerned with the microscopic life in the soil but became extended to include
macroscopic organisms that reside in the soil and participate in soil dynamics
 SOIL MICROBIOLOGY

 Currently included in the soil biota together with unicellar organisms are the soil dwelling small invertebrates called soil
mesofauna.

 These may be either microscopic or macroscopic.

 The micro- and mesofauna play supplementary roles in organic matter transformations, but they lack the wide range of
enzymes capabilities of the soil microflora.
SOIL AS A HABITAT FOR ORGANISMS AND THEIR REACTIONS

 FIVE INTERACTIVE FACTORS IN SOIL FORMATION

 Soil Microorganisms
 Climate
 Topography
 Particle material
 Time
 The physical and chemical breakdown of rocks to fine particles with large surface areas and the accompanying
release of plant nutrients initiate the soil-forming process.
SOIL AS A HABITAT FOR ORGANISMS AND THEIR REACTIONS

 The initial colonizers of soil parent material are usually organisms capable both of photosynthesis and nitrogen
fixation,
 Cyanobacteria (blue-green algae)
 MICROBIAL FLORA OF SOIL
 Fertile soil is inhabited by the root systems of higher plants, by many animal forms (e.g., rodents, insects, and worms), and by
tremendous numbers of microorganisms.
 The conditions that influence the growth of microorganisms in soil
 Amount of nutrients
 Available moisture
 Degree of aeration
 Temperature
 pH
 Root system

 Bacteria, fungi, algae, protozoa, and viruses make up this microscopic menagerie.
 BACTERIA
 The following are likely to be found in soil:
 Autotrophs- produce their own food
 Large numbers of Actinomycetes are present in the
 Heterotrophs-  rely on other organisms
soil.
 Mesophiles- moderate temperature
 Nocardia spp.
 Thermophiles- extreme temperature
 Streptomycetes spp.
 Psychrophiles- low temperature
 Micromonospora spp.
 Aerobs- require oxygen
 Capable to degrade complex organic compounds
 Anaerobs- Does not require oxygen
 Ability to synthesize and excrete antibiotics
 Cellulose degraders- produce cellulase enzymes
 Sulfur oxidizers- oxidize sulfur compounds to sulfate
 Sulphur reducers- reducing elemental sulfur to hydrogen sulfide
 Nitrogen fixers- atmospheric nitrogen into fixed nitrogen
 FUNGI

 Hundreds of different species of fungi inhabit the soil

 They are most abundant near the surface, where aerobic conditions are likely to prevail.
 They exist in both mycelia and spore form.
 They are heterotrophic organisms- require organic compounds for nutrition
 Saprophytes- feed on dead organic matter

 Parasites- fungi cause diseases in plants, animals and humans.

ALGAE, PROTOZOA & VIRUSES

 The population of algae in soil is generally smaller than that of either bacteria or fungi

 Most soil protozoa are flagellates or amoebas. The number per gram of soil ranges from hundred to several
thousand in moist soils rich in organic matter

 Bacterial viruses (bacteriophages), as well as plant and animal viruses, periodically find their way into soils
through additions of plant and animal wastes. Also soil microorganisms themselves may harbour viruses.
 INTERACTIONS AMONG SOIL MICROORGANISMS.

 The microorganisms that inhabit the soil exhibit many different types of associations and they fall mainly into three
categories:
 Neutral: neutralism
 Positive or beneficial: mutualism, commensalism
 Negative or detrimental: antagonism, competition, parasitism, predation
NEUTRAL ASSOCIATIONS

 Neutralism
 It is conceivable that two different species of microorganisms occupy the same environment without affecting
each other.
POSITIVE ASSOCIATIONS
 Mutualism
 Mutualism is an example of a symbiotic relationship in which each organism benefits from the association.
 exchange of nutrients between two species, phenomenon called syntrophism
 collaborate

N2 Air CO2

N2-fixing bacteria CO2-fixing bacteria

B.lacticogenes T.ferrooxidans

Nitrogenous compounds

Organic carbon
POSITIVE ASSOCIATIONS

 Commensalism
 Commensalism refers to a relationship between microorganisms in which one species of a pair benefits and the
other is not affected.
 In soil this occurs commonly with respect to degradation of complex molecules like cellulose and lignin.
 
NEGATIVE ASSOCIATION

 Antagonism
 When one species adversely affects the environment for another species it is said to be antagonistic.
 Such organisms may of great practical importance, since they often produce antibiotics or other inhibitory
substances which affect the normal growth processes or survival of other organisms.
 Antagonistic relationships are quite common in nature.
 For example:
 Both Staphylococcus aureus and Pseudomonas aeruginosa are antagonistic towards Aspergillus terreus. Certain
Pseudomonas pigments inhibit germination of Aspergillus spores. S. aureus produces a diffusible antifungal material that
causes distortions and hyphal swelling in A. terreous.
NEGATIVE ASSOCIATION

 Competition
 A negative association may result from competition among species for essential nutrients.
 In such situations the best adapted microbial species will predominate or, in fact, eliminate other species which
are dependent upon the same limited substrate substance.
NEGATIVE ASSOCIATION

 Parasitism
 Parasitism is defined as a relationship between organisms in which one organism lives in or on another organism.
 The parasite feeds on the cells, tissues, or fluid of another organism, the host, which commonly is harmed in the
process.
 The parasite is dependent upon host and lives in intimate physical and metabolic contact with the host. All major
groups of plants, animals, and microorganisms are susceptible to attack by parasites.
 Bdellovibrio bacteriovorus, which is wide spread in and sewage

 Viruses which attack bacteria, fungi, and algae are strict intracellular parasites since they cannot be cultivated as
free-living forms.
Thank you for your attention