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Practical Guide to Microbial Polyhydroxyalkanoates 1st
Edition Sudesh Digital Instant Download
Author(s): Sudesh, Kumar; Abe, Hideki
ISBN(s): 9781847351197, 1847351190
Edition: 1
File Details: PDF, 9.88 MB
Year: 2010
Language: english
Practical Guide to Microbial
Polyhydroxyalkanoates

Kumar Sudesh
Hideki Abe

FeJSMITHEKS
Smithers
ismithers - A Smithers Group Company

Shawbury, Shrewsbury, Shropshire, SY4 4NR, United Kingdom

Telephone: +44 (0)1939250383 Fax: +44 (0)1939251118
http://www.ismithers.net
 First Published in 2010 by

ismithers
Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK

02010, Smithers Rapra

All rights reserved. Except as permitted under current legislation no part

of this publication may be photocopied, reproduced or distributed in any
form or by any means or stored in a database or retrieval system, without
the prior permission from the copyright holder.

A catalogue record for this book is available from the British Library.

Every effort has been made to contact copyright holders of any material
reproduced within the text and the authors and publishers apologise if
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Typeset by Argil Services

Printed and bound by Lightning Source Inc.
 reface

P
Polyhydroxyalkanoates (PHA) are plastic-like polymers produced naturally by many
types of bacteria. PHA are among the most promising next-generation plastics because
they are bio-based and biodegradable.

The book begins with a few brief chapters on the biology of PHA focusing particularly
on their synthesis by microorganisms. Unlike chemical polymerisation processes,
microbial syntheses involve multiple biological catalysts that convert renewable carbon
sources into monomers, which in turn are polymerised into high-molecular-weight
polymers in an aqueous environment at ambient conditions. This complicated process
is described by giving specific and well-established examples, along with new and
recent findings on the catalytic capabilities of the key enzyme PHA synthase. With
this brief background on the biology of PHA, readers will be exposed to the main
focus of this book: PHA properties. Readers are provided with important information
on the crystalline and solid-state structures of various types of PHA, as well as their
physical and mechanical properties, which are governed by the monomer types, their
composition, and molecular weight. The final objective of this book is to provide
detailed information on the currently understood mechanism of PHA biodegradation
because this is one of the most important factors determining the final applications
of PHA.

We are extremely grateful to Dr Kesaven Bhubalan, Mr Aaron Ooi Wei Yang, Dr Tang
Hui Ying and Ms Rathi Devi Nair Gunasegaven for their invaluable help, particularly
during the final stages of the preparation of this book.
Last but not least, we thank ismithers for publishing this book. The exemplary
support and guidance provided by ismithers staff, in particular Mrs Eleanor Garmson,
expedited completion of the book.

Besides serving as a reference material, we hope that this book will encourage more
people to get involved and contribute to the development of these interesting and
promising microbial polyesters.

Kumar Sudesh
Hideki Abe

...
111
 ontents

C
1. Background ................................................................... 1

2. Polyhydroxyalkanoates (PHA) Types ............................ 5

2.1 Naturally Occurring PHA ................................................... 5
2.2 Unnatural PHA ................................................................... 6

3. Microbiology of Polyhydroxyalkanoate (PHA)

Synthesis ..................................................................... 13

4. Production of Polyhydroxyalkanoates (PHA).............. 17

5. Extraction and Purification of Polyhydroxyalkanoates

(PHA) from Microbial Cells ........................................ 21
6. Crystalline and Solid-state Structures of
Polyhydroxyalkanoates (PHA).................................... 25
6.1 Poly[(R)-3-hydroxybutyrate] (P(3HB)) ............................. 25
6.2 Poly[(R)-3-hydroxyvalerate](P(3HV)).............................. 29
6.3 Medium-chain-length Poly [(R)-3-hydroxyalkanoate]s
(mcl-P(3HA)).................................................................... 30
6.4 Poly(4-hydroxybutyrate) (P(4HB)).................................... 33
6.5 Poly(3-hydroxypropionate)(P(3HP))................................ 36
6.6 Poly[(R)-3-hydroxybutyrate]-basedCopolymers ...............38

V
Practical Guide to Microbial Polyhydroxyalkanoates

7. Physical Properties of Polyhydroxyalkanoates (PHA).. 51

7.1 Crystallisation Kinetics ..................................................... 51
7.2 Thermal Properties ........................................................... 55
7.2.1 Melting Temperature .................................................... 55
7.2.2 Glass Transition Temperature (T, ) ................................ 59
7.2.3 Thermal Degradation Temperature ...............................61
7.3 Mechanical Properties ...................................................... 67
7.3.1 Films and Fibres of P(3HB) Homopolymers .................67
7.3.2 Films and Fibres of P(3HB)-based Copolymers .............74
7.3.3 Mechanical Properties of mcl-P(3HA) and their
Modification Techniques .............................................. 77
7.3.4 P(3HB)-based Polymer Blends ...................................... 77

8. Intracellular degradation (mobilisation)of

Polyhydroxyalkanoates (PHA).................................... 85
8.1 Endogeneous Degradation of PHA ................................... 85
8.2 Intracellular P(3HB)Depolymerases and Degradation
Systems............................................................................. 87
8.3 Intracellular 3HB Oligomer Hydrolases ............................ 91
8.4 Intracellular mcl-PHA Depolymerases .............................. 91

9. Extracellular Degradation of
Polyhydroxyalkanoates (PHA).................................... 97
9.1 Effect of Environmental Conditions on the Degradation
of PHA ............................................................................. 97
9.2 Structure and Properties of PHA-degrading Enzymes........98
9.2.1 Short-chain-length PHA Depolymerase ........................ 99
9.2.2 Medium-chain-length PHA Depolymerase ..................110
9.3 Effect of Chemical Structures on Enzymatic
Degradability .................................................................. 110

vi
 Contents

9.3.1 Enantiomer Selectivity of PHA Depolymerase ............ 110

9.3.2 Substrate Specificity of the Catalytic Domain .............111
9.3.3 Binding Specificity of the Substrate-binding Domain ..114
9.4 Effect of Solid-state Structures on Enzymatic
Degradability .................................................... ..............116
9.5 Molecular Mechanisms of the Enzymatic Degradation
of PHA ........................................................................... 123
9.6 Conclusion and Future Perspectives ................................ 126

Abbreviations.. .................................................................. 137

Index.. ............................................................................... 143

vii
 Background

1
The ability of some microorganisms to synthesise an intriguing plastic-like material
has received much attention from academia and industry over the past 30 years
[1-91. Polyhydroxyalkanoates (PHA)are high-molecular-weight biological polyesters
synthesised by many types of bacteria. PHA serve as carbon- and energy-storage
compound for the bacteria. Figure 1.1 shows the general process of PHA accumulation
by a bacterial cell.

Carbon and Energ

Figure 1.1 PHA accumulation process by a bacterial cell (schematic). If R is CH,,

the PHA is known as poly(3-hydroxybutyrate) [P(3HB)]or PHB. P(3HB) was
the first type of PHA to be discovered. It is also the commonest type of PHA
synthesised by most microorganisms

1
Practical Guide to Microbial Polyhydroxyalkanoates

In conditions that are unfavorable for growth, many bacteria are equipped with
intricate metabolic pathways that convert excess carbon sources into 3-, 4- and
5-hydroxyalkanoyl-CoA [ 10, 111. An enzyme called PHA synthase polymerises, via
a condensation process, these hydroxyalkanoyl-CoA compounds into linear polyester
molecules having number-average molecular masses in the range 0.05-20 MDa. The
resulting amorphous PHA molecules are simultaneously packed into the form of water-
insoluble granules and kept in the cell cytoplasm. The accumulated PHA granules are
usually large enough to be readily viewed under phase-contrast light microscopy.

The entire process from monomer synthesis to polymerisation takes place in an

aqueous environment in the bacterial cell under ambient conditions. Under controlled
cultivation conditions in a fermenter, PHA content in the bacterial cells can increase
to as much as 90 wt% of the dry weight of the cell within 48 hours. In contrast to
the chemical polymerisation process that requires relatively extreme conditions,
biological processes take place in the relatively mild conditions of the cell cytoplasm.
To date, approximately 150 types of monomers have been identified as substrates
for PHA synthase, which is the key enzyme for PHA biosynthesis. This shows
that PHA synthases are a class of enzymes that have broad substrate specificity.
PHA synthases are highly stereospecific. They specifically polymerise only the ( R )
enantiomer of the hydroxyalkanoate monomers. Therefore, microbial PHA are a
source of enantiomerically pure compounds. By hydrolyzing high-molecular-weight
PHA, it is possible to obtain the monomers, which can be used to synthesise useful
compounds.

References
1. A.J. Anderson and E.A. Dawes, Microbiological Reviews, 1990,54,4,450.

2. Y. Doi, Microbial polyesters, VCH, New York, US, 1990.

3. S.Y. Lee, Biotechnology and Bioengineering, 1996,49,1, 1.

4. L.L. Madison and G.W. Huisrnan, Microbiology and Molecular Biology

Reviews, 1999,63, 1,21.

5. Y. Poirier, D.E. Dennis, K. Klomparens and C. Somerville, Science, 1992, 256,

520.

6. A. Steinbuchel in Biomaterials, Ed., D. Byrom, MacMillan Publishers,

Basingstoke, Hampshire, UK, 1991, 125.

2
 Background

7. K. Sudesh, H. Abe and Y. Doi, Progress in Polymer Science, 2000,25, 10,

1503.

8. H.E. Valentin, D.L. Broyles, L.A. Casagrande, S.M. Colburn, W.L. Creely,
P.A. DeLaquil, H.M. Felton, K.A. Gonzalez, K.L. Houmiel, K. Lutke, D.A.
Mahadeo, T.A. Mitsky, S.R. Padgette, S.E. Reiser, S. Slater, D.M. Stark,
R.T. Stock, D.A. Stone, N.B. Taylor, G.M. Thorne, M. Tran and K.J. Gruys,
International Journal of Biological Macromolecules, 1999,25, 1-3, 303.

9. M. Zinn, B. Witholt and T. Egli, Advanced Drug Delivery Reviews, 2001, 53,
1,5.

10. A. Steinbuchel and H.E. Valentin, FEMS Microbiology Letters, 1995, 128, 3,
219.

11. H.E. Valentin, A. Schonebaum and A. Steinbuchel, Applied Microbiology and

Biotechnology, 1996,46,261.

3
 Poly hyd roxyaIkanoates (PHA) Types

2
Studies have shown that there are many types of monomer constituents of PHA. The
various types of PHA monomers can be classified into ‘natural’ and ‘unnatural’. The
former can be found naturally accumulated by microorganisms in the environment.
Unnatural PHA are produced by microorganisms in the laboratory if they are fed
specific chemicals. These chemicals are usually not the normal substrates used by the
microorganisms for growth and survival.

2.1 Naturally Occurring PHA

Poly(3-hydroxybutyrate) or P(3HB)is the commonest among the various types of PHA

that have been reported. The biosynthesis pathway of P(3HB) is shown in Figure 2.1.
P(3HB) was the first type of PHA to be studied and characterised [l,21; it was initially
thought that P(3HB)was the only type of bacterial polyester that existed naturally [3].
This is why, in most early literatures, this bacterial storage polyester was referred to
as ‘P(3HB)’or ‘PHB’, which refers to a polymer of 3-hydroxybutyric acid. It was not
until much later that researchers realised the existence of several types of monomers
that can be incorporated into the storage polyester [4-61. Therefore, the general name
‘PHA’ is more appropriate to refer to this microbial polymeric material.

About 150 types of monomers have been identified as PHA constituents. This variety
of monomers reflects the broad substrate specificity of PHA synthases. Although
the known PHA synthases can polymerise various monomers, bacterial cells cannot
synthesise most of the monomers. Most bacteria can naturally synthesise only a few
of these monomers. Most of the other monomers have to be supplied to the bacteria
in the form of precursor carbon sources for the bacteria to polymerise them.

5
Practical Guide to Microbial Polyhydroxyalkanoates

Acetyl-CoA H3C SCOA H3C~ S C O A

Acetoacetyl-CoA HaCuSCOA

reductase

3-hyd roxybutyry I-CoA H3C

Poly-(R)-3-hydroxybutyrate

Figure 2.1 Schematic pathway for the biosynthesis of P(3HB) in bacteria. This
pathway has been studied in great detail using Cupriavidus necator H16, which
is the model microorganism for studies on PHA biosynthesis. The genes encoding
all the three enzymes have been cloned from various microorganisms and
characterised

2.2 Unnatural PHA

The quest for novel PHA with unique material properties has resulted in the
identification of unnatural PHA. Most of the diverse range of PHA is unnatural.
These ‘exotic’ PHA are synthesised only if structurally related carbon sources or
precursor compounds are provided to the bacterial cells under specific cultivation
conditions. Cells are usually devoid of suitable metabolic pathways to enable them
to use such precursor compounds as sources of carbon andlor energy. Many bacterial
cells can take up a wide range of precursor compounds from the culture medium and
polymerise them into PHA. The key factor that determines the type of PHA that is
produced by a microorganism is the substrate specificity of the polymerising enzyme
PHA synthase [7, 81. Among the types of unnatural PHA monomers produced only
by microorganisms in the laboratory are 4-hydroxybutyrate (four-carbon compound)
[9-181, 4-hydroxyvalerate (five-carbon compound) [ 191, 4-hydroxyhexanoate

6
 Po lyhy d r oxyal ka noates ( PHA) Types

(six-carbon compound) [20], 5-hydroxyhexanoate (six-carbon compound) [21],

4-hydroxyheptanoate (seven-carbon compound) [211and 4-hydroxyoctanoate (eight-
carbon compound) [21].

Regardless of the types of PHA, all of them are accumulated in the form of water-
insoluble granules in microbial cells. Models on the formation of PHA granules have
been proposed. Two well-known models for the in vivo formation of PHA granules
are the ‘micelle model’ [22,23] and ‘budding model’ [24], which both consider the
defined location of the PHA synthase (Figure 2.2).

In the micelle model, a micelle-like structure is formed due to the aggregation of

P(3HB)-linked synthase. In this model, synthase proteins residing on micelle surface
acquire the 3-hydroxybutyrate-CoA substrate from the cytosol, leading to the growing
insoluble PHA chain within the micelle. On the other hand, in the budding model
the synthase proteins adhere to the inner face of the plasma membrane. As the PHA
chain elongates and phasin proteins (specific proteins that are associated with the
PHA granules) are produced, budding of a vesicle with a phospholipid layer could
ultimately lead to granule formation.

This model of granule formation was supported in recent works by two research
groups [25-271. Aside from these two models of formation of PHA granules, Tian
and co-workers noticed that emerging granules arose from the cell centre of wild-type
Cupriavidus necator, which were localised at unknown mediation elements [28,29].
They proposed that these mediation elements may act as nucleation sites for initiation
of P(3HB) granules.

An increasing number of new monomers have been found to be active as substrate

for the PHA synthase. Recently, it has been demonstrated that the PHA synthase can
polymerise even non-PHA polymers such as polythioesters [30-321 and polylactic
acids (PLA) [33-361. In the latter case of lactic acid, a mutant PHA synthase was
used to produce the unnatural copolymer of PHA and PLA. These new findings
further confirm that PHA synthases are interesting enzymes whose potential has
not been fully tapped. However, attempts to crystallise this enzyme and obtain its
crystal structure have proved to be extremely challenging. Therefore, not much is
known about its catalytic mechanism. What is known is that this key enzyme in the
biosynthesis of PHA can polymerise high-molecular-weight water-insoluble polyesters
in an aqueous environment at room temperature [23,24,37- 431. One can imagine
its potential applications if the enzyme mechanism can be understood and exploited
for chemical syntheses.

7
 Practical Guide to Microbial Polyhydroxyalkanoates

~~~ ~

A.
s Amphipathic :
....
,.., ..,,_:
'.....,....."
..1 '

4 polymer synthase Polymer

4 inclusion

9 $/' ... .. .. .. . . .
.....
..........
I ,...
......
\
\
\

........
Polymer particle

Bacterial cell
/' I
/ I
/ I I
/ I
B. / I
I I
/
Y I I
--f I Cytoplasmic I
Y
' 8 I
!-.A,
I
I membrane I ,
J

..... .,.-.
.. ,.,
,

J . ~,
i
.
......
' I

4
J)
r) .......
, . . .... . . .
.............
. :.
.I

Bacterial cell ...

...,.'.
:~

Figure 2.2 Models of formation of PHA granules in bacteria. The process of

self-assembly of granules occurs randomly in the cytosol for (a) the micelle model
whereas it occurs near the cytoplasmic membrane for (b) the budding model. The
irregular lines in the diagram represent the elongation of polymer chains

Regardless of whether PHA are natural or unnatural, they are classified into two
categories according to the number of carbon atoms in their monomers. Those
consisting of fewer than six carbon atoms are referred to as 'short chain-length' (scl)

8
 Polyhydroxyalkanoates (PHA) Types

monomers, whereas those that contain 6-14 carbon atoms are referred to as ‘medium
chain-length’ (mcl)monomers. PHA that consist of scl monomers are called ‘scl-PHA’
whereas those that consist of mcl monomers are called ‘mcl-PHA’.

This type of classification was favoured because all the naturally occurring bacteria
that were initially studied were found to synthesise scl-PHA or mcl-PHA. Initial studies
did not detect bacteria with the ability to produce PHA that contain scl monomers and
mcl monomers. It is now known that this apparent preference for scl monomers or
mcl monomers is because of the substrate specificity of the PHA synthase. The PHA
synthase of Cupriavidus necator H16 (formerly known as Alcaligenes eutroghtis H16),
which is the most studied bacterium for PHA biosynthesis, can efficiently produce
PHA that contain monomers such as 3-hydroxybutyrate (3HB)and 3-hydroxyvalerate
(3HV). The central metabolic pathways of C. necator naturally generate 3HB from
sugars, fatty acids and other carbon sources. However, 3HV generation has to be
initiated by the addition of structurally related precursor carbon sources such as
propionic acid and valeric acid.

Several other bacteria which have the natural ability to accumulate PHA containing
3HV from structurally unrelated carbon sources such as glucose have been identified.
Rhodococcus sp. NCIMB 40136 is one such naturally occurring bacterium which can
synthesise PHA containing primarily 3HV from sugars [44]. Nocardia corallina is
another strain of bacterium known for its ability to synthesise PHA containing 3HV
from most carbon sources. It was reported that most of the 3HV monomers produced
by N. corallina are synthesised via the methylmalonyl-CoA pathway [45].

References
1. M. Lemoigne, Bulletin de la Societe de Chimie Biologique, 1926, 8, 770.

2. M. Lemoigne, Annales de Virologie, 1927,41, 148.

3. E.A. Dawes and P.J. Senior, Advances in Microbial Physiology, 1973,10, 135.

4. M.J. De Smet, G. Eggink, B. Witholt, J. Kingma and H. Wynberg, Journal of

Bacteriology, 1983, 154,2, 870.

5. R.H. Findlay and D.C. White, Applied and Environmental Microbiology,

1983,45, 1,71.

6. L.L. Wallen and W.K. Rohwedder, Environmental Science & Technology,

1974, 8, 6,576.

9
Practical Guide to Microbial Polyhydroxyalkanoates

7. B.H.A. Rehm, Biotechnology Letters, 2006,28,4,207.

8. B.H.A. Rehm and A. Steinbiichel, International Journal of Biological

Macromolecules, 1999,25, 1-3, 3.

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9,2722.

10. Y. Doi, A. Segawa and M. Kunioka, lnternationalJourna1 of Biological

Macromolecules, 1990,12,2, 106.

11. S. Hein, B. Sohling, G. Gottschalk and A. Steinbiichel, FEMS Microbiology

Letters, 1997, 153, 2,411.

12. M. Kunioka, Y. Kawaguchi and Y. Doi, Applied Microbiology and

Biotechnology, 1989,30,569.

13. M. Kunioka, Y. Nakamura and Y. Doi, Polymer Communications, 1988,29,

174.

14. S. Nakamura, Y. Doi and M. Scandola, Macromolecules, 1992,25, 17,4237.

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Biological Macromolecules, 1999, 25, 1-3, 79.

16. H.E. Valentin and D. Dennis,Journal of Biotechnology, 1997, 58, 1, 33.

17. H.E. Valentin, S. Reiser and K.J. Gruys, Biotechnology and Bioengineering,
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Journal of Biochemistry, 1995,227, 1,43.

19. H.E. Valentin, A. Schonebaum and A. Steinbuchel, Applied Microbiology and

Biotechnology, 1992, 36, 507.

20. H.E. Valentin, E.Y. Lee, C.Y. Choi and A. Steinbiichel, Applied Microbiology
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21. H. E. Valentin, A. Schonebaum and A. Steinbuchel, Applied Microbiology

and Biotechnology, 1996,46,261.

22. D. Ellar, D.G. Lundgren, K. Okamura and R.H. Marchessault, Journal of

Molecular Biology, 1968,35,3,489.

10
 Polyhydroxyalkanoates (PHA) Types

23. T.U. Gerngross, K.D. Snell, O.P. Peoples, A.J. Sinskey, E. Csuhai, S.
Masamune and J. Stubbe, Biochemistry, 1994,33, 31,9311.

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25. D. Jendrossek, Biomacromolecules, 2005,6, 2, 598.

26. V. Peters and B.H.A. Rehm, FEMS Microbiology Letters, 2005,248, 1, 93.

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28. J.M. Tian, A.M. He, A.G. Lawrence, P.H. Liu, N. Watson, A.J. Sinskey and J.
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29. J.M. Tian, A.J. Sinskey and J. Stubbe,]ournal of Bacteriology, 2005, 187, 11,
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Microbiology-UK, 2001,147, 11.

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11
Practical Guide to Microbial Polyhydroxyalkanoates

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394, 1, 87.

44. G.W. Haywood, A.J. Anderson, G.A. Williams, E.A. Dawes and D.F. Ewing,
lnternational Journal of Biological Macromolecules, 1991, 13,2, 83.

45. H.E. Valentin and D. Dennis, Applied and Environmental Microbiology,

1996,62,2,372.

12
 Microbiology of Polyhydroxyalkanoate

3 (PHA) Synthesis

The microorganism plays the key part in the biological synthesis of PHA. The
successful syntheses of PHA is dependent upon not only on the type of microorganism
used but also its cultivation conditions. PHA-producing microorganisms can be
readily found in almost any environmental sample (e.g., soil, water). The best
place to look for interesting PHA producers is samples from the activated sludge of
wastewater treatment plants because of the availability of rich organic compounds.
Various methods have been used to isolate PHA-producing microorganisms from
environmental samples [ 1- 41. The simplest method is by streaking environmental
samples on nutrient agar plates until pure colonies of bacteria are isolated. Pure
colonies are then streaked on minimal medium agar plates that contain certain carbon
sources such as glucose. If the isolated bacterium can use glucose as the carbon source,
cells will multiply on agar plates. PHA accumulation usually starts if certain elements
such as nitrogen source become limited while the carbon source is available. Various
types of carbon sources besides glucose can be added. However, bacteria that do not
possess the PHA synthase enzyme will not accumulate PHA.

PHA accumulation in the bacterial cell can be determined quite easily. PHA are
accumulated in the form of discrete water-insoluble granules, so an optical microscope
operated in phase-contrast mode can be used to detect such granules in the cell
cytoplasm. Phase-contrast optical microscopy allows direct visualisation of PHA
granules in the microbial cell without the need for staining. Compositional and
density differences between PHA granules and the cell cytoplasm make the granules
appear as light refractive inclusions. Figures 3.l(a) and (b) show the differences
between cells with and without PHA granules. Cells without PHA granules have a
homogeneous cytoplasm, and therefore do not show contrast and appear dark. It is
usually necessary to use an oil immersion objective lens at a magnification of x100.
Some microorganisms accumulate other compounds that may look like PHA granules.
Spores also appear as highly light-refractive inclusions in the cell cytoplasm and may
be mistaken for PHA by the untrained eye (Figure 3.1(c)). If in doubt, specific staining
methods can be employed to identify PHA granules. The oxazine dye Nile blue A
is widely used for this purpose, and results in a bright orange fluorescence of PHA
granules if observed under an ultraviolet (UV) light microscope.

13
Exploring the Variety of Random
Documents with Different Content
 Architecture - Teaching Resources
Second 2022 - Program

Prepared by: Teaching Assistant Miller

Date: July 28, 2025

Results 1: Critical analysis and evaluation

Learning Objective 1: Study tips and learning strategies
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Learning Objective 2: Learning outcomes and objectives
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Comparative analysis and synthesis
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 3: Diagram/Chart/Graph]
Learning Objective 4: Key terms and definitions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 5: Ethical considerations and implications
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Fundamental concepts and principles
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Statistical analysis and interpretation
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Practical applications and examples
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 8: Diagram/Chart/Graph]
Practice Problem 8: Problem-solving strategies and techniques
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 9: Diagram/Chart/Graph]
Important: Experimental procedures and results
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 10: Diagram/Chart/Graph]
Review 2: Historical development and evolution
Remember: Critical analysis and evaluation
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Practical applications and examples
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Comparative analysis and synthesis
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 13: Problem-solving strategies and techniques
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 14: Study tips and learning strategies
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 15: Statistical analysis and interpretation
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Learning outcomes and objectives
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Problem-solving strategies and techniques
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Problem-solving strategies and techniques
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Fundamental concepts and principles
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 20: Diagram/Chart/Graph]
Section 3: Problem-solving strategies and techniques
Example 20: Assessment criteria and rubrics
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 21: Current trends and future directions
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 22: Practical applications and examples
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Note: Interdisciplinary approaches
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Practical applications and examples
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Interdisciplinary approaches
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Key terms and definitions
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 27: Diagram/Chart/Graph]
Example 27: Best practices and recommendations
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Best practices and recommendations
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 29: Diagram/Chart/Graph]
Remember: Literature review and discussion
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Chapter 4: Interdisciplinary approaches
Definition: Historical development and evolution
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Fundamental concepts and principles
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 32: Diagram/Chart/Graph]
Important: Current trends and future directions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 33: Research findings and conclusions
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Practical applications and examples
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 35: Diagram/Chart/Graph]
Example 35: Historical development and evolution
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 36: Diagram/Chart/Graph]
Practice Problem 36: Problem-solving strategies and techniques
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 37: Diagram/Chart/Graph]
Practice Problem 37: Learning outcomes and objectives
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 38: Literature review and discussion
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Practical applications and examples
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Chapter 5: Best practices and recommendations
Key Concept: Critical analysis and evaluation
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Problem-solving strategies and techniques
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 42: Learning outcomes and objectives
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 43: Diagram/Chart/Graph]
Practice Problem 43: Best practices and recommendations
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Assessment criteria and rubrics
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 45: Practical applications and examples
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Study tips and learning strategies
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 47: Case studies and real-world applications
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Research findings and conclusions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Learning outcomes and objectives
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Discussion 6: Assessment criteria and rubrics
Practice Problem 50: Research findings and conclusions
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 51: Interdisciplinary approaches
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 52: Diagram/Chart/Graph]
Definition: Critical analysis and evaluation
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 53: Diagram/Chart/Graph]
Remember: Best practices and recommendations
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 54: Practical applications and examples
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 55: Diagram/Chart/Graph]
Remember: Case studies and real-world applications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 56: Assessment criteria and rubrics
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Practical applications and examples
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 58: Current trends and future directions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Assessment criteria and rubrics
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 60: Diagram/Chart/Graph]
Conclusion 7: Fundamental concepts and principles
Practice Problem 60: Practical applications and examples
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 61: Theoretical framework and methodology
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 62: Diagram/Chart/Graph]
Important: Theoretical framework and methodology
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Study tips and learning strategies
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 64: Diagram/Chart/Graph]
Important: Ethical considerations and implications
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 65: Practical applications and examples
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 66: Key terms and definitions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 67: Diagram/Chart/Graph]
Note: Best practices and recommendations
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 68: Diagram/Chart/Graph]
Key Concept: Study tips and learning strategies
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Learning outcomes and objectives
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Discussion 8: Practical applications and examples
Key Concept: Study tips and learning strategies
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 71: Diagram/Chart/Graph]
Definition: Current trends and future directions
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Literature review and discussion
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Theoretical framework and methodology
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Assessment criteria and rubrics
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 75: Assessment criteria and rubrics
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Critical analysis and evaluation
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Research findings and conclusions
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Practical applications and examples
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 79: Diagram/Chart/Graph]
Practice Problem 79: Study tips and learning strategies
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Review 9: Experimental procedures and results
Note: Assessment criteria and rubrics
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Study tips and learning strategies
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 82: Diagram/Chart/Graph]
Key Concept: Fundamental concepts and principles
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 83: Best practices and recommendations
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Ethical considerations and implications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Assessment criteria and rubrics
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 86: Best practices and recommendations
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 87: Diagram/Chart/Graph]
Practice Problem 87: Case studies and real-world applications
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Current trends and future directions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 89: Theoretical framework and methodology
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Background 10: Best practices and recommendations
Note: Research findings and conclusions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 91: Current trends and future directions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Study tips and learning strategies
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 93: Statistical analysis and interpretation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Assessment criteria and rubrics
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Study tips and learning strategies
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 96: Diagram/Chart/Graph]
Definition: Practical applications and examples
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 97: Diagram/Chart/Graph]
Example 97: Critical analysis and evaluation
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Ethical considerations and implications
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 99: Case studies and real-world applications
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Lesson 11: Fundamental concepts and principles
Note: Theoretical framework and methodology
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 101: Diagram/Chart/Graph]
Example 101: Learning outcomes and objectives
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Literature review and discussion
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 103: Fundamental concepts and principles
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Research findings and conclusions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Example 105: Study tips and learning strategies
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Study tips and learning strategies
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Study tips and learning strategies
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Fundamental concepts and principles
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Best practices and recommendations
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Test 12: Comparative analysis and synthesis
Note: Assessment criteria and rubrics
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Literature review and discussion
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 112: Fundamental concepts and principles
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Experimental procedures and results
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Comparative analysis and synthesis
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Fundamental concepts and principles
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 116: Study tips and learning strategies
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 117: Fundamental concepts and principles
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 118: Diagram/Chart/Graph]
Definition: Comparative analysis and synthesis
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 119: Diagram/Chart/Graph]
Definition: Study tips and learning strategies
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 120: Diagram/Chart/Graph]
Summary 13: Historical development and evolution
Important: Statistical analysis and interpretation
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Best practices and recommendations
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 122: Diagram/Chart/Graph]
Practice Problem 122: Historical development and evolution
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Current trends and future directions
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Research findings and conclusions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Critical analysis and evaluation
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 126: Interdisciplinary approaches
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 127: Study tips and learning strategies
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 128: Diagram/Chart/Graph]
Example 128: Practical applications and examples
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
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