Corn Straw

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Xylanase, Biochar, Sugarcane, Bagasse, Enzymes, Grains, Soil Temperature, Sweets,

Wheat Bran

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Function and Dysfunction of the Rumi-

nant Forestomach
Walter Grünberg, Peter D. Constable, in Food Animal Practice (Fifth Edition), 2009

Analysis of Rumen Fluid

Color
This is dependent on the diet; corn silage and straw diets produce yellow/brown
rumen contents, concentrates produce an olive/brown color, and pasture produces
a green color. A black/green color usually indicates ruminal stasis, whereas a milky
gray/brown color is often observed in lactic acidosis.

Odor
Rumen fluid normally has a slightly aromatic, unobjectionable odor. Acidic/sour
smells are suggestive of lactic acidosis, whereas rumen putrefaction produces a
rotting odor.

Consistency
Rumen contents are normally slightly viscous. Excessive viscosity indicates signif-
icant saliva contamination, and the sample should be discarded because it is not a
valid representation of rumen fluid. A watery sample with little particulate matter
indicates anorexia and is usually associated with reduced protozoal and bacterial
numbers. Rumen fluid that has numerous stable bubbles that do not coalesce is
indicative of frothy bloat.

pH
Rumen pH is best measured at its lowest value (i.e., 2-4 hours after feeding a
concentrate meal or 4-8 hours after offering a fresh total mixed ration). Rumen
pH is determined using a portable pH meter or pH papers. The normal pH of
grass-fed ruminants is 6-7. A pH value of 5.5-6 is seen in cattle on high-grain
diets or pasture-fed cattle with early lactic acidosis. pH values less than 5.5 are
virtually pathognomonic for lactic acidosis, although feedlot cattle well adapted to
high-grain diets can have rumen pH values approaching 5. A reduced feed intake
of 2 or more days’ duration often increases rumen pH up to 7 or 8. Rumen pH
values exceeding 8 are due to (1) contamination with saliva (in which case the
sample should be discarded), (2) severe putrefaction of ingested protein associated
with prolonged rumen stasis, or (3) urea toxicity. For diagnosing SARA, the most
commonly recommended cut points are a pH = 5.5 for a diagnosis as SARA positive
and a pH = 5.8 for a diagnosis as SARA negative. The suitable number of samples
to be analyzed for a herd or feeding group screening has been determined to be 12
animals of the specific risk group. With three or more of the 12 samples having a
pH of 5.5 or lower, the prevalence of SARA should be regarded as high and thus the
specific group considered as SARA affected.27,29

Methylene blue reduction

This test measures the reducing ability of anaerobic ruminal bacteria. Ten ml of
fresh rumen fluid is added to a test tube containing 0.5 ml of a 0.03% solution of
methylene blue, and the time taken for the solution to clear is measured. In cattle the
clearance time is normally between 2 and 6 minutes, the faster rate being observed
in cattle on high-grain diets. Clearance times greater than 10 minutes indicate
inadequate anaerobic bacterial numbers, and rumen transfaunation is required. The
clearance time is much faster in sheep (usually 1 to 4 minutes). This test is invalid at
rumen pH less than 5.5.

Protozoa
One drop of fresh rumen contents is placed on a slide and examined under low
power (×40). Normal rumen fluid contains greater than 40 protozoa per low-power
field, with actively moving protozoa that can be broadly characterized into three
sizes (small, medium, large). Low protozoal numbers (<8 per low-power field),
nonmotile protozoa, or loss of population heterogeneity all indicate an abnormal
intraruminal environment. Transfaunation is indicated if these abnormalities are
identified, particularly if the methylene blue reduction time is prolonged. It is not
usually necessary to identify the protozoal species present, but occasionally it is
valuable to differentiate Isotrichids (formerly Holotrichs) which are usually the larger
protozoa, from Oligotrichs (Entodiniomorphs), which are smaller and more resistant
to pH values of less than 6. Protozoal energy stores can be assessed by adding one
drop of Lugol’s iodine to one drop of rumen fluid on a slide. Healthy protozoa are
almost uniformly stained by iodine, whereas protozoa that have been starved have
diminished starch stores, evidenced by decreasing numbers of starch granules.

Chloride concentration
The rumen fluid should be centrifuged, and the supernatant submitted for determi-
nation of the chloride concentration, which is normally 10-25 mEq/L in cattle and
less than 15 mEq/L in sheep. Elevated rumen chloride concentrations result from
abomasal reflux (internal vomition), ileus, or high salt intake. High Cl concentrations
are most consistent with an obstruction at or distal to the pylorus. The physical ex-
amination will hopefully provide evidence as to whether this obstruction is functional
or pathologic.

Rumen osmolality
The rumen osmotic pressure is actively controlled by the ruminant and closely
approximates serum osmotic pressure. A constant osmotic pressure ensures home-
ostatic conditions for ruminal microbes, which are susceptible to lysis or swelling if
large and rapid variations in rumen osmolality occur.35

Gram stain
Rumen fluid normally contains a large heterogeneous population of bacteria, which
are predominantly gram negative. Lactic acidosis produces a more uniform bacterial
population, which is predominantly gram positive.

Other tests
A number of other laboratory tests such as sedimentation time, cellulose digestion
test, and total titratable acidity have also been used in the examination of rumen
fluid. These tests seldom add information than that obtained earlier, and their
routine use is not recommended.

> Read full chapter

Carbon-Rich Wastes as Feedstocks for
Biodegradable Polymer (Polyhydrox-
yalkanoate) Production Using Bacteria
Jasmina Nikodinovic-Runic, ... Kevin E. O Connor, in Advances in Applied Microbi-
ology, 2013

8.2 Other lignocellulosic waste and bran

A great portion of agricultural-crop residues is lignocellulosic fibrous waste. As an
example, the yearly cultivation of 600 million tons of palm biomass generates a waste
stream that contains about 90% of the entire palm (empty fruit bunches, cut-offs,
fibers, etc.) (Kumar, Singh, & Singh, 2008). In the last decade, increasing efforts
have been dedicated worldwide to implement biorefinery plants for the conversion
of lignocellulosic and cellulosic waste to starting materials for the biotechnological
production of bioethanol, biopolymers, and a range of fine chemicals (Liu, Abra-
hamson, & Scott, 2012; Menon & Rao, 2012). Lignocellulosic materials consist of
lignin (a complex polyphenolic structure), cellulose ( -d-1,4-glucan), and hemicellu-
lose (d-arabinose, d-xylose, d-mannose, d-glucose, d-galactose, and sugar alcohols)
fibers, in differing portions and represent the most abundant renewable resource
on our planet (Kumar et al., 2008; Menon & Rao, 2012; US Department of Energy,
2011).

Depending on the location and the availability, a number of sources of lignocellulosic

materials can be found: rice straw, corn straw, vegetable waste, pea husks, rice husks,
and dry dead leaves of sugarcane. While some of these are still directly burned in
the fields, contributing to air pollution, controlled burning of these wastes in boilers
for generation of renewable energy (steam and electricity) is a preferred option.
However, utilizing this material as the feedstock for the value-added products is a
much more favorable goal as it generates higher value and more versatile products.
The major obstacles to PHA production from lignocellulose waste are the resistance
of such materials to hydrolytic treatments to release the fermentable hexoses and
pentoses and the presence of inhibitory molecules (e.g., phenolics from lignin)
(Chakar & Ragauskas, 2004; Martinez, Rodriguez, York, Preston, & Ingram, 2000).
Indeed, the utilization of pentoses still poses as a major challenge not only for PHA
production but also for fermentative production of fuel and chemicals (Bertrand,
Ramsay, Ramsay, & Chavarie, 1990; Keenan, Nakas, & Tanenbaum, 2006; Lee, 1998;
Meijnen, de Winde, & Ruijssenaars, 2008).

Bran is the hard outer layer of cereals and is often produced as a by-product of
milling in the production of refined grains. Bran is present in and may be milled
from any cereal grain, including rice, corn (maize), wheat, oats, barley, and others.
Due to the high volumes of cereal production worldwide, bran was identified as a
by-product that could provide a good accessible carbon source for PHA production
(Akaraonye, Keshavarz, & Roy, 2010; Van-Thuoc et al., 2008). Van-Thuoc et al. suc-
cessfully grew Halomonas boliviensis LC1 on wheat bran hydrolysate to produce a
total biomass of 3.2 g/l, a PHB concentration of 1.1 g/l (33.8% cdw) (Van-Thuoc et al.,
2008). In addition, inexpensive extruded rice bran supplemented with extruded corn
starch (1:8 ratio) supported Haloferax mediterranei during fed-batch fermentation in
5 l reactor growth of 140 g/l, P(3HB-co-3HV) concentration of 77.8 g/l and polymer
content of 55.6% cdw (Huang et al., 2006).

> Read full chapter

The Effects of Mulch and Nitrogen Fer-

tilizer on the Soil Environment of Crop
Plants
Xiukang Wang, ... Zhanbin Li, in Advances in Agronomy, 2019

2.2 Soil Temperature

Both the composition and quantity of surface mulch affect how it modifies the
soil temperature. Here, we discuss how mulch composed of different materials
influences the soil temperature. Crop residue mulch significantly decreased the
maximum soil temperature by approximately 2–7 °C at a depth of 15 cm in the early
growing season (Olasantan, 1999), while corn straw mulch applied to the soil surface
reduced the maximum temperature but increased the minimum diurnal soil tem-
perature (Cadavid et al., 1998; Jun et al., 2014). Liu et al. (2014e) reported lower soil
temperatures during the warmer seasons and higher temperatures during the colder
seasons when rice straw mulch was applied compared to the no-mulch treatment.
The application of plastic mulch has been found to significantly increase the soil
temperature in the subsoil layers compared to the bare surface (Gan et al., 2013; Li et
al., 2017; Steinmetz et al., 2016; Sui et al., 1992). Plastic mulch increased soil temper-
atures, and the highest soil temperatures were observed when the soil was covered
with black plastic mulch (Filipović et al., 2016). Jiang et al. (2016) reported that plastic
film mulching increased the near-surface soil temperatures by approximately 1 °C,
while partial plastic film mulching with furrow-ridge tillage was found to increase
the soil temperature by 1.3 °C compared with non-mulching treatments (Gong et
al., 2015). Gravel mulch can increase or decrease soil temperatures; specifically,
soil temperatures have been found to increase during warmer periods and decrease
during cold months. An experiment conducted using tea plants showed that gravel
mulch increased the soil temperature in the first 2 years of growth, but no change
was observed when the tea canopy had developed to 40% coverage (Othieno and
Ahn, 1980). Gravel mulch was shown to cause significant increases of 0.97 °C at a
depth of 3 cm and 1.5 °C at 10 cm (Nachtergaele et al., 1998). Quadratic and cubic
relationships between the particle size of gravel mulch and soil temperature were
observed in an experiment with watermelon (Xie et al., 2010). Gravel and plastic
film mulching can significantly increase topsoil temperatures (Bu et al., 2013; Li,
2003; Luo et al., 2015). In the tropics, soil temperatures were found to decrease
with mulching treatments, increasing crop yields (Awan, 1964). Furthermore, the
application of thick newspaper mulch reduced the soil temperature compared to
the bare soil surface (Monks et al., 1997), while mulching with dark-colored paper
increased the temperature of the soil above that attained with light-colored paper
mulch (Haapala et al., 2015).

Plastic film is the principal mulch type, as it can increase the near-surface soil
temperature by up to 6 °C and provides other benefits for crop growth during the
growing seasons. Black plastic mulches greatly increase soil temperatures because
they can absorb and re-emit infrared radiation as heat energy or long-wavelength
thermal infrared radiation (Filipović et al., 2016; Lamont, 2005; Zhang et al., 2015),
while white plastic mulches are generally used in high-temperature regions to obtain
slightly higher soil temperatures compared to bare land (Briassoulis et al., 2015;
Heißner et al., 2005). However, the effects of mulching on soil temperatures are
limited by the soil depth, becoming non-significant below 40 cm (Díaz-Hernández
and Salmerón, 2012; Li et al., 1999; Yang et al., 2006). There are few references on
the effects of nitrogen fertilizer on soil temperature, but nitrogen fertilizer has been
shown to indirectly increase the soil temperature by increasing SOM decomposition
rates or fertilizer decomposition (Brackin et al., 2015; Paustian et al., 2000). Meng
et al. (2005) reported that basal and supplementary nitrogen fertilizer application
did not significantly change the soil temperature. Soil temperatures increase when
polymer-coated urea fertilizers are used because they release more nitrogen than
other granular fertilizers (Halvorson et al., 2014). However, nitrogen release patterns
and rates may change depending on the soil moisture and other field conditions. We
might conclude that there was no significant effect of nitrogen fertilizer application
on soil temperature. There are currently quite few studies on the interactive effects
of mulching and nitrogen fertilizer on soil temperature, and there is therefore much
work to do in the future.

> Read full chapter

Carbohydrate-based food processing
wastes as biomass for biorefining of bio-
fuels and chemicals
B. Kamm, in Handbook of Waste Management and Co-Product Recovery in Food
Processing, Volume 2, 2009

20.8.2 Lignocellulosic feedstock biorefinery

Among the potential large-scale industrial biorefineries, the lignocellulose feedstock
(LCF)-biorefinery will most probably be pushed through with highest success. On the
one hand, the raw material is optimal (straw, reed, grass, wood, paper-waste etc.), on
the other hand, conversion products are in a good position on the traditional petro-
chemical as well as on the future bio-based product market. An important point for
utilisation of biomass as chemical raw material is the cost of raw material. Currently,
the costs for corn stover or straw are: 30 USD/tonne; for corn 110 USD/tonne (3
USD/bushel) (Dale, 2002).

Lignocellulose materials consist of three primary chemical fractions or precursors:

a) hemicellulose/polyoses, a sugar polymer of predominantly pentoses; b) cellulose,
a glucose polymer; and c) lignin, a polymer of phenols (Fig. 20.11).

Fig. 20.11. A possible general equation of conversion at the lignocellulose feedstock

biorefinery.

The lignocellulosic biorefinery regime has a distinct advantage for genealogical trees
in that the natural structural and structural elements are preserved, the raw materials
are also cheap, and a large variety of products is possible (Fig. 20.12). Nevertheless,
there is still a need for development and optimisation of these technologies, e.g.
in the field of separation of cellulose, hemicellulose and lignin as well as lignin
utilisation in the chemical industry.
Fig. 20.12. Lignocellulose feedstock biorefinery (Kromus et al., 2006).

Furfural and hydroxymethylfurfural, in particular, are interesting products. Furfural

is the starting material for the production of nylon 6.6 and nylon 6. The original
process for the production of nylon 6.6 was based on furfural. The last of these
production plants was closed in 1961 in the USA for economic reasons (the artificially
low price of petroleum). Nevertheless, the market for nylon 6 remains large.

However, there are still some unsatisfactory parts within the LCF process, such as
utilisation of lignin as fuel, adhesive or binder, because the lignin scaffold contains
considerable amounts of mono-aromatic hydrocarbons, which, if isolated in an
economically efficient way, could add significantly to the value of the primary
processes. It should be noticed that there are obviously no natural enzymes to split
the naturally formed lignin into basic monomers as easily as for the also naturally
formed polymeric carbohydrates or proteins (Ringpfeil, 2001).

An attractive accompanying process to the biomass nylon process is the already

mentioned hydrolysis of cellulose to glucose and the production of ethanol. Certain
yeasts give a disproportionate yield of the glucose molecule during their gener-
ation of ethanol to glucose which practically shifts their entire reduction ability
into ethanol and makes the latter obtainable in 90% yield (w/w; according to the
theoretical stoichiometric calculation).

Based on recent technologies, a plant was conceived for the production of the main
products furfural and ethanol from LC feedstock for the area West Central Missouri,
USA. Optimal profitability can be reached with a daily consumption of about 4360
tonne of feedstock. Annually, the plant produces 47.5 million gallon (180 million l)
of ethanol and 323 000 tonne of furfural (Van Dyne, 1996).

Ethanol may be used as a fuel additive and is also a connecting product for a
petrochemical refinery. Ethanol can be converted into ethene by chemical methods.
As is well known, petrochemically produced ethene is the basis today of a whole
series of large-scale technical chemical syntheses for the production of important
commodities, such as polyethylene, or polyvinylacetate. Further petrochemically
produced substances can similarly be manufactured by microbial substantial conver-
sion of glucose, such as hydrogen, methane, propanol, acetone, butanol, butandiol,
itaconic acid, succinic acid (Zeikus et al., 1999; Vorlop et al., 2006; Werpy et al., 2005).
DuPont has entered a six-year alliance with Diversa in a biorefinery to produce sugar
from husks, straw and stovers, and to develop processes to co-produce bioethanol
and value-added chemicals, such as 1,3-propandiol (Chem World, 2003).

Through metabolic engineering, an Eschericia coli K12 micro-organism produces

1,3-propandiol (PDO), in a simple glucose fermentation process developed by
DuPont and Genencor. In a pilot plant operated by Tate & Lyle, the PDO yield reaches
135 g l−1 at the rate of 4 g l−1 h−1. PDO is used for the production of polytrimethylene
terephthalate (PTT), a new polymer which is used for the production of high-quality
fibres branded Sorona (DuPont, 2004). Production is predicted to reach 500 kt per
year by 2010.

> Read full chapter

Nutrition and Fertilization

Godofredo Cesar Vitti, ... Wellington Sacco Altran, in Sugarcane, 2015

Ratoon fertilization: N–K2O and B fertilization

Prior to defining the amounts of N and K2O, it is necessary to know about the areas’
administration as far as harvesting, burnt cane and raw cane.

a. Burnt-cane: N fertilization is based on the harvested yield, while K2O, is

based on yield and soil assessment (ratoon sample), whenever available, using
approximately 1.0 kg/t of sugarcane and between 1.3 and 1.5 kg/t of sugarcane
for K2O, keeping the reason between N/K2O in the range of 1.0 for 1.3–1.5,
according to Tables 4.24 and 4.25.Table 4.24. Mineral fertilization of burnt
ratoon in relation to yield expectations.Yield Expectations (t/ha)N (kg/ha)K2O (kg/ha)-
65–8080100–12081–100100130–150&gt;100120160–180Table 4.25. Potassium fertil-
ization recommendation for burnt cane-ratoon based on soil assessments.K
(mmolc/dm3)K2O (kg/ha)&lt;1.5150–1801.6–3.0110–140&gt;3.080
b. Raw-cane: In the raw cane harvesting system (without straw burning), a 10–15-
 t/ha raw matter (RM) build-up occurs. In Table 4.26, the RM mass for raw cane
straw and the respective amounts of nutrients and structural carbohydrates
are represented.Table 4.26. Raw matter mass for raw sugarcane straw, amount
of nutrients and structural carbohydrates, on samples collected in 1996 and in
the remainder straw in 1997.YearRMNPKCaMgSCHemicelluloseCelluloseLigninCel-
lular ContentC/NC/SC/P(t/ha)(kg/ha)199613.9 to64 to6.6 to66 to25 to13 to9 to6.255 to3.747
to5.376 to1.043 to3.227 to97 to695947199710.8 b53 to6.6 to10 b14 b8 b8 to3.642 b943 b6.619
to1.053 to2.961 b68 b455552Source: Oliveira
et al. (1999).
The data shown in Table 4.26 point out a differentiated crop management for ratoon
fertilization for the following reasons:

High level relationship between C/N, C/P and C/S, pointing at low mineral-
ization of straw organic matter, even one year after the cutting;
Paralyzing of anions (N, P and S) inside the straw;
Mineralization of the mineral cationic nutrients (K, Ca and Mg), particularly K,
giving approximately 50 kg/ha of K back to the soil system; and
Clear increase in microbe activity, particularly ureases, responsible for NH3
losses in urea through volatilization.

Due to these considerations regarding the supply, especially the supply of N, two
factors should be paid attention to: dose and source.

Regarding dose, it should be increased by at least 30%, i.e., 3 kg of N/t of produced
sugarcane. As to the source of N, the use of urea on the surface is prohibitive, since
losses may go up to 70%, as observed by Lara Cabezas et al. (1997) when urea is
superficially applied on corn straw. Therefore, regarding the use of urea there are
only three options:

Partial burying of urea;

Usage of “protected” urea, with urease inhibitors; or
Mixing of urea with ammonium sulfate, for example formulation 32-00-00-12,
resulting from the equitable mixture of those two sources.

Regarding potassium fertilization, it has been observed that the straw releases from
40 to 50 kg/ha of K, which is deducted from mineral fertilization. Thus, the K2O doses
in raw cane may be of the order of 0.8–1.0 kg/t of harvested cane K2O, resulting in
the relation N/K2O of 1.0–1.3/1.0. Thus, for a production of 100 t/ha of raw harvested
cane, the doses of N and K2O would be, respectively, 130 and 80 to 100 kg/ha.

For ratoon, the supply of the micronutrient boron is essential and, to a certain
degree, so is molybdenum; these micronutrients have mass flow mechanisms (travel
with water) similar to N and K2O.

Phosphate-rich fertilization in ratoon is widely argued, mainly due to the absence or

low mobility of in the soil, as well as due to the acidification of the rood zone. An
adequate supply of P2O5 during the implementation of the crop is necessary, both in
full area and in the furrow, in order to make it expandable in the ratoon. Neverthe-
less, for areas where P has not been adequately managed during implementation,
we suggest applying about 30 kg/ha of P2O5, but in soil conditions with acidity duly
corrected (V%>50) and when P in the soil is less than 15 mg/dm3 in resin.

Sugarcane fertilization with sulfur is paramount, particularly in areas with greater

response probabilities, i.e.:

Areas with no organic residue application nor gypsum;

Areas further from the mill (without SO2 from bagasse burning); and
Sandier soils with low levels of organic matter.

When gypsum is applied, the 1.0 t/ha dose, for example, is enough for the supply
of S for at least two cuts and its application should be renewed in ratoon after the
third cut, when the levels of S in the soil for the 25- to 50-cm layers are lower than
15 mg/dm3.

In addition to this option, sulfur may be supplied during planting, through P2O5,
sources such as simple superphosphate (12% S), thermophosphate and magnesium
multiphosphate containing S. For ratoon, particularly when raw, that has a high C/S
relationship, S may be supplied through nitrogen-rich sources: ammonium sulfate
(24% S), a mixture of urea with ammonium sulfate, (12% S) and, in the event of
liquid fertilization, with vinasse, ajifer, or sulfuran (4% S).

> Read full chapter

Lignin
Fachuang Lu, John Ralph, in Cereal Straw as a Resource for Sustainable Biomaterials
and Biofuels, 2010

6.5.2 p-Coumarates in Grass Lignins

Studies on lignin have showed that lignins in some plants are acylated by various
acids: acetates in all hardwoods but at high levels in palms, kenaf, abaca, and sisal [-
32, 195, 196, 249]; p-hydroxybenzoates in palms, and Populus species (willow, aspen,
poplar) [33, 263–268]; and p-coumarates in all grasses [74, 269–271]. p-Coumarate
in grass lignins is mainly esterified to the -position of phenylpropanoid sidechains
of S units in lignin as demonstrated by NMR studies with corn and wheat straw
[74, 93]. Although some p-coumarates are esterified to arabinoxylans in immature
tissues, most p-coumarate accretion occurs in tandem with lignification [272, 273],
making p-coumarate accumulation a convenient indicator of lignin deposition in
grasses.
There are several pathways possible for introducing such acylation. However it has
now been rather unequivocally demonstrated for acetates in kenaf that acylation
occurs at the monolignol stage [32]. The acylation by other acids (p-hydroxyben-
zoate, p-coumarate) logically follow a similar pathway. Thus, sinapyl p-coumarate in
grass cell walls is then incorporated into the lignin polymer by polymerization and
copolymerization with the traditional monolignols. Acylated monolignols implicate
transferase enzymes in their synthesis. Researchers seem to be getting close to
obtaining the enzymes involved and their genes. Monolignol p-coumaroylation in
maize is via p-coumaroyl-CoA [274].

Acetylated lignin units may consists of up to 60% in kenaf lignin and p-coumarate
content may reach up to 18% in corn lignin. It is quite evident that these monolignol
conjugates can comprise a very high portion of the monomer pool for those lignins.
Therefore, these acylated monolignols should be considered as lignin precursors in
those plants in addition to the three monolignols (Fig. 6.2).

Acylation is, generally, heavy on syringyl units and little guaiacyl acetylation is
detected [57, 177, 275, 276]. The acylation of the monolignol -OH does not greatly
affect the course of the radical coupling reactions, but does markedly influence
postcoupling rearomatization reactions of the resulting quinone methide interme-
diates QM (Fig. 6.9) [277] particularly in the cases where the -OH would nor-
mally be involved in that rearomatization (via intramolecular nucleophilic trapping
reactions). For example, although homo-dehydrodimerization reactions are usually
greatly favored over cross-coupling reactions (due to simple chemical compatibility),
in vitro peroxidase-H2O2 initiated coupling of equimolar sinapyl alcohol (MS) and
sinapyl acetate MSE (R = acetate) produces an essentially statistical distribution
(1:2:1) of the - -dimers syringaresinol (S- - -S, from sinapyl alcohol + sinapyl
alcohol), tetrahydrofurans T-1a and T-2a (from sinapyl alcohol + sinapyl acetate), and
tetrahydrofurans T-3a and T-4a (from sinapyl acetate + sinapyl acetate) (Fig. 6.9) [32].

One initially surprising feature of p-coumarates on grass lignins is that they are
simply terminal pendant groups. Their free-phenolic nature is readily evident from
13C-NMR spectra [74]. Despite their phenolic nature, p-coumarate esters on lignin

units form few, if any, cross-linked structures mediated by radical coupling reactions
of the p-coumarate moiety. On their own, in vitro or under conditions where radical
generation capability is not limiting, p-coumarates will undergo radical coupling.
But, there is no evidence that they do so in grass cell walls. The reason for this is that
although p-coumarates interact with peroxidase to generate radicals, they quickly
undergo radical transfer reactions with other phenolics, particularly sinapyl alcohol
and syringyl units, producing more stable radicals (Fig. 6.18) [47, 277].
FIGURE 6.18. A diagram illustrating the possible mechanism by which p-coumarate
acts as an oxidative shuttle to enhance the oxidation of sinapyl alcohol during
lignification of grass cell walls

Adapted from Hatfield et al. [277]

Whether this facilitation of sinapyl alcohol oxidation is a primary role for p-

-coumarates in grasses (and analogously, p-hydroxybenzoates in aspen, poplar,
willow, and palms) is unknown. Although the above-mentioned in vitro studies
indicate that p-coumarate esters enhance the oxidation of sinapyl alcohol [279–281],
the artificial polymerization of syringyl-rich lignins into primary maize walls was
at times depressed by sinapyl p-coumarate because of accelerated inactivation of
peroxidase and disruption of ferulate–lignin cross-linking [282].

Regardless, the p-coumarate radical's preference to undergo radical transfer to

another phenolic present during lignification in grasses, over its radical coupling
propensity, results in p-coumarates that are indeed part of the lignin polymer, but
are not integrally incorporated into the polymer chains via radical coupling reactions
– at least in the typical syringyl-guaiacyl lignins in grasses, they remain simply as
free-phenolic pendant groups acylating the primary -OH of the lignin sidechain,
albeit on a variety of lignin structures.

> Read full chapter

Advances in Agronomy
Ren-kou Xu, ... Jun Jiang, in Advances in Agronomy, 2016

5.4 Effects on Soil Acidity

Considering their very heterogeneous properties, biochars of different types have
similar effects on the soil pH. Biochar-induced pHs are mostly neutral-to-basic and
studies have demonstrated that pH ranges between 6.2 and 9.6 with an average
of 8.1 (Chan and Xu, 2009). The liming effect of biochars has been discussed in
the literature as one of the most likely mechanisms behind increases in plant
productivity after biochar applications (Verheijen et al., 2009). Lower pH values in
soils (greater acidity) often reduce the CEC and thereby the nutrient availability. In
addition, for many of the soils of the humid tropics studied, reduced aluminum
toxicity by reducing the acidity is proposed as the likely chemical mechanism behind
plant productivity increases.

Statistically significant positive effects of biochar application to soils, especially

to acidic free-draining soils, on plant productivity suggest that one of the main
mechanisms behind them may be the liming effect. This would imply that biochars
with greater liming capacity can provide greater benefits to arable soils that require
liming, by being applied more frequently at lower application rates.

In two recent studies, biochars were prepared from nine plant residues – the
nonlegume materials of canola straw, wheat straw, corn straw, rice straw, rice hull,
and the legume straws of soybean, peanut, fava bean, and mung bean – and their
ameliorating effects on Ultisols and Oxisols were compared with incubation experi-
ments (Yuan and Xu, 2011, 2012). The pH of these biochars ranged from 6.4 to 10.4.
Incubation of the soils with the biochars increased soil pH. The ameliorating effects
of the biochars on soil acidity were correlated with their alkalinity, giving a good
linear correlation between soil pH and biochar alkalinity (R2 = 0.95) (Yuan and Xu,
2011), showing that biochar alkalinity was a key factor controlling its ameliorating
effect on acid soils. The biochars from the legume materials had greater ameliorating
effects than those from the nonlegume materials as a result of their higher alkalinity,
and are thus more appropriate as amendments for acid soils.
The carbonates and organic anions were two major forms of alkali in crop straw
biochars studied by Yuan et al. (2011a). Organic anions consumed proton through
association with H+ when acid was the input. The contribution of carbonates to
biochar alkalinity increased with rising pyrolytic temperature of the biochar, while
the contribution of organic anions decreased correspondingly (Yuan et al., 2011a).
Biochar alkalinity was also increased with rising pyrolytic temperature and thus
the ameliorating effect of biochars on acid soils increased with rising pyrolytic
temperature of the biochars (Yuan et al., 2011a). The optimum pyrolysis temperature
of 500°C was suggested for producing biochar amendments from crop straws for
acid soils because at this temperature, both organic anions and carbonates can
contribute significantly to the alkalinity of the biochars, and the biochars have
greater amelioration effects on soil acidity than those produced at 300°C (Yuan et al.,
2011a). In addition, at this temperature (500°C) a medium biochar production rate
can be achieved because the biochar production rate decreased with rising pyrolytic
temperature.

Biochars contain base cations of Ca2+, Mg2+, K+, and Na+, and the incorporation of
biochars increased exchangeable base cations of acid variable charge soils through
cation exchange reaction of these base cations with exchangeable H+ and Al3+ on soil
exchange sites, which also decreased soil exchangeable acidity (Yuan and Xu, 2011,
2012). When exchangeable H+ and Al3+ were released into soil solution, the alkaline
substances in biochars neutralized the acidity (H+ and Al3+). It was found that the
content of base cations in legume straw biochars was superior to that in nonlegume
straw biochars and thus incorporation of legume straw biochars into acid soils led
to more increase in soil exchangeable base cations (Yuan and Xu, 2012).

Ca, Mg, and K are nutrients for plants and are normally deficient in acid soils.
Therefore, the incorporation of crop straw biochars has not only corrected soil acidity
but has also improved the fertility of acidic variable charge soils. Biochar made from
the wood of white lead trees (Leucaena leucocephala (Lam.) de Wit) has an extremely
high pH (>9.0) and a high liming effect on acid soils (Jien and Wang, 2013). Applying
biochar improved the physicochemical and biological properties of highly weathered
soils, including significant increases in soil pH from 3.9 to 5.1, CEC from 7.41 to
10.8 cmol(+) kg−1, base cation percentage from 6% to 26%, and microbial biomass
carbon from 835 to 1262 mg kg−1.

Biochar's physical and chemical characteristics may significantly alter key soil
physical properties and processes and are, therefore, important to consider prior
to its application to soil. Most biochars have neutral-to-basic pH and many field
experiments show an increase in soil pH after biochar application when the initial
pH was low. Biochar have good potential to improve the CEC of soil. However,
the effectiveness and duration of this effect after addition to soils remain not well
understood.
> Read full chapter

FUNGI IN FRESHWATER HABITATS

CAROL A. SHEARER, ... JOYCE E. LONGCORE, in Biodiversity of Fungi, 2004

Collection
Chytrids are microscopic, cannot be collected by unaided sight, and usually are not
detected with standard microbiological methods such as dilution plating. They can
be found by examining their natural substrata under a microscope, but this can be
time-consuming and suitable natural substrata are not always easily found during
a collecting trip. Therefore, “baits” are commonly used to provide chytrids and
hyphochytrids with a fresh substratum to colonize. Baits are selected to represent
naturally occurring organic debris in pieces that are thin enough to allow viewing
with transmitted light on a microscope slide. Commonly used baits include chitin
(bits of purified shrimp exoskeleton or insect wings); cellulose (cellophane, lens
paper, white onion skin, and bleached corn straw); keratin (defatted, blond baby
hair; pieces of snake skin; or defatted wool fibers); and pollen grains of various
types, especially, but not limited to, pine (Pinus species), spruce (Picea species), and
sweetgum (Liquidambar species). Those baits, except pollen, are boiled from 3 to
5 minutes before using. Larger baits, such as seeds, fruits, and twigs, are used to
attract members of Blastocladiales and Monoblepharidales. Collection and baiting
need to be tailored to the group of fungi sought; detailed methods for use with
members of the various orders of chytrids and hyphochytrids are found in Fuller and
Jaworski (1987). Whisler (1987) offered advice regarding the Monoblepharidales and
Blastocladiales, and W. W. Martin (1987) explained methods of collecting parasites
of aquatic insects. Later in this chapter, we include methods that we have used
successfully for baiting for Chytridiales, Spizellomycetales, and Hyphochytriales.
Investigators should consult D. J. S. Barr (1987), Sparrow (1960), Emerson (1950),
and Couch (1939) for additional insights.

Organic debris and water can be collected from a study site and baited in the
laboratory. A small amount of debris (bottom organic matter amounting to one or
two waterlogged leaves, a small aquatic plant, or more than 10 cc of detritus) and
water from the same habitat are added to a deep, glass Petri plate or finger bowl.
Two pieces (about 0.5–1 cm × 0.5–1 cm) each of cellophane, onion skin, and chitin,
plus a thin shower of pollen, are added as bait. The baited collection is known as
a gross culture and should be incubated at a temperature near that of the water
at the collection site. The gross culture should consist mostly of water with a small
amount of bait and organic debris because excess organic matter encourages growth
of bacteria instead of chytrids. Chytrids usually appear on pollen grains after 1–2 days
and on the other baits after several days. Many cellulosic chytrids have generation
times of 4–7 days, and a longer time may elapse before sporangia appear. Gross
cultures may continue to yield fungi for weeks and should be examined periodically.

If knowledge of the phenology of the mycota is important, chytrids are collected

by placing baits enclosed in a weighted (sterilized glass marbles work well), nylon
net bag into the habitat being studied. Pollen is wrapped in lens paper so that
the grains become enmeshed among cellulose fibers and are retained in the bag.
The net bag is fastened in an inconspicuous place and left at the site for about 1
week if temperatures are higher than 20°C and up to a month if temperatures are
below 10°C. Bait bags and surrounding water are retrieved into a sterile container
and transported to the laboratory, where baits and water are transferred to glass
Petri plates or finger bowls. Baits then are examined for chytrids using a compound
microscope. Additional baits can be added, if needed, to encourage development of
populations of fungi.

Chytrids can be found and isolated directly from natural substrata. Such substrata
include algae, senescent plants, waterlogged wood, and insect exuviae. Algae are
carried to the laboratory in the water in which they are growing. A subsample (the
amount that can be picked up with forceps) is removed, dipped in 50°C water for
about 1 minute (D. J. S. Barr, personal communication), and returned to the gross
culture at a location where it can be found later. The temperature shock kills the
algae without completely disrupting membranes, and the newly dead algae attract
chytrids that occur on naturally senescent algae.

Some aquatic plants have leaves that are a few cell layers thick. Such leaves easily
can be examined for fungi by placing them on a microscope slide. If leaves are
thicker, epidermal peels can be examined. Chytrids and peronosporomycetous fungi
occur most frequently on senescent leaves, and hyphomycetes most commonly
occur in more decomposed materials. If no newly senescent leaves are present, a
small sample of leaves can be heat-treated as for algae. If fungi are not seen in
collected aquatic plants, onion skin can be used as a substitute substratum. We have
isolated chytrids directly from Eriocaulon, Typha, Utricularia, and Potamogeton (J. E.
Longcore, unpublished data). Exuviae of aquatic insects also harbor many interesting
chytrids and can be examined easily using a compound microscope.

Waterlogged wood supports the growth of members of the Monoblepharidales and

Blastocladiales. Waterlogged twigs that still retain bark are collected and packed into
jars with water from the collection site. Storage of these collections for weeks or
months at 5°C before subsamples are brought to room temperature enhances the
likelihood of finding monoblepharidalean fungi. The fungi can be detected by using
a dissecting microscope to locate tufts of hyphae, which often grow as an understory
beneath peronosporomycetes.

> Read full chapter

Low-Cost Enzymes and Their Applica-

tions in Bioenergy Sector ‡
V.L. Queiroz, ... L. Tasic, in Agro-Industrial Wastes as Feedstock for Enzyme Produc-
tion, 2016

Lignocellulose Biomass
Agricultural and agro-industrial waste salvage is growing rapidly as a consequence
of efforts for diminishing the environmental impacts caused by industrial and urban
activities (Tsukamoto et al., 2013b). These wastes are composed of lignocellulosic
biomass and serve as an attractive feedstock for 2G-ethanol production because
they are cost effective, renewable, and abundant. Moreover, crops such as corn and
sugarcane are primarily used as food and feedstock, therefore being unable to meet
the global demand of bioethanol production (Sarkar et al., 2012). Conventionally,
sugarcane bagasse is utilized as feedstock for bioethanol production (Soccol et al.,
2010). Other agro-industrial residues are important to consider, for example, orange
waste (Awan et al., 2013), rice straw, wheat straw, and corn straw (Sarkar et al., 2012)
as these are major agricultural wastes in terms of their quantity as well as availability
(Awan et al., 2013).

The structural material in the cell wall of woody and nonwoody plants (Hon and
Shiraishi, 2000; Menon and Rao, 2012) is known as lignocellulose, which provides
support, strength, and shape to the plant. This biomass is being explored for
2G-ethanol production, and its chemical composition directly influences the type
and the amount of enzymes employed for hydrolysis. The perfect enzyme combi-
nation in an enzyme cocktail is not only needed for successful biomass digestion
in an industrial process but also required and constantly produced and secreted
in nature by the native microorganisms that habit this biomass. Therefore, it is
fundamental to know chemical composition and exact proportions of lignocellulosic
biomass components in order to understand how to prepare an adequate enzyme
cocktail. Lignocellulosic biomass mainly contains pectin, cellulose, hemicelluloses,
and lignin (Wyman, 1996) as is shown in Fig. 5.1. Different parts of the plant have
different proportions of these components. The outer wall is mainly composed of
carbohydrates and is called primary cell wall. It surrounds growing and dividing
plant cells. Next is the much thicker and stronger secondary cell wall, which accounts
for most of the lignin part in biomass (Willats et al., 2001; Sticklen, 2008). The
secondary wall usually consists of three sublayers: S1 (outer), S2 (middle), and S3
(inner). The formation of these three distinctive layers in secondary cell walls is a
result of changes in the orientation of cellulose microfibrils during their deposition.
The S1 and S3 layers are typically thin and have cellulose microfibrils oriented in a
flat helix relative to the elongation axis of the cell (Zhong and Ye, 2009). The S2 layer
is thick and has cellulose microfibrils that account for 75–85% of the total thickness
of the cell wall (Plomion et al., 2001). It is the S2 layer that largely determines the
mechanical strength of fibers in wood. Hemicelluloses and lignin are also present
in each of these layers (Plomion et al., 2001; Zhao et al., 2012).

Figure 5.1. Representation of cell wall containing cellulose microfibrils, hemicellu-

lose, pectin, and lignin and their molecular structures.

Pectin is an important component of cell wall and is a polysaccharide rich in

galacturonic acid and galacturonic acid methyl ester units. It is a soluble fiber that
combines with proteins and other polysaccharides and forms skeletal tissue that is
responsible for the chemical stability and physical strength of plants (Willats et al.,
2006). Cellulose is a high-molecular-weight linear insoluble polymer of d-glucose
units connected via -(1,4)-glycosidic bonds. Cellulose, hemicellulose, and lignin
are often found in close association with each other. Usually, cellulose does not
occur alone in a free threadlike chain, instead it has a supramolecular structure
consisting of both crystalline and amorphous regions made of fibrillary units, and in
the majority of cases, the crystalline region is made up of several sheets of long-chain
fibers united by both intra- and intermolecular hydrogen bonds (Mansfield et al.,
1999).

The structure of cellulose can be cleaved by hydrolysis of -(1,4)-glycosidic bond

catalyzed with acids or enzymes such as cellulases. However, the hydrogen bonds
incorporated within long chains of cellulose in a crystalline structure inhibit biomass
hydrolysis and demand harsh pretreatment conditions (Gardner and Blackwell,
1974). It has been investigated and widely accepted that the higher the crystalline
content of cellulose, the more difficult is the enzymatic attack to hydrolyze this
polymer. The amorphous regions, due to their high accessibility to enzymes, can
be hydrolyzed easily, while in the crystalline areas enzyme contact efficiency is
decreased (Chang and Holtzapple, 2000; Carvalho, 2009).

Unlike cellulose, hemicellulose polymers are chemically heterogeneous and mostly

amorphous with lower degrees of polymerization. That is why hemicellulose chains
are more easily hydrolyzed into monomeric units as compared to cellulose chains
(Wyman, 1999). Hemicellulose is a heteropolysaccharide consisting of arabinose,
galactose, glucose, mannose, and xylose, with minor amounts of a few other
compounds. There are many suitable pretreatments that first help in removing
hemicelluloses and later facilitate the cellulose digestion (Saha et al., 2005).

Lignin, however, is not a polysaccharide and does not have a well-defined chemical
structure. Lignin is a cross-linked macromolecular material based on a phenyl-
propanoid monomer structure (Doherty et al., 2011). Lignin adds strength and
rigidity to cell walls and is more resistant to enzyme attack compared to cellulose
and other structural polysaccharides (Kirk, 1971; Baurhoo et al., 2008). Lignin is
able to form covalent bonds with hemicelluloses. Covalent bonds incorporated in
lignin and carbohydrates mostly consist of benzyl esters, benzyl ethers, and phenyl
glycosides (Smook, 2002). In this way, lignin provides integrity, structural rigidity,
and prevention of swelling of lignocelluloses. It is commonly accepted as one of
the major factors responsible for biomass recalcitrance to enzymatic hydrolysis. It
implies steric hindrance and prevents fibers from swelling (Mansfield et al., 1999;
Carvalho, 2009).

In order to use the lignocellulosic biomass in the production of biofuels, it is

important to convert biomass polymers in monomeric sugars, and to accomplish
that, pretreatment is a key step to guarantee efficient hydrolysis and high yield
of d-glucose. Pretreatment of lignocellulosic materials is s an important step since
it reduces the time of hydrolysis (Sun and Cheng, 2002). The main objectives of
pretreatment are: reducing the crystallinity of cellulose, lignin removal, increasing
the porosity of the lignocellulosic material, and increasing the susceptibility to
enzymatic substrate action compared with the original material. The conversion
steps of lignocellulosic biomass into 2G ethanol consist of a minimum of four steps:

1. Pretreatment for increasing the degradation capacity of the biomass.

2. Hydrolysis of lignocelluloses to simple monomeric sugars.

3. Fermentation of sugars to 2G ethanol.

4. Recovery of 2G ethanol by distillation/evaporation.

> Read full chapter

Industrial enzyme production for the
food and beverage industries: process
scale up and scale down
S.M. Stocks, in Microbial Production of Food Ingredients, Enzymes and Nutraceu-
ticals, 2013

7.1 Introduction
There are many articles and book chapters on the subject of ‘scale up’; why yet
another? Readers of this chapter, in this particular book, almost certainly have
no need for an introduction to biotechnology, or what is now established as the
standard way to produce almost any of the products established in the biotech
market, that is submerged aerobic fermentation (almost universally adopted). This
subject has been covered comprehensively in other texts such as Stanbury et al.,
(1995). There are also many well-regarded courses on the subject of fermentation
and biotechnology, which an interested reader may well have attended (for example,
Berovic and Nienow, 2005). Design of bioreactors has been extensively covered
too (examples include Schugerl, 1991; Van’t Riet and Tramper, 1991; or Atkinson
and Mavituna, 1991). As such, scale up is something about which a great deal has
been written, but there appears to be no one universally agreed approach. This lack
of agreement leads to some confusion for those entering the discipline; what to
believe?

The lack of agreement probably reflects the differences in physiology and genetic or
metabolic regulation around production of the desired product, that is to say, there
can be no agreement when the products and methods of production are necessarily
so diverse. For this reason the scope of this chapter will be limited to the subject
of submerged aerobic fermentation for the production of industrial enzymes, those
enzymes sold from business to business in large quantities, amounting to several
tens of thousands of tons each year (more precise figures can possibly be extracted
from the financial reports of Novozymes A/S and its competitors). The bulk of these
enzyme preparations end up in laundry detergents and an increasing amount is
used for the production of fuel from corn or agricultural residues like corn stover or
straw. Somewhere in between and representing a significant and strong business
for enzyme production companies, are a diverse number of enzyme preparations
sold to the food sector for the degradation of starch into sweeter substances, there
isomerisation and modification, for juice or oil extraction, wine production, brewing
and so on. Proteases are also an obvious class of use to the food industry, for
de-bittering or flavour modification.
Juice and wine production is probably the most traditional area where enzymes have
improved juice yields, or reduced the residual starch in the production of ‘lite’ brews,
or improved clarity or productivity. There is now a range of enzymes which allows
beer production without malt, which is good news for brewers facing a shortage
of quality malts. There are enzymes for baking too, which can increase shelf life of
bread or cake by altering starch crystallisation, slowing the rate of staling, or which
can remove asparagine before the cooking process, to reduce acrylamide formation.
Lipases are also very interesting for bakers, they can also be used in the dairy for
better control of the cheese making process. And, since meat is also a big part of the
food industry, we might include enzymes added to animal feeds in this book chapter;
these enzymes improve the nutritional value of the feed by, for example, freeing
phosphate from phytate, allowing reduced inorganic phosphate consumption at the
farm, and therefore reduced amounts in waste water, yielding financial benefits for
the farmer and also the consumer.

Generally speaking, enzymes are used when some customer benefit is obtained that
cannot be obtained more economically by other means. Enzymes are so successful
because they are relatively inexpensive natural products with great potency that
eliminate or reduce the need for chemical additives or high temperatures and
pressures seen in other process industries. Enzymes are a natural choice and they
are big business. If further information on enzymes themselves is required, then the
reader is encouraged to consult a well-known Novozymes information book (Olsen,
2004).

There is nothing new about the enzyme business (Aunstrup, 1979). In fact, the only
changes since the cited 1979 review article, seem to be the ones predicted within it:
proliferation and commoditisation of genetic information and computational power
has made it possible to produce almost any desired enzyme in one host organism or
another, and through similarly enabled automation of enzyme and strain screening
technology, production in larger quantities with higher yields is now possible. The
enzyme producer is still faced with the challenge of producing more of everything as
each year passes: thousands of large scale batches each year, from a diverse portfolio.
This means that each piece of production equipment should be either in use or under
brief planned maintenance 100% of the time and every batch should result at least in
the planned amount of product, so the chosen technology for production needs to
be robust and easily serviceable. Also a large optimisation effort is applied to avoid
the need for constant investment in new production capacity. This constant drive
for capacity increase at low cost probably explains the prevalence of the stirred tank
reactor for enzyme production. It is a well established and reliable technology with
proven commercial success that is very versatile, easily understood and operated and
large versions are well supported by a diverse number of engineering companies.
More exotic forms of production equipment are perhaps best left to more exotic
types of business.

Also, it is not surprising that production of enzymes for food, feed, or even technical
applications, is still done with GRAS (generally regarded as safe) microorganisms
(not E.coli which features heavily in the literature on fermentation technology).
Choosing a GRAS organism may increase the workload in R&D, and development
as off the shelf tools or texts are harder to find or create than those for the molecular
biologists’ most favourite work horse (E.coli). GRAS organisms include Aspergillus
oryzae (famous for its role in soy sauce production), Aspegillus niger (citric acid
production) and harmless Bacillus species that can be found in a variety of fermented
foods.

Where does scale up come into this? In this context, the above-mentioned produc-
tion equipment is considered ‘large scale’ and, as already mentioned, nothing stands
still in a competitive industry. In order to make the most of the capital investment
and to stay competitive on price, constant improvements in yields and productivity
are needed, as well as the introduction of new products. Thus, scale up, or perhaps
more appropriately, scale down, is required for optimisation of existing products
and for development of new ones. Of course, scale up as a discipline is not confined
to an existing enzyme production business. Many new start up companies or young
researchers with good candidate products may struggle with scale up – and this is an
important point in dealing with the contradictions and confusion surrounding scale
up that exists in the literature – it is one thing to scale up or down surrounded by
professionals in a successful company that has existing and well-defined production
equipment with a high number of product introductions each year (i.e. a large
experienced peer group can be called upon) and quite another situation to face the
problem almost alone and for the first time, without existing production equipment
in mind and with the barrier of secrecy agreements preventing an open discussion
with potential help.

In the following sections I hope to bring some of the perspective gained in my few
years working in the pilot plant at Novozymes, a large pilot plant based at head
office in Denmark, serving the existing factories and business of Novozymes A/S. The
overall aim will be to help clarify and explain some of the apparent contradictions
in the scale up literature to a general reader based upon my direct experience. By
way of a disclaimer, the content of these sections represents the personal views of
the author and not necessarily the consensus of my peer group of experienced and
talented colleagues.

> Read full chapter

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