Introduction to the Biotechnology 1

of Bacillus
COLIN R. HARWOOD

Bacteria of the genus Bacillus are aerobic, endospore-forming, gram-positive rods.

The genus was created in 1872 by Ferdinand Cohn, who changed the name of
Ehrenberg's (1835) "Vibrio subtilis" to Bacillus subtilis. The Cohn strain, B. subtilis
Cohn 1872, 174, remains the type species of the genus.
The genus is one of the most diverse and commercially useful groups of micro-
organisms. Representatives of this genus are widely distributed in soil, air, and
water where they are involved in a range of chemical transformations that rival
those of the Pseudomonads and Actinomycetes (see Chapter 3). Although primarily
saprophytes, at least one species (B. schlegelii) shows facultative chemolithotrophic
activity in an 02/C02/H2 or 02/C02 atmosphere (Schenk and Aragno, 1979) and
two species (B. macerans and B. polymyxa) are able to fix nitrogen (Hino and Wilson,
1958; Witz et al., 1967). The ability of certain strains to tolerate high or low tem-
perature and high and low pH has made them a particularly important source of
commercial enzymes (Norris et al., 1981).
The metabolic diversity of Bacillus spp., together with low reported incidence
of pathogenicity, has led to representatives of this group being used in a wide range
of industrial processes. One of the oldest recorded uses is the fermentation of
soybeans into natto, a tempe-like fermentation that uses a strain of Bacillus now
recognized as a variant of B. subtilis. Some 6 X 106 kg of natto are consumed
annually in Japan (Djien and Hesseltine, 1979), surely the finest testimony to the
suitability of this organism for the production of products for the food and phar-
maceutical industries. Nowadays strains of Bacillus are used for the production of
four main types of product: (1) enzymes, (2) antibiotics, (3) fine biochemicals in-
cluding flavor enhancers and food supplements, and (4) insecticides (see Chapters
10 and 11).
It is the enzyme market that currently accounts for the main commercial im-
portance of members of the genus. In particular they are the major source of
hydrolyzing enzymes for the food and detergent markets. Subtilisin, an alkaline
protease isolated from B. subtilis and closely related species, represents some 25% of

COLIN R. HARWOOD • Microbial Technology Group, Department of Microbiology,

The Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom.

C. R. Harwood (ed.), Bacillus

© Springer Science+Business Media New York 1989
2 COLIN R. HARWOOD

the world enzyme market and is calculated to be worth about $26 million per
annum in the United States alone (Towalski and Rothman, 1986). The continual
requirement of the detergent industry for proteases with greater heat and alkaline
tolerance has resulted in two quite separate approaches to the production of im-
proved enzymes. The more traditional approach has been to isolate organisms from
increasingly alkaIiphilic and thermophilic environments. Although this approach is
initially very successful, the law of diminishing returns means that the rate of
discovery of "improved" enzymes decreases with time, particularly when conven-
tional screening programs are employed. However, we are now beginning to see the
wider application of modern selective isolation techniques in which a large amount
of phenetic data, accumulated in computer databases, are employed both to devise
the selection regimes and to analyze the resulting isolates for genuine "novelty." To
exploit these newer methods to the full, progress will need to be made on some of
the problems currently besetting Bacillus taxonomy (see Chapter 2).
The alternative approach to improving enzyme performance, and one that has
exciting possibilities for many other systems, is that of enzyme engineering. This
approach involves engineering precise changes in the nucleotide sequence encod-
ing the enzyme in question to alter particular amino acid residues in its primary
sequence. Fersht and colleagues (Thomas et al., 1985), for example, studied the
effects of changes in surface charge on the pH dependence of subtilisin BPN' by
making individual amino acid substitutions that influence the protonation of His64
at the active site of this enzyme.
The production of microbial pesticides like the delta-endotoxins produced
during sporulation by B. thuringiensis, B. popilliae, and B. lentimorbus represents a
potentially elegant solution to increasing resistance to chemical pesticides and their
toxic effects in man and the environment. The production of microbial pesticides is
currently low, worth at most some $10 million per annum compared with some $13
billion for chemical alternatives (Hacking, 1986). However, it is to be hoped that
their particular advantages of lack of general toxicity to man and the environment,
their lack of persistence, and their specificity for particular insect pests will out-
weigh their significant cost disadvantage in an increasingly environmentally con-
scious world.
More recently, with the development of a variety of host/vector systems (see
Chapters 6 and 7), recombinant DNA technology has expanded considerably the
potential commercial importance of B. subtilis and other representatives of the
genus. This point was first brought home to me by Frank Young (1980), appropri-
ately in the Eighth Griffith Memorial Lecture of the Society for General Micro-
biology. Until this time I, and probably countless others, had regarded B. subtilis
primarily as an amenable "model" microorganism in which to study the physiology
and molecular biology of such processes as sporulation and hadn't foreseen its
potential to rival Escherichia coli as a commercial producer of the products of genetic
engineering.
Our understanding of the genetics and physiology of B. subtilis is second only to
that of E. coli and surely will be the second living organism to have its entire
nucleotide sequence determined. The adoption of this organism for so many inves-
tigations relates directly to the pioneering work of Spizizen (1958), whose demon-
stration of transformation in B. subtilis was the first for any nonpathogenic micro-
organism. Exploitation of transformation together with other subsequently