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Risk Assessment of Bacillus amyloliquefaciens subsp. amyloliquefaciens for 

Consideration of Addition to the List of Eligible Recipient Microorganisms for the 

5(h)4 Tiered Exemptions from MCAN Reporting Requirements 

I. INTRODUCTION

Bacillus amyloliquefaciens is a ubiquitous bacterium commonly isolated from soil 

and plants. It is capable of survival in the environment because it can produce an 

endospore that withstands adverse conditions such as heat or desiccation. B. 

amyloliquefaciens produces a variety of enzymes, particularly α-amylase and proteases, 

and it accounts for the majority of industrial production of traditional α-amylase. B. 

amyloliquefaciens is not considered pathogenic to humans, other animals, or plants. 

Recently, it has been proposed that there are two subspecies within the species B. 

amyloliquefaciens, B. amyloliquefaciens subsp. amyloliquefaciens and B. 

amyloliquefaciens subsp. plantarum. The former subspecies includes the type strain 

and likely most, if not all, of the industrial strains of B. amyloliquefaciens used for 

enzyme production. The latter subspecies consists of plant-associated strains used as 

biocontrol agents since they produce a number of antifungal lipopeptide and 

antibacterial polyketide toxins. This exemption is being restricted to only those strains 

of B. amyloliquefaciens that would fall into the subspecies B. amyloliquefaciens subsp. 

amyloliquefaciens as proposed by Borriss et al. (2010). This exemption does not apply 

to any strain of B. amyloliquefaciens that would fall into the proposed subspecies B. 

amyloliquefaciens subsp. plantarum. The potential risk associated with the use of 

industrial strains of B. amyloliquefaciens subsp. amyloliquefaciens for enzyme or 

specialty chemical production in fermentation facilities is low. 

History of Commercial Use and Products Subject to TSCA Jurisdiction

The primary TSCA application of B. amyloliquefaciens is the production of αamylase, an extracellular enzyme which hydrolyzes the internal 1,4-α linkages in 

amylose and amylopectin. The resulting products of starch hydrolysis are maltooligosaccharides of two to six units in length (Priest, 1989). The α-amylase produced by 

B. amyloliquefaciens is the traditional industrial amylase (Priest, 1989). B. 

amyloliquefaciens is the primary source of α-amylase whereas a closely related 

species, B. licheniformis, produces α-amylase with better thermostable properties. 

Although α-amylase historically has been used predominately as an aid to digestion or 

in food processing (Priest, 1989), the primary TSCA use of α-amylase is as a 

component of laundry detergents. In addition to use in the food, brewing, and distilling 

industries, α-amylase is also used for starch saccharification and in textile processing 

(Gangadharan et al., 2009). In addition to α-amylase, B. amyloliquefaciens is used for 

the production of other enzymes such as proteases, carbohydrases, xylanases, 

nucleases, and phosphatases (Priest, 1977). Specific enzymes obtained from B. 

amyloliquefaciens include glucanase, glucosidase, pullulanase, L-pyrrolidone 
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carboxylate peptidase, L-pyrrolidonyl peptidase, and the restriction endonuclease 

BamHI barnase (Harwood, 1992). 

In addition, B. amyloliquefaciens is used to produce an antibacterial surfactant 

called surfactin which is used as an antibiotic, but it possesses antivirus and 

antimycoplasma activities as well (Vollenbroich et al, 1997a; Vollenbroich et al., 1997b). 

Surfactin has physical/chemical characteristics similar to those of traditional chemical 

surfactants. Hence its potential use as an alternative to chemical surfactants is being 

investigated (Reznik, 2010). 

B. amyloliquefaciens is used for the industrial production of the specialty 

chemical guanosine, and for the production of ribavirin which is an antiviral drug (Zhang 

et al., 2011). It is also used for the industrial production of poly-γ-glutamic acid which 

has applications in medicine, cosmetics, and food, but it also functions as a biopolymer 

flocculant in wastewater treatment (Geng et al., 2011). 

II. IDENTIFICATION AND TAXONOMY

A. Overview 

 B. amyloliquefaciens is commonly isolated from soils and plants, but has also 

been isolated from water and air. Its importance in the environment is well-documented 

for the biological control strains of B. amyloliquefaciens where the bacterium is 

endophytic or rhizosphere-associated and promotes plant growth through inhibition of 

growth of various fungal plant pathogens or by plant hormone production. However, 

other members of the B. amyloliquefaciens are soil isolates that, like other bacilli, likely 

exist predominately in the inactive endospore stage except when conditions favorable 

for vegetative growth exist. Under those conditions, the bacterium contributes to 

nutrient cycling because of the numerous enzymes it is capable of producing. 

B. Taxonomy and Characterization 

1. The Genus Bacillus 

The genus Bacillus consists of a large number of diverse, rod-shaped Gram 

positive (or positive only in early stages of growth) bacteria that are motile by 

peritrichous flagella and are aerobic. Members of the genus are capable of producing 

endospores that are highly resistant to unfavorable environmental conditions (Claus and 

Berkeley, 1986). Historically, the genus consisted of a highly diverse group of 

organisms as evidenced by the wide range of DNA base ratios of approximately 32 to 

69 mol% G + C (Claus and Berkeley, 1986), which was far wider than that usually 

considered reasonable for a genus (Norris et al., 1981). Thus, numerous revisions of 

the genus were made. Many species formerly in the genus have been transferred, 

based on phylogenetic information that the species were not closely related enough to 

remain in the same genus, to newly formed genera or to other existing genera. Some of 
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the genera to which well-known members of the former genus Bacillus were transferred 

include Acyclobacillus, Brevibacillis, Geobacillus, Paenibacillus, and Sporosarcina. 

 2. The Species B. amyloliquefaciens 

Historically, there has been confusion about the taxonomic placement of B. 

amyloliquefaciens as a separate species. The name Bacillus amyloliquefaciens was 

first proposed by Fukomoto in 1943 for those strains of Bacillus subtilis that produced 

50 to 150 times more α-amylase than typical strains of B. subtilis (Priest et al., 1987). 

However, prior to the monographs of Smith in 1946 and 1952, B. subtilis was the name 

given to all aerobic endospore-forming bacilli (Logan, 1988). Thus, for several decades 

strains capable of producing large quantities of α-amylase either were considered 

variants of B. subtilis, or were given subspecies status, i.e., B. subtilis subsp. 

amyloliquefaciens. Phenotypically, B. amyloliquefaciens is very closely related to B. 

subtilis which currently has three subspecies, B. subtilis subsp. subtilis, the type species 

of the genus, B. subtilis subsp. spizizenii (Nakamura et al., 1999), and B. subtilis subsp. 

inaquosorum proposed by Rooney et al. (2009). 

Mounting evidence over several decades on the genetic dissimilarities between 

B. amyloliquefaciens and B. subtilis led to the revival of the name B. amyloliquefaciens

as a species separate from B. subtilis. Welker and Campbell (1967) first observed that 

DNA homology between B. subtilis and B. amyloliquefaciens was only about 15%, when 

according to Priest et al. (1987), typically members within a single species show at least 

50-60% DNA homology. The current ‘gold standard’ for species separation requires 

that strains within a species share 70% or greater homology (Wayne et al., 1987). 

Welker and Campbell (1967) also found several phenotypic properties that differed 

between those B. subtilis strains capable of producing copious quantities of α-amylase 

and those that didn’t, such as growth in 10% NaCl, growth on potato tubers, and ability 

to ferment lactose with associated acid production. They maintained that strains 

characterized by greater α-amylase production were not merely variants of B. subtilis 

because there was more than one characteristic shown to be different, and those 

characteristics were stable with no reversion back to the B. subtilis traits for at least a 5-

year period. In addition, studies on transformation between the species and 

experiments with transducing phages both indicated that the species do not contribute 

to, or share in, a common gene pool and that they were not closely related (Welker and 

Campbell, 1967). Later, O’Donnell et al. (1980) found that the four species in the B. 

subtilis group, B. subtilis, B. amyloliquefaciens, B. licheniformis, and B. pumilus, could 

be differentiated by three methods, pyrolysis gas-liquid chromatography, numerical 

analysis of phenotypic features, and API identification systems although B. 

amyloliquefaciens and B. subtilis could not be separated solely by classical biochemical 

tests. 

Thus, the name B. amyloliquefaciens was revived and recommended for 

separate species status by Priest et al. (1987) based on differences found between the 

two bacilli. Later, Priest et al. (1988) published a comprehensive study with all the 
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supporting data of all Bacillus species existing at that time along in which the separation 

of B. amyloliquefaciens from B. subtilis was re-iterated. As a result, B. 

amyloliquefaciens was re-established as a name with standing in bacterial 

nomenclature. 

Years after its revival as a separate species, it was suggested that there were 

two different ecotypes of B. amyloliquefaciens. One ecotype consisted of strains

isolated from stems and roots of plants that produced antibacterial and antifungal 

compounds which lessened certain plant diseases. The other ecotype included strains 

that were soil-associated rather than plant-associated. Strains in this ecotype produced 

greater quantities of α-amylase and generally did not produce antifungal toxins. It was 

Reva et al. (2004) who first reported that plant-associated strains of Bacillus were very 

similar, but not identical to, the type strain of B. amyloliquefaciens, DSM 7T

. By analysis 

of 16S rRNA, the gyrase A gene, and the histidine kinase gene, they found that 7 of 17 

strains of Bacillus isolated from plant roots and soils formed a cluster closely related to, 

but distinct from the type strain of B. amyloliquefaciens. These strains were shown to 

be better adapted at colonization of plant rhizospheres, and the authors suggested that 

these B. amyloliquefaciens strains were a distinct ecotype. Recently, it was proposed 

that there are two subspecies of B. amyloliquefaciens, B. amyloliquefaciens subsp. 

amyloliquefaciens, and B. amyloliquefaciens subsp. plantarum (Borriss et al., 2010). 

They stated that the results of DNA:DNA hybridization studies, microarray-based 

comparative genome hybridization, and MALDI-TOF mass spectrometry of cellular 

components justified the proposed creation of subspecies for the two ecovars of B. 

amyloliquefaciens strains. Currently, the two subspecies, B. amyloliquefaciens subsp. 

amyloliquefaciens and B. amyloliquefaciens subsp. plantarum, appear as separate 

entries in the List of Prokaryotic Names with Standing in Nomenclature 

(http://www.bacterio.cict.fr/b/bacillus.html) which is indicative of the likely acceptance of 

the proposal for the two subspecies. 

The type strain DSM 7T and related industrial strains capable of producing 

copious amounts of extracellular α-amylase and different proteases would belong in the 

amyloliquefaciens subspecies, whereas the plant-associated B. amyloliquefaciens 

strains capable of producing a number of antifungal and antibacterial toxins, exemplified 

by the common biocontrol strain FZB42, would belong in the plantarum subspecies. 

Additional justification for the separation into subspecies for the two ecovars is genetic 

evidence presented by Rückert et al. (2011) that the type strain of B. amyloliquefaciens, 

DSM 7T, and the biocontrol strain of B. amyloliquefaciens, FZB42, differed in their ability 

to synthesize enzymes and the secondary metabolites inhibitory to plant fungal 

pathogens. They found a diminished capacity in DSM 7T for the non-ribosomal 

synthesis of antifungal lipopeptides, except for surfactin. This type strain also had a 

decreased ability to produce polyketides compared to the biocontrol strain of B. 

amyloliquefaciens subsp. plantarum, FZB42. In addition, it was shown that DSM 7T

produces a different amylase (amyA) than the plant-associated strain, which produces a 

B. subtilis-like amylase (amyE). 
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There is another species of Bacillus that is considered a synonym for B. 

amyloliquefaciens. An isolate from the brackish water of the river Vélez in southern 

Spain that was shown to be a member of the B. subtilis group was given the name B. 

velezensis (Ruiz-Garcia et al., 2005). However, it was later determined, based on 16S 

rRNA gene sequence similarity and a lack of distinctive phenotypic characteristics, that 

B. velezensis is a heterotypic synonym of Bacillus amyloliquefaciens and does not 

deserve separate species status (Wang et al., 2008). This strain is more similar to the 

strains that would fall in the B. amyloliquefaciens subsp. plantarum subspecies. 

C. Related Species of Concern

There are several species of the genus Bacillus that are known pathogens. 

These include B. anthracis which is pathogenic to humans and other animals, and B. 

cereus which is a common cause of food poisoning (Claus and Berkeley, 1986; Norris 

et al., 1981). B. thuringiensis, B. larvae, B. lentimorbus, B. popilliae, and some strains 

of B. sphaericus are pathogenic or toxigenic to certain insects. 

Bacillus amyloliquefaciens is securely included within the B. subtilis group of 

related species. Many of these species have a long history of safe industrial use 

including B. subtilis and B. licheniformis. These species are distinguished from the B. 

cereus group which contains some species with significant human health hazard 

potential, including B. anthracis and some strains of B. cereus. The B. cereus group is 

readily differentiated from other species in the genus Bacillus, although separation 

among the various members of the B. cereus group is more difficult than separation 

among the members of the B. subtilis group. The latter can be easily differentiated by 

genetic methods. Given the ready separation of the B. cereus group from other bacilli, it 

appears that B. amyloliquefaciens is unlikely to be confused with any member of that 

potentially pathogenic group of taxa. 

III. HAZARD ASSESSMENT 

A. Human Health Hazards 

 1. Colonization and Pathogenicity 

Bacillus amyloliquefaciens is not pathogenic to humans and there are no reports 

in the literature associating B. amyloliquefaciens with infection or disease in humans. B. 

amyloliquefaciens has been categorized as a Risk Group 1 microorganism which means 

that it is not known to cause disease in healthy adults (NIH, 2011). There have been 

animal toxicity studies performed with a biological control strain of B. amyloliquefaciens, 

FZB24, to support its registration as a biopesticide 

(http://www.epa.gov/oppbppd1/biopesticides/ingredients/index_ab.htm). It was tested 

for acute oral toxicity/pathogenicity, acute pulmonary toxicity/pathogenicity, and acute 

injection toxicity/pathogenicity. Results of these tests showed little to no adverse 
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effects, which indicate a low mammalian toxicity and a lack of pathogenicity/infectivity. 

B. amyloliquefaciens has a long history of safe use for the production of enzymes 

with both food and industrial uses with no incidences associated with human 

pathogenicity. In response to a petition from the Enzyme Technical Association, the 

FDA affirmed that carbohydrase enzyme preparations and protease enzyme 

preparations derived from either B. subtilis or B. amyloliquefaciens are generally 

recognized as safe (GRAS) for use as direct food ingredients (FDA, 1999). This 

conclusion was based on information available in the literature rather than on actual 

toxicity tests. The European Food Safety Authority (EFSA) has put B. 

amyloliquefaciens on their list of bacteria that have a “qualified presumption of safety” 

(QPS) (EFSA, 2008). QPS bacteria have a long history of apparent safe use in food 

and feed production and do not require extensive safety assessment. It was, however, 

put on the QPS list with a qualifier that only strains of B. amyloliquefaciens that do not 

have toxigenic potential be used. 

In a review article on the safety of B. subtilis and B. amyloliquefaciens de Boer 

and Diderichsen (1991) stated that there have been no cases demonstrating invasive 

properties of either of these bacteria. No specific information is available in the 

literature on the ability of B. amyloliquefaciens to colonize the human body. However, 

B. amyloliquefaciens is not known to have any specialized attachment mechanisms 

typically found in organisms capable of colonizing humans. Given its ubiquity in nature 

and the environmental conditions under which it is capable of surviving, B. 

amyloliquefaciens undoubtedly comes into frequent contact with humans without 

colonizing and infecting the human body. B. amyloliquefaciens, like other closely 

related species in the genus, B. subtilis, B. licheniformis, and B. pumilus, have been 

shown to be capable of producing lecithinase, an enzyme which disrupts membranes of 

mammalian cells. Even though lecithinase is considered a virulence factor, there is no 

evidence of human pathogenicity from B. amyloliquefaciens. 

 2. Toxin and Secondary Metabolite Production 

There is one report that a strain of B. amyloliquefaciens has the potential to 

produce food poisoning toxins (Phelps and McKillip, 2002). Food poisoning is typically 

due to B. cereus which causes two types of food poisoning, the emetic and diarrheal 

syndromes, and a variety of local and systemic infections (Schoeni and Wong. 2005). 

The three main food poisoning toxins produced by B. cereus are the emetic toxin, 

cereulide, the tripartite hemolysin BL (HBL) which is a diarrheal protein enterotoxin, and 

a nonhemolytic enterotoxin (Nhe) that has also been associated with the diarrheal 

syndrome. Phelps and McKillip (2002) reported the presence of genes encoding B. 

cereus enterotoxin virulence factors/toxins (HBL and Nhe) in strains belonging to 

several other Bacillus spp. including one strain of B. amyloliquefaciens isolated from air. 

The HBL and Nhe toxins were shown to be produced in this B. amyloliquefaciens strain 

in a model system maximized for toxin expression. On the contrary, there are three 

different research groups (Sergeev et al., 2006; Matarante et al., 2004; De Jonghe et 
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al., 2010) who have reported that strains of B. amyloliquefaciens do not have B. cereus 

enterotoxin genes. De Jonghe et al. (2010) also reported that the strain of B. 

amyloliquefaciens did not have the B. cereus emetic toxin synthase genes (ces). 

However, in an in vitro cytotoxicity assay, it was shown to produce a heat-stable 

cytotoxin component other than the emetic toxin. Generally, the risk of food-borne 

disease caused by bacilli other than B. cereus is considered to be low (Matarante et al., 

2004). Moreover, Pedersen et al. (2002) concluded that industrial strains of Bacillus 

species belonging to the B. subtilis group do not express B. cereus toxins and are safe. 

This conclusion was based on cytotoxicity testing of one industrial strain of B. subtilis 

and one strain of B. amyloliquefaciens that both produced α-amylase, and on two 

strains of B. licheniformis that produced other enzymes. 

 

 As previously mentioned, some strains of B. amyloliquefaciens, particularly those 

in the subspecies B. amyloliquefaciens subsp. plantarum, have been shown to produce 

bioactive cyclic lipopeptide metabolites such as iturin, surfactin, fengycin, and 

bacillomycin D (Winfried et al., 1998; Yoshida et al., 2001; Hiradate et al., 2002; 

Koumoutsi et al., 2004), and the polyketide toxins macrolactin, bacillanene, and 

difficidin. The cyclical lipoprotein biosurfactants are produced by non-ribosomal peptide 

synthesis. Arguelles-Arias et al. (2009) described these antifungal compounds. The 

cyclic lipopeptides are composed of seven (surfactin and iturin A) or 10 (fengycins) αamino acids linked to a β-hydroxy (surfactins and fengycins) fatty acid which varies from 

C-13 to C-16 for surfactins, C-14 to C-17 for iturins, and C-14 to C-18 for fengycins. 

 Although these biosurfactant compounds may have some mammalian toxic 

potential, they are used in cosmetic applications since they have been shown to have 

low toxicity. For example, one surfactin isomer, surfactin C, has been tested for toxicity 

in mammals. In an acute toxicity test of surfactin C, no lethality in mice was seen after 

administration of 2500 mg/kg which means that the LD50 was >2500 mg/kg (Park et al., 

2006). In a 28- day repeat dose oral gavage study of surfactin C, administration of 1000 

mg/kg-day resulted in increases in relative liver weight, increases in serum liver 

enzymes, and zonal liver necrosis. The no observable adverse effect level (NOAEL) in 

this study was 500 mg/kg-day (Hwang et al., 2009). These effect levels support the 

conclusion that surfactin C has low toxicity, however, surfactin C may possibly cause 

injury to mucosal membranes.

There are several reports in the literature of the isolation of B. amyloliquefaciens 

from water-damaged buildings in which occupants were suffering ill health symptoms. 

Andersson et al. (1997) were the first to report the isolation of B. amyloliquefaciens from 

a water-damaged area of a building, but the bacterium was not isolated from 

undamaged areas. There were 18 strains of B. cereus, 18 strains of B. 

amyloliquefaciens, and three strains of B. pumilus isolated from dust settled on surfaces 

or from water-damaged construction materials in the water-damaged area. All of the 

Bacillus isolates selected from agar plates for further examination in this study were 

ones that exhibited antifungal activity. The biomass of these strains from agar plates 

were extracted with methanol, and the methanol extracts were then assessed for the 
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toxicity endpoints of boar spermatozoa viability parameters of motility, plasma 

membrane integrity, and depolarization of the mitochondrial membrane potential. All of 

the B. cereus strains and the B. amyloliquefaciens strains exhibited cytotoxicity as they 

inhibited the motility of boar spermatozoa. The type and level of toxicity of the isolated 

B. cereus strains were indistinguishable from those of B. cereus strains known to 

produce the emetic food poisoning toxin cereulide. The B. amyloliquefaciens strains 

affected boar spermatozoa differently from the indoor B. cereus isolates and the 

reference food-poisoning strain. The methanol-soluble mitochondrial toxin extracted 

from biomass of B. amyloliquefaciens inhibited the motility of boar spermatozoa and 

mitochondria were depolarized. At a concentration 100-fold higher, the methanol 

extracts also damaged the plasma membrane permeability barrier. 

Further studies on these strains were conducted by Andersson et al. (2002). 

Their results showed that the B. amyloliquefaciens isolates produced a toxin(s) that 

depolarized mitochondria with a potency close to that of the highly hydrophobic 

depsipeptide cereulide toxin of B. cereus, but by a different mechanism. According to 

the authors, hydrophobic peptides of small molecular size would be expected to be 

readily sorbed from the respiratory tract of humans into their blood stream, and could 

possibly result in the suppression of the immune system. 

The same research group further studied the fungicidal isolates of Bacillus

amyloliquefaciens from moisture-damaged buildings. Mikkola et al. (2004) reported that 

five strains of B. amyloliquefaciens produced a methanol-extractable, heat-stable, nonprotein substance that inhibited the motility of boar spermatozoa, and also killed fetal 

feline lung cells at a high dilution. In boar sperm and in human neural cells, the toxicity 

of this substance was apparently due to a depolarization of the transmembrane 

potential of mitochondria and the plasma membrane. This toxin was shown to form 

cation-specific channels permeable to K+

, Na+, and Ca2+ with different selectivities. A 

similarly prepared methanol extract from the type strain of B. amyloliquefaciens, 

DSM 7T, did not exhibit toxicity or affect the spermatozoa viability parameters. 

 A different toxin substance from these indoor B. amyloliquefaciens strains 

exhibited toxicity by damaging the plasma membrane (Mikkola et al., 2004). This 

substance was identified as a heptalipopeptide identical to surfactin with respect to 

mass patterns, tandem spectrum patterns, and amino acid composition. The authors 

concluded that their results suggested that spores of toxin-producing B. 

amyloliquefaciens strains may be a potential respiratory health risk for humans 

inhabiting moisture-damaged buildings. However, these isolates of B. 

amyloliquefaciens strains tested in this study, again, were specifically selected because 

of their fungicidal activity. 

Mikkola et al. (2007) further studied the toxins isolated by Mikkola et al. (2004) 

from B. amyloliquefaciens strains from water-damaged buildings. The authors termed 

the first toxin reported by Mikkola et al. (2004) amylosin. This is a cation channelforming peptide with a higher selectivity for potassium ions than sodium ions. The 
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channel-forming amylosin and the ionophores caused swelling of the boar spermatozoa 

mitochondria due to the uptake of potassium ions and a loss of membrane potential. 

Normally, the mitochondrial membrane is not permeable to potassium ions. Further 

study of this toxin by Saris et al. (2009) showed that amylosin also exhibited inhibition of 

maximal respiration rates and inhibition of ATP synthesis. Saris et al. (2009) stated that 

the amylosin from B. amyloliquefaciens is similar to the peptaibol toxins produced by 

Trichoderma harzianum in that this toxin forms cation channels with a high affinity for 

potassium ions. 

The only other instance of mammalian toxin production by B. amyloliquefaciens 

comes from an episode in the 1980s with the commercial production of tryptophan by a 

genetically engineered strain of B. amyloliquefaciens, strain IAM 1521. The 

consumption of the tryptophan food supplement from various retail lots from one 

specific company resulted in an epidemic of a disease known as eosinophilia-myalgia 

syndrome (EMS) in which 1,511 were sickened and 37 people died (Mayeno and 

Gleich, 1994). Although this disease incidence was widely studied, the cause of the 

disease was never confirmed. It was thought to be due to the consumption of a 

chemical constituent that was associated with the company’s specific tryptophan 

manufacturing processes. This included the combination of using reduced quantities of 

powdered carbon for a purification step with the use of a ‘new’ strain of B. 

amyloliquefaciens called Strain V (Belongia et al., 1990). There supposedly was some 

chemical substance produced as a result of the genetic engineering of this certain 

strain. The toxicity of the tryptophan was not attributed to the parental strain of B. 

amyloliquefaciens, but only to this one genetically modified strain used with the 

particular manufacturing processes as not all production batches were toxic. 

Although there are isolated reports of toxin production in several environmental 

isolates of B. amyloliquefaciens, there are also reports on the safety and lack of toxicity 

of B. amyloliquefaciens strains used industrially for enzyme production. de Boer and 

Diderichsen (1991) conducted a literature search and published a paper on the safety of 

industrial strains of B. subtilis and B. amyloliquefaciens. In Europe, the toxicity of B. 

amyloliquefaciens strains DSM9553 and DSM9554 for production of α-amylase and 

bacillolysin was assessed by the Scientific Panel on Additives and Products or 

Substances used in Animal Feed upon a request from the Commission on the safety of 

the product Kemzyme W Dry (http://www.efsa.europa.eu./de/scdos/doc/118.pdf). The 

cytotoxicity was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium 

bromide (MTT) color change in metabolically active Chinese hamster ovary cells(CHO 

K1) due to a mitochondrial reaction. This MTT test was stated as being a wellestablished short-term cytotoxicity assay. Neither of the two B. amyloliquefaciens 

strains produced any evidence of toxicity in the MTT test or in another test studying the 

immunological cross-reaction with B. cereus enterotoxins. The FEEDAP Panel 

concluded that the B. amyloliquefaciens “production strains DSM9553 and DSM9554 

when used as a source of extracellular enzyme do not present a toxigenic risk”. As 

mentioned previously, Pedersen et al. (2002) also studied the toxicity of industrial 

strains of B. amyloliquefaciens, B. subtilis, and B. licheniformis used for large-scale 
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enzyme production. These industrial strains did not exhibit any cytotoxicity in the 

Chinese hamster ovary MMT test as described above. 

It also appears that it is the biocontrol strains of B. amyloliquefaciens that have 

been shown to produce the toxins in question. The reports of Andersson et al. (1997), 

Andersson et al. (2002), Mikkola et al. (2004), Mikkola et al. (2007), and Saris et al. 

(2009), who are all members of the same research group, claimed mammalian toxicity 

with B. amyloliquefaciens isolated from water-damaged buildings. However, all of the 

strains they studied were initially selected for further study because they exhibited 

antifungal activity on the agar plates. Rückert et al. (2011) compared the genome of the 

type strain of B. amyloliquefaciens DSM 7T

 (now B. amyloliquefaciens subsp. 

amyloliquefaciens) and the biocontrol strain FZB42. They found that even though the 

core genomes of the two subspecies were very similar, the subsp. plantarum had 

genomic islands that had prophage sequences, transposases, integrases, and 

recombinases which the type species did not have. As previously mentioned, the 

DSM 7T

 type strain was found to lack the ability to non-ribosomally synthesize some 

secondary metabolites with antifungal and antibacterial activities. The type strain could 

not produce the polyketides difficidin or macrolantin, and could not produce lipopeptides 

such as iturin, macrolantin, and other compounds, except for the compound surfactin. 

The authors stated that the differences in toxin-production ability observed in the 

genomes of the type strain and the plant-associated strain FZB42 is further justification 

for the designation of the two subspecies, amyloliquefaciens and plantarum, for the 

species B. amyloliquefaciens.

 3. Human Health Hazards Conclusions 

 B. amyloliquefaciens is not pathogenic to humans. There is one publication on 

the detection of B. cereus enterotoxins in a strain of B. amyloliquefaciens in a model 

system. However, there are no cases of food poisoning being caused by B. 

amyloliquefaciens. There are these five reports on the production of secondary 

metabolites toxic to mammalian cells by strains of B. amyloliquefaciens isolated from 

water-damaged buildings. However, as already stated, these isolates were selected for 

further study because of their antifungal properties and fungicidal strains are known to 

produce a wide array of secondary metabolites. 

Given B. amyloliquefaciens’s widespread distribution in the environment, its long 

history of use in industrial fermentation, the absence of reports on pathogenicity, and 

the limited reports of mammalian cytotoxicity by only a few fungicidal strains, there is 

not a concern for adverse human health effects with the use of strains of B. 

amyloliquefaciens subsp. amyloliquefaciens in fermentation facilities for production of 

enzymes or specialty chemicals. 

 

 

 B. Environmental Hazards 
11

 1. Hazards to Animals

 A search of the literature did not reveal any reports suggesting that B. 

amyloliquefaciens is pathogenic or toxic to domesticated animals or wildlife. 

 

 2. Hazards to Plants 

 

 B. amyloliquefaciens is not pathogenic to plants. There are, however, two 

reports in the literature by the same researchers that indicated an indirect adverse effect 

on stored plant material. B. amyloliquefaciens was shown to stimulate aflatoxin 

production by the fungus Aspergillus flavus in irradiated maize and rice grains (Cuero et 

al., 1987; Smith et al., 1988). B. amyloliquefaciens is apparently a common 

contaminant on stored grains which is not surprising given the widespread distribution of 

spores of many members of the genus Bacillus in the environment. The relevance of 

these studies on aflatoxin production is questionable, however, since these studies were 

conducted using a heavy application of vegetatively growing B. amyloliquefaciens and 

Aspergillus flavus cells on sterilized grain, where under normal grain storage conditions, 

there is a succession of microorganisms on the grain determined by competition and 

antagonism among the various bacterial and fungal contaminants (Cuero et al., 1987). 

 

As discussed above, there are plant-associated strains of B. amyloliquefaciens 

that are beneficial to plants. Earlier publications referred to these as a specific ecovar, 

but they are now thought to be the subspecies B. amyloliquefaciens subsp. plantarum 

(Borriss et al., 2010). One of the first publications on biocontrol activity of B. 

amyloliquefaciens was that by Yu et al. (2002) who reported suppression of the fungal 

pathogen Rhizoctonia solani by iturin A produced by B. amyloliquefaciens. Of three 

endophytic Bacillus groups isolated from cabbage, the B. amyloliquefaciens group was 

found to be the most effective in reducing black rot caused by Xanthomonas campestris 

pv. campestris (Wulff et al., 2002). These B. amyloliquefaciens isolates produced 

surfactin, iturin, bacillomycin and/or azalomycin F which were metabolites antagonistic 

to the plant pathogen. Goryluk et al. (2009) reported that B. amyloliquefaciens was an 

endophyte in the stems of Chelidonium majus L. (greater celandine) that exhibited 

antifungal properties. Sun et al. (2006) isolated an endophytic strain of B. 

amyloliquefaciens from the Chinese medicinal plant Scutellaria baicalensis Georgi that 

produced two families of secondary metabolites with broad-spectrum antibacterial and 

antifungal activities. These compounds were shown to be fengycin and surfactin 

homologues which had the potential for antagonism to fungal plant pathogens and food 

spoilage bacteria. In another study B. amyloliquefaciens was shown to confer 

protection of oilseed rape from the fungal pathogens Alternaria brassicae, Botyris 

cinerea, Leptosphaeria maculans, and Verticillium longisporum (Danielsson et al., 

2006). Lisboa et al. (2006) reported a bacteriocin-like substance produced by B. 

amyloliquefaciens isolated from a Brazilian forest which was inhibitory to fungal plant 

pathogens and to food-spoilage bacteria such as Listeria monocytogenes, B. cereus, 
12

Serratia marcescens, and Pasteurella haemolyticus. The lipopeptide iturin A produced 

by a strain of B. amyloliquefaciens was shown to be the principal inhibitor of seven 

fungal pathogens of citrus (Arrebola et al., 2010). Koumoutsi et al. (2004) found that 

some of the antifungal lipopeptides produced by B. amyloliquefaciens had synergistic 

effects. A double mutant of the biocontrol strain FZB42 lacking both bacillomycin D and 

fengycin was greatly diminished in its capacity to inhibit the growth of plant pathogenic 

fungi. León et al. (2009) isolated a strain of B. amyloliquefaciens from the rhizosphere 

of soybean plants that was capable of protecting soybean plants from damping-off 

disease caused by Pythium ultimum. It also increased seedling emergence when 

inoculated onto soybean seeds. 

Chen et al. (2009) studied the genome of the well-known B. amyloliquefaciens 

biocontrol strain FZB42. The genome had five gene clusters that coded for direct, nonribosomal synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an 

unknown peptide, and the iron siderophore bacillibactin. In addition, there were three 

gene clusters that coded for the antibacterial polyketides macrolactin, bacillaene, and 

difficidin. It was found that about 340 kb, or 8.5% of the total genome of strain FZB42, 

was devoted to the synthesis of antifungal and antibacterial secondary metabolites. 

The same five non-ribosomally synthesized lipopeptides were also shown to be 

produced by a different biological control strain of B. amyloliquefaciens, GA1 (ArguellesArias et al., 2009). This strain produced the same antibacterial polyketides, however, 

genes involved in the ribosome-dependent synthesis of bacteriocins and other peptides 

in FZB42 were not found in strain GA1. In a strain of B. amyloliquefaciens with 

antagonistic activity against the fungi Aspergillus spp., Fusarium spp., and Bipolaris 

sorokiniana, PCR analysis showed the presence of the gene ituD which codes for iturin, 

but not the presence of the gene sfp, that codes for surfactin (Benitez et al., 2010). The 

antimicrobial substances isolated and purified from the bacterium were shown to be 

iturin-like and fengycin-like peptides. These peptides were shown to be resistant to 

treatment with proteolytic enzymes and heat. Romano et al. (2011) reported the 

isolation of three new antifungal lipopeptides from a strain of B. amyloliquefaciens. 

These three compounds were identified as members of the iturin family, all of which 

showed strong and dose-dependent antifungal activity against the fungal pathogen 

Fusarium oxysporum. 

 Unlike most other antifungal compounds produced by bacteria that are usually 

peptides or ring structures, an antifungal protein called baciamin was produced by a 

strain of B. amyloliquefaciens (Wong et al., 2008). This protein was found to exhibit a 

broad spectrum of antifungal activity. It was relatively thermostable and pH-stable, and 

baciamin was shown to retain its antifungal activity even after incubation with trypsin 

and EDTA. It induced membrane permeabilization in fungi, but not in rabbit 

erythrocytes. 

In addition to the ability of B. amyloliquefaciens to produce antifungal and 

antibacterial compounds, the bacterium is known as a plant growth-promoting 

rhizobacterium (Idris et al., 2007; Calvo et al., 2010; Adesemoye et al., 2009). Idris et 
13

al. (2009) showed that the biocontrol strain of B. amyloliquefaciens, strain FZB42, 

produced the phytohormone indole-3-acetic acid (IAA). Dilute suspensions of the 

bacterium or culture filtrates resulted in significant increases in fresh weight of Lemna 

minor (duck weed). Mutant strains with diminished levels of IAA production did not 

promote growth as well as FZB42. Calvo et al. (2010) found that the majority of plant 

growth-promoting strains of Bacillus in the rhizosphere of potatoes in Andrean soils of 

Peru were B. amyloliquefaciens. Many of these rhizosphere bacilli showed not only 

antifungal activity, but also IAA production. In addition, more than half the strains also 

promoted plant growth through solubilization of tricalcium phosphate. Adesemoye et al. 

(2009) showed that less chemical fertilizer was needed to achieve the same growth of 

tomato when the plants were inoculated with B. amyloliquefaciens. 

 

 3. Environmental Hazards Conclusions 

There are no reports in the literature indicating that B. amyloliquefaciens is 

pathogenic to animals or plants, nor that it causes any adverse effects in the 

environment. It is frequently isolated from soil and from plant rhizospheres. Some 

members of the species produce a number of antifungal and antibacterial secondary 

metabolites which makes them useful for biocontrol agents of fungal plant pathogens. 

However, the typical industrial strains of B. amyloliquefaciens that are similar to the type 

strain, DSM 7T

, appear to have a diminished capacity for producing many of these 

secondary metabolites. 

IV. EXPOSURE ASSESSMENT

 Estimates of occupational exposure and releases of the microorganism from an 

enzyme fermentation facility with the resulting exposures to the environment and to the 

general human population are given below. Note that these numbers have been 

revised since the worker exposure and environmental release and exposure estimates 

used in the risk assessments of the original ten eligible recipient microorganisms listed 

at §725.420 in the Final Microbial Biotechnology Rule promulgated in 1997. Information 

gleaned from Microbial Commercial Activity Notices received over the years since the 

rule was published has allowed for better estimates of exposures using a generic 

scenario (Chemical Engineering Branch, 2011). For this generic scenario, it is assumed 

that a large-scale closed system enzyme fermentation plant operates 24 hours per day 

for 350 days/yr and that there are 100 batches/yr. 

A. Worker Exposure

 No data were available for assessing the releases and exposures specifically for 

enzyme fermentation facilities using B. amyloliquefaciens. Therefore, the potential 

worker exposures and routine releases to the environment from large-scale, 

conventional fermentation processes were estimated using information available from a 

number of submissions to EPA under TSCA Section 5 and from published information 
14

collected from non-engineered microorganisms. These values are based on reasonable 

worst-case scenarios and typical ranges or values are given for comparison. 

 During fermentation processes, worker exposure is possible during laboratory 

pipetting, inoculation, sampling, harvesting, extraction, processing, and decontamination 

procedures. A typical fermentation facility has a total of 36 workers with four workers 

per shift involving fermentation, four workers per shift for recovery operations, three 

workers per shift for laboratory procedures, and one supervisory worker, all with three 

shifts per day. It is assumed that the plant operates 24 hours/day for 350 days per year 

with 100 batches of enzyme per year. 

 The National Institute for Occupational Safety and Health (NIOSH) has 

conducted walk-through surveys of several fermentation facilities in the enzyme industry 

and monitored for microbial air contamination. These particular facilities were not using 

recombinant microorganisms, but the processes were considered typical of fermentation 

process technology. Typically, area monitoring data would not be used to estimate 

occupational exposure levels since the correlation between area concentrations and 

worker exposure is highly uncertain. However, personal sampling data are not available 

at the present time. Thus, area sampling data have been the only means of assessing 

exposures for previous microbial biotechnology submissions. Applying these NIOSH 

data to the workers in the various plant areas (laboratory, fermentation, recovery, 

control room) yields a range of potential inhalation exposures of 81 to 9800 colonyforming units (cfu)/day. The uncertainty associated with this estimated range of 

exposures is not known. 

 The potential dermal exposure for workers in the various plant areas ranges from 

6.0 x 1010 to 1.8 x 1011 cfu/day. However, the typical personal protection equipment 

(PPE) used in these types of facilities (i.e., gloves and lab coats) is expected to be 

adequate to protect against dermal exposure to the microorganism. In the unlikely 

event that dermal exposure was to occur, unbroken skin is considered to be a protective 

barrier to entry of the microorganism into the body. Even in the event of dermal contact 

with broken skin, there is little risk of infection because this microorganism is not 

pathogenic to humans. 

B. Releases and Environmental and General Population Exposure 

 Estimates of the number of B. amyloliquefaciens cells released during typical 

enzyme production are tabulated in Table 1. The uncontrolled/untreated scenario 

assumes no control features for the fermentor off-gases, and no inactivation of the 

fermentation broth for the liquid and solid waste releases. The containment criteria 

required for the full exemption scenario assume the use of features or equipment that 

minimizes the number of viable cells in the aerosol releases. They also assume 

inactivation procedures resulting in a validated 6-log reduction of the number of viable 

microorganisms in the liquid and solid wastes relative to the maximum cell density of the 

fermentation broth. 
15

 TABLE 1. Estimated Number of Viable Bacillus amyloliquefaciens

 Cells Released During Production 

_____________________________________________________________________ 

 Uncontrolled/ Full 

Release Media Untreated Exemption Release 

 (cfu/day) (cfu/day) (days/yr) 

AIR 

 Fermentor exhaust 2 x 109 < 2 x109

 350 

 Rotary Drum Filter 7 x 109

 < 7 x 109 100 

 

WATER - to WWT 

 Fermentor cleaning 1.4 x 1017 1.4 x 1011 100 

LAND - to landfill 

 Filter cakes 7 x 1018 7 x 1012 100 

____________________________________________________________________ 

These are "worst-case" estimates for releases from the facility that assume a maximum 

cell density in the fermentation broth of 1011 cfu/ml for bacteria, a fermentor size of 

70,000 liters, and a separation efficiency for the rotary drum filter of 99 percent. For the 

Full Exemption there is also a release to wastewater treatment (WWT) 100 days/yr of 

6.9 x 109 cfu/day each from treatment of fermentor and rotary drum exhausts. 

 1. Releases to Air 

 Specific data which indicate the survivability of B. amyloliquefaciens in the 

atmosphere after release are currently unavailable. Survival of vegetative cells during 

aerosolization is typically limited due to stresses such as shear forces, desiccation, 

temperature, and UV light exposure. However, human exposure may occur via 

inhalation if the organisms are dispersed in the atmosphere attached to dust particles, 

or lofted through mechanical or air disturbance. 

 Air releases from fermentor off-gas and rotary drum filtration could potentially 

result in nonoccupational inhalation exposures due to point source releases. To 

estimate exposures from this source, the sector averaging form of the Gaussian 

algorithm described in Turner (1970) was used. For purposes of this assessment, a 

release height of 3 meters and downward contact at a distance of 100 meters were 

assumed. Assuming that there is no removal of organisms by controls/equipment for 

aerosol releases, the potential human inhalation dose rates are estimated to be 5.11 x 

103

 cfu/yr from fermentor off-gas and 1.46 x 103 cfu/yr from rotary drum filtration 

exhaust for the uncontrolled/untreated scenario, and less than those for systems 

meeting the full exemption criteria. It should be noted that these estimates represent 

hypothetical exposures under reasonable worst-case conditions. 
16

 2. Releases to Water 

The concentrations of B. amyloliquefaciens in surface water were estimated 

using stream flow values for water bodies receiving process wastewater discharges 

from facilities within SIC Code 283 (drugs, medicinal chemicals, and pharmaceuticals). 

The surface water release data (cfu/day) tabulated in Table 1 were divided by the 

stream flow values to yield a surface water concentration of the organism (cfu/l). The 

stream flow values for SIC Code 283 were based on discharger location data retrieved 

from the Industrial Facilities Dischargers (IFD) database on December 5, 1991, and 

surface water flow data retrieved from the RXGAGE database. Flow values were 

obtained for water bodies receiving wastewater discharges from 154 indirect (facilities 

that send their waste to a POTW) and direct dischargers facilities that have a NPDES 

permit to discharge to surface water). Tenth percentile values indicate flows for smaller 

rivers within this distribution of 154 receiving water flows and 50th percentile values 

indicate flows for more average rivers. The flow value expressed as 7Q10 is the lowest 

flow observed over seven consecutive days during a 10-year period. The use of this 

methodology to estimate concentrations of B. amyloliquefaciens in surface water 

assumes that all of the discharged organisms survive wastewater treatment and that 

growth is not enhanced by any component of the treatment process. Estimated 

concentrations of B. amyloliquefaciens in surface water for the uncontrolled/untreated 

and the full exemption scenarios are tabulated in Table 2. 

TABLE 2. Bacillus amyloliquefaciens Concentrations in Surface Water 

_________________________________________________________________ 

 Receiving 

 Flow Stream Flow Organisms 

 (MLD*) (cfu/l) 

 ______________________________________________ 

 Mean 7Q10 Mean 7Q10 

_________________________________________________________________ 

Uncontrolled/Untreated 

 10th Percentile 156 5.60 8.97 x 108

 2.50 x 1010

 50th Percentile 768 68.13 1.82 x 108

 2.05 x 109

Full Exemption 

 10th Percentile 156 5.60 8.97 x 10-2 2.50 x 104

 50th Percentile 768 68.13 1.82 x 10-2 2.05 x 103

_________________________________________________________________ 

*MLD = million liters per day 

 

 
17

C. Release and Exposure Summary 

 Although direct monitoring data are unavailable, worst-case estimates do not 

suggest high levels of exposure of B. amyloliquefaciens to either workers or the public 

resulting from normal fermentation operations. 

 D. Survival of the Organism in the Environment 

 As previously mentioned, the natural habitat for B. amyloliquefaciens is in soil, on 

plant roots, or as an endophyte within the roots or stems of plants. Therefore, survival 

in soil or on roots may be expected to occur, at least for some duration. However, like 

other bacilli, survival in soil may occur predominately as the resistant endospore rather 

than as active vegetative cells. There are two studies in the literature that have 

assessed the survival of B. amyloliquefaciens. 

 Providenti et al. (2009) conducted survival studies of several bacilli, B. 

amyloliquefaciens, B. licheniformis, B. circulans, B. subtilis, B. megaterium, B. 

thuringiensis, and Paenibacillus polymyxa, in intact soil-core microcosms. Using PCR 

with a detection limit of 102 cfu/g of soil, they showed that B. amyloliquefaciens, like the 

three strains of B. subtilis, B. licheniformis, and P. polymyxa, decreased in numbers 

from an initial inoculum densities of 106 to 109 cfu (in 1 to 5 ml of bacterial suspension) 

to below the detection limit by days 25 - 105. B. amyloliquefaciens decreased to below 

the detection limit by about day 25 - 30. 

 A genetically modified B. amyloliquefaciens sprayed onto the leaf surfaces of 

grass showed a decline from 1.5 × 108 to 3.1 × 102 cfu per gram over the course of a 70 

day incubation period (Wendt-Potthoff et al., 1994). Active vegetative cells of this 

modified bacterium died off much more rapidly in soils and roots. Active cells were not 

detected in soil 2 - 6 days post application and declined from approximately 3.2 × 106 to 

3.2 × 102

 cfu per gram in the roots after 27 days (Wendt-Potthoff et al., 1994). The slow 

decline of the vegetative form of the modified B. amyloliquefaciens sprayed onto blades 

of grass as compared to the native soil suggests that survival conditions on vegetative 

surfaces may be more favorable than in soils for both the wild type and genetically 

modified microorganism. 

 Data regarding the survivability of B. amyloliquefaciens in natural waters are not 

available; however, this bacterium is typically not isolated from water. A study 

conducted on the very closely related species B. subtilis showed that it failed to survive 

in sewage samples and in lake water beyond a few days following their release (Liang 

et al. 1982). Vegetative B. subtilis cells fell from an initial value of 4.1 × 105 cells per ml 

to 8 cells per ml two days following its introduction into non-sterile lake water, but during 

the same time period, the spore density increased. 

 

V. INTEGRATION OF RISK 
18

Bacillus amyloliquefaciens exists widespread in the environment as a saprophyte 

in soil or as a plant-associated rhizobacterium. In bulk soil, the organism most 

frequently exists in a resting endospore state, however, it may contribute to the nutrient 

cycling in soil during active growth by virtue of its ability to produce various enzymes. 

The plant-associated strains are known to produce numerous antifungal and 

antibacterial secondary metabolites, and therefore, have utility as biocontrol agents of 

fungal plant pathogens. 

The ability to produce copious amounts of α-amylase, and several other industrial 

enzymes, makes B. amyloliquefaciens a candidate as a host organism for genetic 

modification to produce elevated levels of selected enzymes. To date, B. 

amyloliquefaciens has been used for commercial production of α-amylase, proteases, 

carbohydrases, xylanases, nucleases, phosphatases, and other enzymes. 

There are no reports in the literature that suggest that B. amyloliquefaciens is 

pathogenic to humans. The potential for human infection from B. amyloliquefaciens in 

an industrial setting where exposure to the bacterium is expected to be low and where 

good industrial practices are followed also should be quite low. It would appear that the 

antifungal strains of B. amyloliquefaciens are capable of producing a number of 

secondary metabolites such as lipopeptides and polyketides that apparently the type 

strain of the species and related industrial strains with a history of safe use are either 

genetically diminished in their capability to produce, or simply, are just not produced 

under the conditions used during enzyme fermentation. Although B. amyloliquefaciens 

has not been shown to be toxic to humans, fungicidal strains of B. amyloliquefaciens 

apparently can produce toxins, that when purified and dosed at high enough 

concentrations, can exhibit cytotoxicity in mammalian cells. This presents the possibility 

that some strains may be capable of causing adverse human health effects. It is 

precisely for this reason that the biocontrol strains of B. amyloliquefaciens that would be 

assigned to the subspecies proposed by Borriss et al. (2010), B. amyloliquefaciens 

subsp. plantarum, are not eligible for this Tiered Exemption. The one report of the 

presence of B. cereus-like enterotoxins in a strain of B. amyloliquefaciens is of little 

concern as there are no reports of food-poisoning caused by B. amyloliquefaciens. In 

addition, the enzymes produced for TSCA uses are not for ingestion by humans or 

animals. 

Likewise, the potential for adverse ecological effects resulting from the use of B. 

amyloliquefaciens in fermentation facilities is low. There are no reports in the literature 

indicating that B. amyloliquefaciens is pathogenic or toxigenic to animals. There are no 

reports in the literature indicating any pathogenicity of B. amyloliquefaciens to plants. B. 

amyloliquefaciens is typically thought of as beneficial to plants because it produces 

secondary metabolites inhibitory to the growth of fungal plant pathogens. Also, some 

strains have other plant-growth promoting effects such as IAA production. However, 

these beneficial strains are likely the ones that would fall into the subspecies B. 
19

amyloliquefaciens subsp. plantarum which are not eligible as a recipient microorganism 

for this exemption. 

In conclusion, a thorough review of the literature failed to identify any cases of 

disease caused by B. amyloliquefaciens in humans, animals, or plants despite its 

widespread occurrence in the environment, particularly in soils and on plants. Industrial 

strains of B. amyloliquefaciens in fermentation facilities for the production of enzymes 

have a long history of safe use. With the exemption limited to only the subspecies B. 

amyloliquefaciens subsp. amyloliquefaciens, the concern for cytotoxicity by biocontrol 

strains is mitigated. In addition, enzyme manufacturers routinely test for the presence 

of toxins in their enzyme products as part of their good manufacturing practices, and the 

use of toxin-producing strains would be avoided. There are low hazards presented by 

the bacterium B. amyloliquefaciens subsp. amyloliquefaciens, and there is low exposure 

expected to workers, the general population, and the environment from the use of this 

bacterium in enzyme production. Therefore, there is low risk associated with the use of 

industrial strains of B. amyloliquefaciens subsp. amyloliquefaciens in submerged 

standard industrial fermentation for enzyme production. 

Recommendations

Strains of Bacillus amyloliquefaciens that would fall into the subspecies Bacillus 

amyloliquefaciens subsp. amyloliquefaciens as defined by Borriss et al. (2010) are 

recommended as a recipient microorganism for the 5(h)4 Tiered Exemption. 
20

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