1 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 fr om 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 conditi ons 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

. a myloliquefaciens 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 cons ists of plant-associated strains used as biocontrol agents since they produce a number of antifungal lipopeptide and antibacterial polyketide toxins. This exempti on 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 fe rmentation facilities is low.

History of Commercial Use and Products Subject to TSCA Jurisdiction

The primary TSCA application of B. amyloliquefaciens is the production of α

amylase, an extracellular enzyme wh ich hydrolyzes the internal 1,4- α linkages in amylose and amylopectin. The resulting pr oducts 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-pyrrolidone2 carboxylate peptidase, L-pyrrolidonyl pept idase, 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 antib iotic, but it possesses antivirus and antimycoplasma activities as well (Vollenbroi ch et al, 1997a; Vollenbroich et al., 1997b). Surfactin has physical/chemical characteristi cs similar to those of traditional chemical surfactants. Hence its potential use as an al ternative 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 f ood, 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 import ance in the environment is well-documented for the biological control strains of B. amyloliquefaciens where the bacterium is endophytic or rhizosphere-associated and promot es 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 endospor e stage except when conditions favorable for vegetative growth exist. Under those conditions, the bacterium contributes to nutrient cycling because of the numerous en zymes 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. Mem bers of the genus are capable of producing endospores that are highly resistant to unf avorable 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 gener a or to other existing genera. Some of3 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 monogr aphs 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 seve ral 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 pot ato tubers, and ability to ferment lactose with associated acid pr oduction. 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 indica ted that the species do not contribute to, or share in, a common gene pool and that t hey 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, pyrol ysis 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 the4 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 spec ies, it was suggested that there were two different ecotypes of B. amyloliquefaciens . One ecotype consisted of strains isolated from stems and roots of plants t hat produced antibacterial and antifungal compounds which lessened certain plant diseas es. The other ecotype included strains that were soil-associated rather than plant-asso ciated. 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 report ed that plant-associated strains of Bacillus were very similar, but not identical to, the type strain of B. amyloliquefaciens , DSM 7 T

. By analysis of 16S rRNA, the gyrase A gene, and the histid ine 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 rh izospheres, 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 MALD I-TOF mass spectrometry of cellular components justified the propos ed 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 Prokaryoti c 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 7 T 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 antif ungal 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 7 T, 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 7 T for the non-ribosomal synthesis of antifungal lipopeptides, except for surfactin. This type strain also had a decreased ability to produce polyketides co mpared to the biocontrol strain of B. amyloliquefaciens subsp. plantarum, FZB42. In addition, it was shown that DSM 7 T
produces a different amylase ( amyA ) than the plant-associated strain, which produces a

B. subtilis- like amylase ( amyE)

.5 There is another species of Bacillus that is considered a synonym for B. amyloliquefaciens . An isolate from the brackish wa ter 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 dist inctive 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/ingredient s/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 adverse6 effects, which indicate a low mammalian toxici ty 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 wit h no incidences associated with human pathogenicity. In response to a petition from the Enzyme Technical Association, the FDA affirmed that carbohydrase en zyme 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 l ong 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 qualif ier 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 ca ses 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 coloni zing humans. Given its ubiquity in nature

and the environmental conditions under wh ich 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 lecithinas e, 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 poi soning, 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) repor ted 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 et7 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. Gener ally, 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; Yo shida et al., 2001; Hiradate et al., 2002; Koumoutsi et al., 2004), and the polyketide toxins macrolactin, bacillanene, and difficidin. The cyclical lipoprotein biosur factants are produced by non-ribosomal peptide synthesis

. Arguelles-Arias et al. (2009) descri bed these antifungal compounds. The cyclic lipopeptides are composed of seven (s urfactin and iturin A) or 10 (fengycins) α

amino 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 fo r iturins, and C-14 to C-18 for fengycins.

Although these biosurfactant compounds may have some mammalian toxic potential, they are used in cosmetic applicat ions since they have been shown to have low toxicity. For example, one surfactin isom er, surfactin C, has been tested for toxicity in mammals. In an acute toxicity test of su rfactin C, no lethality in mice was seen after administration of 2500 mg/kg which means that the LD 50 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 occ upants were suffering ill health symptoms. Andersson et al. (1997) were the fi rst 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 ma terials in the water-damaged area. All of the

Bacillus isolates selected from agar plates for fu rther examination in this study were ones that exhibited antifungal activity. The bi omass of these strains from agar plates were extracted with methanol, and the methanol extracts were then assessed for the8 toxicity endpoints of boar spermatozoa vi ability parameters of motility, plasma membrane integrity, and depolariz ation 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. T he 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 poisoni ng toxin cereulide. The B. amyloliquefaciens strains affected boar spermatozoa differently from the indoor B. cereus isolates and the reference food-poisoning strain. The met hanol-soluble mitochondrial toxin extracted

from biomass of B. amyloliquefaciens inhibited the motility of boar spermatozoa and mitochondria were depolarized. At a c oncentration 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 clos e 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 st udied the fungicidal isolates of Bacillus amyloliquefaciens from moisture-damaged buildings. Mikkola et al. (2004) reported that

five strains of B. amyloliquefaciens produced a methanol-extract able, heat-stable, nonprotein substance that inhibi ted 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 Ca 2+ with different selectivities. A similarly prepared methanol extrac t from the type strain of B. amyloliquefaciens

, DSM 7 T , 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 suggest ed 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 to xins 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. The9 channel-forming amylosin and the ionophores c aused 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) show ed 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 supplem ent 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 incidenc e was widely studied, the cause of the

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

chemical constituent that was associat ed with the company’s specific tryptophan manufacturing processes. This included the combination of using reduced quantities of powdered carbon for a purification step wit h 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 to xin 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 reques t 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-dimet hylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) color change in metabolically active Chinese hamster ovary cells(CHOK1) 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 t he 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 en zyme 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-scale10 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 re search group, claimed mammalian toxicity

with B. amyloliquefaciens isolated from water-damaged buildings. However, all of the strains they studied were initially select ed for further study because they exhibited antifungal activity on the agar plates. Rücke rt et al. (2011) compared the genome of the type strain of B. amyloliquefaciens DSM 7 T (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 sequenc es, transposases, integrases, and recombinases which the type species did not have. As previously mentioned, the

DSM 7 T type strain was found to lack the ability to non-ribosomally synthesize some secondary metabolites with antifungal and antibacte rial activities. The type strain could not produce the polyketides difficidin or ma crolantin, and could not produce lipopeptides such as iturin, macrolantin, and other com pounds, 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-associ ated 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 proper ties 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 f ungicidal 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 Hazards11 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 researc hers 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 su rprising 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 questi onable, however, since these studies were conducted using a heavy applicati on 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 f ungal 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) w ho 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 azalom ycin 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 antagoni sm 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 biocontro l strain FZB42 lacking both bacillomycin D and fengycin was greatly diminished in its capacit y 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 pr otecting 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 t he genome of the well-known B. amyloliquefaciensbiocontrol 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 bacillibac tin. 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 t he same antibacterial polyketides, however, genes involved in the ribosome-dependent synthes is 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 is olated 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 mem bers of the iturin family, all of which showed strong and dose-dependent antifungal acti vity 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 et13 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 dimi nished 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 7 T , 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 resulti ng 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 env ironmental release and exposure estimates used in the risk assessments of the original t en eligible recipient microorganisms listed at §725.420 in the Final Microbial Biotechnology Rule promulgated in 1997. Information gleaned from Microbial Commercial Activity No tices received over the years since the rule was published has allowed for better estimates of exposures using a generic scenario (Chemical Engineering Branch, 2011). Fo r this generic scenario, it is assumed that a large-scale closed system enzyme fe rmentation 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 es timated using information available from a

number of submissions to EPA under TSCA Section 5 and from published information14 collected from non-engineered micr oorganisms. 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 work ers 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 pl ant operates 24 hours/day for 350 days per year with 100 batches of enzyme per year.

The National Institute for Occupati onal Safety and Health (NIOSH) has conducted walk-through surveys of several ferm entation facilities in the enzyme industry

and monitored for microbial air contamination. These particular facilities were not using recombinant microorganisms, but the processe s were considered typical of fermentation process technology. Typically, area monito ring data would not be used to estimate occupational exposure levels since the correlation between area concentrations and worker exposure is highly uncertain. Howeve r, personal sampling data are not available at the present time. Thus, area sampli ng data have been the only means of assessing exposures for previous microbial biotec hnology submissions. Applying these NIOSH data to the workers in the various plant areas (laboratory, fermentation, recovery, control room) yields a range of potential i nhalation 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 10 10 to 1.8 x 10 11 cfu/day. However, the typi cal 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, unbr oken skin is considered to be a protective barrier to entry of the microorganism into t he body. Even in the event of dermal contact with broken skin, there is little risk of in fection 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 ferm entor off-gases, and no inactivation of the fermentation broth for the liquid and solid wa ste 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 t he aerosol releases. They also assume inactivation procedures resulting in a validat ed 6-log reduction of the number of viable microorganisms in the liquid and solid wastes re lative 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 10 9 < 2 x10 9 350 Rotary Drum Filter 7 x 10 9 < 7 x 10 9 100

WATER - to WWT Fermentor cleaning 1.4 x 10 17 1.4 x 10 11 100

LAND - to landfill

Filter cakes 7 x 10 18 7 x 10 12 100

____________________________________________________________________ These are "worst-case" estimates for releases from the facility that assume a maximum cell density in the fermentation broth of 10 11 cfu/ml for bacteria, a fermentor size of 70,000 liters, and a separation efficiency for the ro tary 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 10 9 cfu/day each from treatment of fe rmentor and rotary drum exhausts.

1. Releases to Air

Specific data which indicate the survivability of B. amyloliquefaciens in the atmosphere after release are currently unavail able. 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 exposur es due to point source releases. To estimate exposures from this source, t he sector averaging form of the Gaussian algorithm described in Turner (1970) was us ed. 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 remo val of organisms by controls/equipment for aerosol releases, the potential human inhalat ion dose rates are estimated to be 5.11 x

10 3 cfu/yr from fermentor off-gas and 1.46 x 10 3 cfu/yr from rotary drum filtration

exhaust for the uncontrolled/untreated sc enario, and less than those for systems meeting the full exemption criteria. It s hould 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, m edicinal 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 wate r 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 Discharger s (IFD) database on December 5, 1991, and

surface water flow data retrieved from t he 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 rece iving 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 dur ing a 10-year period. The use of this methodology to estimate concentrations of B. amyloliquefaciens in surface water assumes that all of the discharged organism s 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 10 8 2.50 x 10 10

50th Percentile 768 68.13 1.82 x 10 8 2.05 x 10 9

Full Exemption

10th Percentile 156 5.60 8.97 x 10 -2 2.50 x 10 4

50th Percentile 768 68.13 1.82 x 10 -2 2.05 x 10 3

_________________________________________________________________ *MLD = million liters per day17 C. Release and Exposure Summary

Although direct monitoring data are unava ilable, 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 wit hin the roots or stems of pl ants. 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 predom inately as the resistant endospore rather than as active vegetative cells. There ar e two studies in the literature that have

assessed the survival of B. amyloliquefaciens.

Providenti et al. (2009) conducted su rvival studies of several bacilli, B. amyloliquefaciens, B. licheniformis, B. ci rculans, B. subtilis, B. megaterium, B. thuringiensis, and Paenibacillus polymyxa, in intact soil-core microcosms. Using PCR

with a detection limit of 10 2 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 10 6 to 10 9 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 × 10 8 to 3.1 × 10 2 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 applicati on and declined from approximately 3.2 × 10 6 to

3.2 × 10 2 cfu per gram in the roots after 27 da ys (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 sugges ts 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 typi cally 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 × 10 5 cells per ml to 8 cells per ml two days following its introduc tion into non-sterile lake water, but during the same time period, the spore density increased.

V. INTEGRATION OF RISK18 Bacillus amyloliquefaciens exists widespread in the environment as a saprophyte in soil or as a plant-associated rhizobacte rium. 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 virt ue of its ability to produce various enzymes. The plant-associated strains are k nown to produce numerous antifungal and antibacterial secondary metabolites, and theref ore, 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 bac terium 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 st rains 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 ma mmalian cells. This presents the possibility that some strains may be capable of caus ing 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 ecologi cal 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 i ndicating 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 gr owth of fungal plant pathogens. Also, some strains have other plant-growth promoting e ffects 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 elig ible as a recipient microorganism for this exemption.

In conclusion, a thorough review of the lit erature failed to identify any cases of disease caused by B. amyloliquefaciens in humans, animals, or plants despite its widespread occurrence in the environment, particu larly 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 m anufacturers 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 avoi ded. 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 fermentat ion 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 VI. REFERENCES

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