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Risk Assessment of Trichoderma reesei for Consideration of Addition to the List 

of Eligible Microorganisms for the Tiered 5(h)4 Exemptions from MCAN 

Reporting Requirements 

I. INTRODUCTION

Trichoderma reesei is an ascomycetous wood-rot fungus that has been used for 

decades for the production of enzymes for agricultural, food, pharmaceutical, and 

industrial uses. It is the anamorph (asexual reproduction through mitotic division of 

conidia) of the teleomorph (sexual reproduction through the formation of ascospores) 

known as Hypocrea jecorina. QM6a is the designation given to the original strain of T. 

reesei isolated in 1944 from decaying tent fabric on the Solomon Islands in the South 

Pacific during World War II. According to Nevalainen et al. (1994) almost all, if not all, 

industrial strains of T. reesei are derivatives of QM6a. Recent taxonomic studies have 

shown that actually the species T. reesei consists only of this single isolate and its 

derivatives. Many other strains of this fungus isolated from other places, which have 

sometimes been referred to in the literature as T. reesei, have now been proposed as 

belonging to the newly named species, T. parareesei (Druzhinina et al., 2010). These 

other isolates differ from T. reesei in habitat, sporulation, and metabolic versatility. T. 

reesei has been shown to belong to a single species now referred to as H. jecorina/T. 

reesei (QM6a) which reflects its relationship to its teleomorph H. jecorina (Atanasova et 

al., 2010). The only anamorphic strains within the species H. jecorina/T. reesei are 

those of QM6a and its derivatives. Thus, given these recent taxonomic publications, all 

strains of T. reesei are, by definition, QM6a or a derivative (Druzhinina et al., 2010; 

Atanasova et al., 2010). 

T. reesei is not pathogenic to humans, other animals, or plants. However, T. 

reesei can produce a secondary metabolite called paracelsin which is a compound 

known as a peptaibol. Peptaibols are small linear peptides characterized by a high 

content of the non-proteinogenic amino acid α-aminoisobutyric acid, with an acetylated 

N terminus, and a C-terminus that is linked to an amino alcohol. Paracelsin, like other 

peptaibols, modifies cell membranes of various organisms through the formation of ion 

channels which may result in loss of osmotic balance and in leakage of cell contents. 

Paracelsin is inhibitory to the growth of Gram-positive bacteria and to fungi, but it also 

has been shown to exhibit cytotoxicity to mammalian cells and to aquatic indicator 

species. However, paracelsin is apparently not produced by the fungus under the 

conditions of submerged standard industrial fermentation used for enzyme production. 

There is a long history of safe use of T. reesei for production of enzymes for food and 

animal feed. Data submitted in Generally Recognized as Safe (GRAS) petitions to the 

Food and Drug Administration (FDA) for numerous enzyme preparations from T. reesei 

for human and animal consumption demonstrate that the enzymes are nontoxic. Given 

the long history of safe use of T. reesei for production of enzymes, it is appropriate to 
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consider this microorganism eligible as a recipient microorganism for the 5(h)4 Tiered 

Exemptions from Microbial Commercial Activity Notice (MCAN) reporting requirements if 

this fungus was to be used in submerged standard industrial fermentation for enzyme 

production. However, scenarios have been proposed for use of T. reesei in cellulosic 

biofuel facilities under conditions not typical of submerged standard industrial 

fermentation. Publications have suggested on-site cellulase production at biofuel 

facilities by the growth of T. reesei directly on the pre-treated cellulosic hydrolysate (i.e., 

delignified plant material) for degradation of the plant material into sugars that are 

subsequently fermented into ethanol (Wooley et al., 1999; Kazi et al., 2010). The 

growth of T. reesei directly on plant material in biofuel facilities poses concern because 

paracelsin may be produced under these conditions. Historically, the conditions used 

by researchers to optimize peptaibol production by Trichoderma species, including T. 

reesei, have typically involved the addition of insoluble sources of carbon such as 

cellulose to the fermentation broth, the presence of solid surfaces in the fermentation 

broth, or the growth of the fungus on a solid surface. Presently there is no information 

on the quantities of paracelsin that could be formed if T. reesei was grown on pretreated plant material in the saccharification tanks. There is no information on the 

stability of paracelsin, if formed, during the entire process stream of ethanol production 

in cellulosic biofuel facilities. There is no information available on its persistence or 

degradation during wastewater treatment of spent fermentation broth, nor its 

persistence and resulting potential effects if it made its way into the environment. 

Therefore, given paracelsin’s demonstrated biological activity to bacteria, fungi, 

mammalian cells, and aquatic indicator species, T. reesei cannot be recommended as a 

recipient microorganism for the 5(h)4 Tiered Exemption if used under conditions other 

than those typical of submerged standard industrial fermentation for enzyme production. 

History of Commercial Use and Products Subject to TSCA Jurisdiction 

There is a long history of safe use of enzymes produced by various species of 

Trichoderma in agricultural, food, pharmaceutical, and industrial applications. 

According to the Enzyme Technical Association (ETA), Trichoderma enzymes are 

currently used in brewing processes (β-glucanses), in potable alcohol manufacture 

(glucoamylases), for maceration in fruit juice production (pectinases, cellulases, 

hemicellulases), as an additive to livestock feed (xylanases), and for pet food 

processing (ETA, 2010). In addition to their use for enzyme production, some species 

of Trichoderma are used as biocontrol agents of fungal plant pathogens, predominately 

strains of T. harzianum and T. viride (Verma et al., 2007). 

 The species T. reesei has been used extensively for enzyme production in both 

foods and in industrial applications, mainly for production of cellulases. Cellulases have 

been manufactured commercially in Japan since the 1960’s (Toyama, 1969, as cited by 

Nevalainen et al., 1994). Cellulases are used in the food industry for baking, malting, 

brewing, and in grain alcohol production (Nevalainen et al., 1994; Olempska-Beer et al., 

2006). Several cellulases produced by T. reesei have been approved for food use by 
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FDA in GRAS petitions (21 CFR §184.1250). GRAS notices for enzymes produced 

from T. reesei used in food include cellulase, hemicellulase, transglucosidase, pectin 

lyase, and acid fungal protease (http://www.fda.gov/Food/FoodIngredientsPackaging/ 

GenerallyRecognizedasSafeGRAS/default.htm). Cellulases produced by T. reesei have 

also been used in the pharmaceutical industry as digestive aids, and in the animal feed 

industry to increase the digestibility of feed (Malmos, 1977, as cited by Nevalainen et 

al., 1994). 

T. reesei produces a number of different enzymes that have TSCA uses, 

especially cellulases. Historically, T. reesei cellulases have been used in the wood 

processing and textile industries (Nevalainen et al., 1994). More recently, however, 

there has been an enormous interest in producing T. reesei cellulases for use in the 

saccharification step of plant material in cellulosic biofuel production facilities. T. reesei 

also produces numerous extracellular proteases (Kredics et al., 2005). T. reesei may 

also be used for production of hydrophobins, which are proteins with characteristics for 

potential use as biosurfactants (Askolin et al., 2001; Wösten, 2001). To date, the 

Agency has reviewed numerous Microbial Commercial Activity Notices of T. reesei for 

production of enzymes with TSCA applications such as α-amylase, β-glucosidase, acid 

protease, glucoamylase, phytase, laccase, and a number of cellulose-degrading 

preparations. 

 

II. IDENTIFICATION AND TAXONOMY 

 A. Characterization of the Genus Trichoderma 

The genus Trichoderma, which was first named by Persoon in 1794, is a member 

of the Hypocreales (fungi, Ascomycota). It is an anamorphic fungus that reproduces 

solely asexually through mitotic division of conidia. Hypocrea is the genus name of the 

teleomorph that reproduces sexually through the formation of ascospores. Members of 

the genus Trichoderma are ubiquitous in nature. They are geographically widely 

distributed and have been observed in a broad range of habitats because they can 

colonize a wide variety of substrates. Most Trichoderma are aerobic mesophilic wood 

rot fungi found in soils and decaying wood and vegetative materials (Gams and Bissett, 

1998). Trichoderma spp. are very common in agricultural, prairie, forest, and desert 

soils and in salt marshes with a variety of climates (Klein and Eveleigh, 1998). They are 

saprophytic fungi that produce copious amounts of degradative enzymes including 

cellulases, xylanases, and chitinases which enables them to live on a wide variety of 

organic materials and transform many xenobiotic compounds (Klein and Eveleigh, 

1998). The production of cellulases and xylanases by various species, particularly T. 

reesei, has made them important industrial microorganisms. Members of the genus 

Trichoderma produce a vast array of secondary metabolites such as peptaibols and 

peptide antibiotics active against fungi and bacteria. Mycotoxins such as trichodermin, 

trichotoxin A, and trichothecenes are produced by some species of the genus. Some 
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Trichoderma strains produce chitinases and possess antagonistic properties to other 

fungi, and are thus, used as biocontrol agents (Jaklitsch, 2009). Species used as fungal 

biocontrol agents are predominately strains of T. harzianum and T. viride, but also 

include T. lignorum, T. virens, T. hamatum, T. koningii, T. pseudokoningii, T. 

aureoviride, T. longibrachiatum, and T. asperellum (Verma et al., 2007). The 

antagonistic properties to fungal plant pathogens by some Trichoderma strains are due 

to multiple mechanisms such as competition for nutrients and space, modification of 

environmental conditions, promotion of plant growth and plant defense mechanisms, 

and by mycoparasitism (Haran et al., 1996; Benitez et al., 2004; Harman et al., 2004; 

Woo et al., 2006; Verma et al., 2007). Some endophytic species confer plant 

protection, most likely through the production of secondary metabolites (Degenkolb et 

al., 2006). There are also Trichoderma biocontrol agents against bacteria, insects and 

nematodes, and even plants (weeds) (Verma et al., 2007; Bokari, 2009). 

Other species of Trichoderma, such as T. aggressivum, T. pleuroticola, T. 

pleurotom, T. pleuotrophilum, and T. fulvidum are pathogenic to cultivated mushrooms 

such as Agaricus bisporus and Pleurotus ostreatus (Samuels et al., 2002; KomońZelazowska et al., 2007a; Kubicek et al., 2007). 

There is one species in the genus that is considered an opportunistic human 

pathogen, T. longibrachiatum. It has been shown to cause fungal mycosis in 

immunocompromised individuals (Pfaller and Diekema, 2010; Druzhinina et al., 2005). 

B. Taxonomy of the genus Trichoderma 

Currently, there are more than one hundred different Trichoderma species that 

have been molecularly characterized. However, the taxonomy of Trichoderma has 

been problematic and assignment of an accurate species name to an isolate may be 

challenging. Before the discovery and development of molecular biological identification 

methods, fungi were classified based solely on morphological characteristics, often 

incorrectly so. According to Druzhinina et al. (2005), approximately 50% of the 

Trichoderma isolates currently in culture collections under names based solely on 

morphological analysis are wrong. With the advent of molecular techniques for 

identification, the internal transcribed spacer (ITS) 1 and 2 fragments were first used for 

assignment of an isolate into a species. However, Druzhinina et al. (2005) stated that 

the ITS1 and ITS2 sequences do not provide sufficient phylogenetic resolution even 

though these sequences are still commonly used by some researchers. Proper 

identification methods are available however. The currently accepted mode of 

identification for Trichoderma and Hypocrea is a more comprehensive approach 

employing criteria of the Genealogical Concordance Phylogenetic Species Recognition 

(GCPSR) concept, which often demands the use of genealogies of three or four genes, 

not just the spacer regions (Taylor et al., 2000). The GCPSR was meant to provide a 

common approach to classifying new strains of Trichoderma. Druzhinina et al. (2005) 

proposed an online identification tool using an oligonucleotide barcode, i.e., a diagnostic 
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combination of several oligonucleotides specifically allocated within the ITS1 and ITS2 

sequences of the rDNA repeat which they called TrichOKEY. The latest version of this 

tool is available via the International Subcommission on Trichoderma and Hypocrea 

Taxonomy (ISTH) website at http://www.isth.info/index.php. More recently, Druzhinina 

et al. (2010) indicated that additional markers besides those in the GCPSR protocol are 

essential for differentiating among H. jecorina and its close relatives. In any case, these 

methods appear to be best suited for research laboratories doing Trichoderma

systematics rather than routine identification methods needed by commercial entities or 

others. Without these protocols, it appears that differentiation of these closely related 

species will be difficult. Even though identification of new species of Trichoderma may 

be challenging, the lineage of all commercial strains of T. reesei from the original QM6a 

isolate has been established. 

C. The Species Trichoderma reesei, strain QM6a and its Derivatives

 1. The species Trichoderma reesei 

 

The history of the well-known T. reesei QM6a strain is presented in Kubicek and 

Harman (1998). During World War II, because of the military’s interest in determining 

what was causing the rapid rotting of canvas tent material and fabric in the South Pacific 

tropics, research on members of the genus Trichoderma accelerated greatly. A very 

cellulolytic fungal strain was isolated from an island, reported variously as either 

Bougainville or Guadalcanal, in the chain known as the Solomon Islands (now politically 

part of Papua New Guinea). This isolate was designated as Trichoderma viride QM6a. 

At that time there was only one species of Trichoderma which was T. viride (Druzhinina 

et al., 2005). Approximately 20 years later, QM6a was re-named by Simmons as 

Trichoderma reesei after Elwyn T. Reese who conducted the majority of investigations 

on this fungal strain (Kubicek and Harman, 1988). The American Type Culture 

Collection designation for this original strain of T. reesei QM6a (Simmons 1977) is 

ATCC13631. 

As previously stated, Trichoderma reesei is the anamorphic form (asexual 

reproduction) of the fungus which also has a teleomorphic form (sexual reproduction) 

known as Hypocrea jecorina (Eveleigh, 1985). Kuhls et al. (1996) presented molecular 

evidence that T. reesei is indistinguishable from H. jecorina based on sequences of 

internal transcribed spacer (ITS) regions. In addition, failed attempts to cross T. reesei 

with wild type H. jecorina strains led Kuhls et al. (1996) to believe that T. reesei was a 

clonal derivative of H. jecorina. 

Later Lieckfeldt et al. (2000) were successful in isolating anamorphic H. jecorina

from South American soils and vegetation, rather than just the usual teleomorphic H. 

jecorina from ascospores. They demonstrated that H. jecorina was a heterothallic 

ascomycete having two mating types, with ascospores that exhibited bipolar 

segregation within the ascus, the sac containing the sexual spores during sexual 
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reproduction. Using ITS regions, identical sequences between these new anamorphic 

H. jecorina isolates and collection-based cultures of H. jecorina and T. reesei QM6a 

were observed. Thus, it appeared that H. jecorina and T. reesei had a 

teleomorph/anamorph relationship, sharing habitats and gene sequences. However, 

Lieckfeldt et al. (2000) noted differences in metabolic phenotype between strain QM6a 

and other putative H. jecorina (T. reesei) in that all of the new isolates utilized sucrose, 

nitrate, and nitrite, but QM6a did not. 

In the years that followed there were several other reports in the literature on the 

isolation of T. reesei from geographical regions other than the Solomon Islands. Kar et 

al. (2006) reported the isolation of T. reesei from soil in West Bengal, India. Another 

paper reported the isolation of T. reesei from sea mud in the tideland in Lianyungang, 

Jiangsu Province, China (Sun et al., 2006). However, the identification of the fungal 

isolate as T. reesei was questionable as the method of identification used was not 

specified. There is one other article reporting the isolation of T. reesei from an area 

south of the Caspian Sea in Iran (Roodsari et al., 2007). Although robust molecular 

identification methods appear to have been used in the identification of the 22 species 

of Trichoderma found on wood or in soils from Iran, this report exists only as an abstract 

from a presentation at a meeting, and the data have not been published. It could also 

be that in these papers the authors used the better known species name of T. reesei 

rather than H. jecorina. 

Later, mating experiments between T. reesei and H. jecorina conducted by Seidl 

et al. (2009) led to the discovery that T. reesei (strain QM6a) and H. jecorina were not 

identical in mating types. T. reesei has a single mating type known as MAT1-2, 

whereas H. jecorina possesses either a MAT1-1 or a MAT1-2 mating type. Thus, in 

experiments T. reesei was a successful male donor in mating with opposite type H. 

jecorina, but was unable to form female structures when undergoing mating with H. 

jecorina, or with an artificially modified T. reesei having opposite mating type. These 

results led Seidl et al. (2009) to a conclusion different from that of Kuhls et al. (1996) 

which was that T. reesei is not a clonal derivative of H. jecorina, but rather a cryptic 

species. Cryptic species are morphologically similar, or even virtually identical, but 

have been reproductively isolated. In this case Seidl et al. (2009) speculated that the 

reproductive isolation may be related to non-functional or absent genes required for 

female fruiting bodies. Therefore, Seidl et al. (2009) stated that T. reesei is a true 

anamorph of H. jecorina, i.e., a true member of the same species. 

In the literature the names T. reesei and H. jecorina have been used 

synonymously as some researchers consider them to be identical fungi (except for 

mating type). Others think that the anamorph and teleomorph should be referred to as 

separate species. However, a recent taxonomic publication has now proposed a split of 

H. jecorina into as many as four species (Druzhinina et al., 2010). They proposed the 

name T. parareesei for those fungal strains of H. jecorina that were isolated as 

anamorphs from soils, and showed that the majority of these anamorphic strains were 
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genetically isolated from H. jecorina/T. reesei. They also suggested that the remaining 

strains that do not belong to H. jecorina/T. reesei probably belonged to at least one 

other species, but the number of isolates studied was so limited they did not propose 

species names for those few isolates. Later that year, building upon the work of 

Druzhinina et al. (2010), Atanasova et al. (2010) indicated that there were actually three 

species equivalents within H. jecorina sensu lato (H. jecorina in the broad sense). They 

provided data further justifying the proposal for the new species T. parareesei based 

upon differences in habitat, sporulation, and metabolic capabilities of these strains. H. 

jecorina/T. reesei strains were differentiated from T. parareesei by being more sensitive 

to light, having a narrower substrate range, growing less robustly at higher temperatures 

on many carbon substrates, and being less competitive with other fungi. Although more 

light tolerant, T. parareesei was viewed as primarily a soil fungus while H. jecorina/T. 

reesei seemed to be better adapted to plant surfaces. These papers therefore 

proposed that the species T. parareesei would consist of those strains formerly referred 

to as T. reesei from soils in Latin America, Africa, and Southeast Asia. These included 

fungal isolates from soils of subtropical and tropical areas in the South American 

countries of Brazil, Argentina, and Columbia, the Central American country of Mexico, 

from Ghana and Ethiopia in Africa, and from India (Druzhinina et al., 2010). Atanasova 

et al. (2010) also presented data showing that strains within the species T. parareesei 

had lost the ability to reproduce sexually. They stated that although T. parareesei 

strains contain both the MAT1-1 and MAT1-2 mating types, the MAT1-2 locus in T. 

parareesei was altered, and was thus defective compared to that found in H. jecorina. 

These recent taxonomic papers also showed that T. reesei sensu stricto (QM6a) 

was in the same clade as almost all teleomorphic strains of H. jecorina (Atanasova et 

al., 2010; Druzhinina et al., 2010). T. reesei QM6a was the only anamorphic strain 

within that clade, termed H. jecorina sensu stricto. Atanasova et al. (2010) stated that 

the strain QM6a (plus derivatives) and teleomorphic strains of H. jecorina are a single 

species now referred to as H. jecorina/T. reesei (QM6a). This, of course, implies that 

the species T. reesei consists solely of the strain QM6a and its derivatives. 

In summary, recently Druzhinina et al. (2010) proposed splitting up some T. 

reesei/H. jecorina isolates into several new species, one of which was called T. 

parareesei. Atanosova et al. (2010) continued with that work and characterized the 

species T. parareesei, which would consist of those H. jecorina strains isolated from 

soils around the globe as an anamorph which differ from the original QM6a strain in 

sporulation, photosensitivity, substrate utilization, and habitat. Since this new species, 

T. parareesei, was formally described by Atanasova et al. (2010), it now awaits 

acceptance from the scientific community. Upon acceptance, the species H. jecorina 

will consist of the teleomorphic strains remaining after those called T. parareesei and 

some as yet to be named other isolates are removed, plus the single anamorphic isolate 

called T. reesei which encompasses strain QM6a and its derivatives. Only the latter 

has a stable taxonomic status at this time and can be unequivocally identified due to 

well-established culture collection provenance. Therefore, this exemption is being 
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considered only for T. reesei strain QM6a and its derivatives, and not for other potential 

or existing species of Trichoderma or Hypocrea. 

 

2. Derivatives of QM6a 

At the U.S. Army Natick Laboratories in Natick, Massachusetts, researchers 

created mutants of QM6a with increased cellulase production. Conidia of the strain T. 

reesei QM6a (called T. viride QM6a at the time) were suspended in distilled water at 

20°C and were irradiated with high energy electrons from a 24-million electron volt, 18-

kw linear accelerator (Mandels et al., 1971). A dose of 0.05 megarads killed more than 

95% of the conidia, and all were killed with radiation above 0.2 megarads. Survivors 

(0.05 - 0.2 megarads) formed small colonies on deoxycholate agar even after extended 

incubation periods, but then sporulated and were transferred to potato dextrose agar 

where normal growth resumed. One isolate, designated QM9123 (dose, 0.05 

megarads), was found to produce twice as much cellulase as the wild type QM6a strain. 

According to Morawetz et al. (1992), this T. reesei mutant strain QM9123 was 

apparently subjected to another radiation step to produce the mutant strain T. reesei 

QM9414. However, the literature is not clear in exactly what mutagenesis was 

conducted to arrive at strain QM9414. Nevalainen et al. (1994) says that this 

mutagenesis also took place at the Natick Laboratory and references the paper by 

Mandels et al. (1971) for this mutagenesis. The Mandels et al. (1971) paper, however, 

does not mention strain QM9414, but only QM9123 and another mutant obtained that 

was deficient in cellulase production designated QM9136. Montenecourt and Eveleigh 

(1977) stated that QM9414 was derived by a 2-step mutational process from QM6a, but 

again, there are no specifics regarding the second mutation. Brückner et al. (1984) 

stated that QM9414 was obtained from a second irradiation of the conidia of QM6a, but 

these authors reference the Mandels et al. (1971) paper and Montenecourt and 

Eveleigh (1977), neither of which specifically report exactly what the second mutational 

step consisted of. Morawetz et al. (1992) further stated that “the nature of the mutations 

leading from the original isolate QM6a (Simmons 1977) to QM9414 have never been 

elucidated”. It is apparently only in an abstract of a meeting where Simmons (1977) 

described QM9414, but this abstract is not available. The American Type Culture 

Collection (ATCC) gives a history of QM9414 as a mutant of QM9123, and says that it 

was originally Mandels’ strain 3019. The strain QM9123 from which QM9414 was 

derived was described by Mandels et al. (1971) as a mutant that appeared to only have 

an increased quantity of cellulase production and that other carbohydrases were not 

affected. Mandels et al. (1971) further reported that QM9123’s increased cellulase 

activity was stable for more than two years in subcultures and through a lyophilized 

state. Obviously the mutations of QM9123 to arrive at QM9414 resulted in a further 

increase in cellulase production. ATCC says that QM9414 produces 1.5-2.0 times more 

cellulase on cellulose medium than strain QM9123. 

In an article on the safety of T. reesei in enzyme fermentation, Nevalainen et al. 

(1994) say that most, if not all, of the T. reesei strains used industrially are derived from 
9

the T. reesei QM6a originally described by Mandels and Reese (1957). The authors 

present a figure showing the genealogy of the different high cellulase-producing T. 

reesei strains in use throughout the world. All of the following strains were apparently 

derived from the mutant QM9414: MCG77 (Natick Laboratory), the MG Series 

(Gulbenkian Institute of Science), the VTT-D Series (Technical Research Center, 

Finland), the L Series (Cetus Corporation, USA), the MHC Series (Slovak Academy for 

Sciences, Slovakia), the D1 Series (Indian Institute of Technology, India), the Kyowa 

Series (Kyowa Hakko Kogyo Co. Ltd., Japan), and the CL Series (Societe Cayla, 

France). The only series of T. reesei that was not derived from this QM9414 mutant is 

the Rut Series (Rutgers University, USA) which is a derivative directly from the wild type 

QM6a strain. The additional mutations associated with these various high cellulaseproducing T. reesei series were not specified. 

D. Related Species of Concern 

There are a few species within the genus Trichoderma that present human health 

or ecological concerns. One of these is Trichoderma longibrachiatum, which has been 

referred to as a serious opportunistic pathogen that can cause human fungal infections 

(Pfaller and Diekema, 2010). Druzhinina et al. (2008) stated that T. longibrachiatum is 

the species most commonly associated with Trichoderma mycoses, which often has 

unfavorable outcomes. In addition to this species, T. citrinoviride is thought of as a 

potentially pathogenic species (Kuhls et al., 1999). Earlier reports on the isolation of T. 

koningii and T. pseudokoningii from clinical samples were found to be incorrect when 

the sequence analysis of the internal transcribed spacer (ITS) region was examined 

according to Antal et al. (2006). Invasive potential within the genus is apparently 

restricted to T. longibrachiatum and T. citrinoviride based on recent molecular 

taxonomic analyses (Pfaller and Diekema, 2010). Infection by these species has been 

associated with immunocompromised individuals, and those undergoing peritoneal 

dialysis (as summarized by Pfaller and Diekema, 2010). The species T. 

longibrachiatum is apparently closely related to Hypocrea orientalis, which is a member 

of the H. schweinitzii complex, even though it is not the anamorph of H. orientalis 

(Samuels et al., 1998, as cited by Antal et al., 2006). 

In addition to potential infections from some strains of Trichoderma, other strains 

present human health concerns because of the production of mycotoxins or other 

secondary metabolites, some of which are highly toxic (Lübeck et al., 2000). The 

mycotoxin trichodermin is a strong inhibitor of protein synthesis, and thus, is highly toxic 

to mammalian cells (Wei et al., 1974). T. viride has been shown to produce a 

mycotoxin trichotoxin A (Hou et al., 1972) with a median LD50 (lethal dose required to kill 

50% of the population) of 4.36 mg/kg in mice. Degenkolb et al. (2008b) stated that T. 

harzianum and T. viride, two biocontrol strains that are known to produce trichothecene 

toxins, have been re-identified as the closely related species T. brevicompactum and T. 

arundinaceum. Both of these species belong to the T. brevicompactum complex, a 

complex with members that have been proven to produce trichothecene-like mycotoxins 
10

such as trichodermin and harzianum A (Degenkolb et al., 2008a). Some Trichoderma 

species and their toxins are associated with “sick building syndrome” from water 

damaged buildings (Lübeck et al., 2000). T. longibrachiatum, T. citrinoviride, T. 

harzianum, T. viride, T. hamatum, and T. atroviride were all isolated from waterdamaged building materials in various Danish buildings (Lübeck et al., 2000). 

 

 In addition to human health concerns, species producing mycotoxins may 

present concerns for animals other than humans, including aquatic species, and also 

pose concerns for plants. For instance, trichodermin is a potent inhibitor of protein 

synthesis, so it is toxic to all eukaryotes, both animals and plants. 

Some members of the genus Trichoderma are pathogens of commercial 

mushrooms. Earlier reports in the literature stated that the species T. harzianum

caused disease in commercial mushrooms (Gams and Bissett, 1998). Later, new 

names were proposed for the species of Trichoderma that causes green mold of the 

commercial white button mushroom, Agaricus bisporus (Samuels et al., 2002). The 

new names proposed were T. aggressivum var. aggressivum, and T. aggressivum var. 

europeaeum for the fungi responsible for disease of commercial mushrooms in North 

American and Europe, respectively. The former designations were T. harzianum 

biotypes Th2 and Th4, respectively (Samuels et al., 2002). More recently, studies have 

reported that a number of genetically closely related, but phenotypically divergent 

species of Trichoderma are responsible for disease on the oyster mushroom, Pleurotus 

ostreatus, and a number of new species have been described (as summarized by 

Komoń-Zelazowska et al., 2007a). The ability of these strains to attack mushrooms is in 

part due to the production of chitinase that can degrade chitin, an important component 

of fungal cell walls, and mushrooms are fungi. Also, the ability of these particular 

species to cause disease in mushrooms is also due to the production of antifungal 

compounds. T. aggressivum, T. pleuotrophilum, and T. fulvidum are responsible for 

significant loss of the mushroom crops of Agaricus bisporus and Pleurotus ostreatus

(Kubicek et al., 2007). 

III. HAZARD ASSESSMENT 

A. Human Health Hazards 

 

 1. Colonization and Pathogenicity 

Trichoderma reesei is not a human pathogen. There is only one report in the 

literature associating T. reesei with an infection in an immunocompromised human. T. 

reesei is not the species thought to be of increasing importance with infection of 

immunocompromised individuals, which is T. longibrachiatum. The other species of the 

genus thought to have invasive potential is T. citrinoviride (Pfaller and Diekema, 2010). 

T. reesei has a long history of safe use for production of enzymes with food, agricultural, 
11

pharmaceutical, and industrial uses with no incidences associated with human disease. 

Because of its long history of enzyme production, and due to the use of some of these 

enzymes in food and pharmaceuticals, the potential for T. reesei to cause disease and 

the potential toxicity of its enzyme products has been tested a number of times. 

 An elaborate series of studies designed to elucidate both consumer and 

occupational health effects were conducted over the period of 1978 - 1982 on a 

cellulase produced by Novo Industries used as a food processing aid (Hjortkjaer et al., 

1986). The T. reesei microorganism used to produce an enzyme product known as 

Celluclast was tested for its pathogenic potential and for the production of toxins and 

antibiotics. Mice, guinea pigs, and rabbits were used for investigating the potential 

pathogenicity of the T. reesei strain. However, the animals were immunosuppressed 

first with a high dose of cortisone prior to inoculation with either viable or killed spores to 

facilitate survival and multiplication of the fungus in the animals. Nonpathogenic 

microorganisms are typically eliminated readily from a healthy animal by its immune 

system defenses. Preliminary studies for determining the lethality of the fungus in mice 

revealed that the median lethal dose (LD50) was greater than 107 viable spores with 

interperitoneal (ip) injection in mice. In the main study, the immunosuppressed animals 

were injected either intravenously (iv) or ip with 105 to 107 viable spores, or with 107 

killed spores. No effects were seen with ip injection of 105 viable spores, but 

microorganisms were found in different organs 40 days after iv injection of 105 or 107 

viable spores or ip injection of 107 viable spores. In all animals, granulomatous 

inflammation was observed with histological examination. Additional studies on mice 

conducted to further elucidate the chronic nature of the lesions led the authors to 

conclude that T. reesei was pathogenic to cortisone-treated mice. However, 

interpretation of these data by Nevalainen et al. (1994) was that T. reesei should be 

regarded as non-pathogenic because it was only under the circumstances of using high 

doses of spores in immunocompromised animals that T. reesei behaved as a pathogen. 

A pathogenicity study on a strain of T. reesei was conducted by Genencor that 

was described in a GRAS notice for an enzyme submitted to the FDA (GRN No. 333). 

With an ip dose of 2.2 x 107 viable spores of T. reesei strain A83 in Sprague-Dawley 

(SD) rats, T. reesei was judged to be non-pathogenic and non-toxic as no animals died, 

no adverse clinical symptoms were observed, and clearance of the microorganism from 

the rat spleen was observed. A different study in the same GRAS notice using an ip 

dose of 5 x 106 colony-forming units (cfus) in Charles River Sprague-Dawley (CD) rats 

also demonstrated a lack of pathogenicity and clearance of the fungus from the rats. 

Data on the potential pathogenicity of T. reesei are available in another GRAS 

petition (GRN No. 32) for the enzyme pectin lyase submitted by Röhm Enzyme GmbH. 

A genetically modified T. reesei strain, ALKO 2224, and its parental strain were iv 

injected into Naval Medical Research Institute (NMRI) rats at a dosage of 5 x 106 viable 

spores. Even with the injection of cortisone acetate to one group of animals two days 

prior to inoculation to immunosuppress them, no pathogenicity of the T. reesei strains 
12

was observed in any of the rats. 

 In GRAS notice number 315 from Genencor for the enzyme transglucosidase, 

data from another pathogenicity study were reported for T. reesei strain EG1-EP9 (GRN 

No. 315). CD rats were ip injected with 5.6 x 106

 cfu viable microorganisms or with 

heat-killed cells. No deaths occurred and no adverse clinical observations were 

reported. Initial necroscopy on days 7 and 21 revealed enlarged spleens and 

decreased liver weight, however, the animals completely recovered thereafter. 

There is no information available on the presence of any virulence factors in T. 

reesei that would enable it to be pathogenic. Potential fungal virulence factors include 

adherence factors, penetration factors, necrotic factors (enzymes), melanin pigments, 

toxins, and the ability to grow at elevated temperatures. T. reesei is a mesophilic 

fungus that has generally been cultured in vitro in the range of 25-30C, and been 

grown between 25 and 28C for optimal enzyme production. There is one report in the 

literature stating that T. reesei can grow near human body temperature at 36C, 

however, the body of evidence suggests that T. reesei does not pose concerns 

regarding human pathogenicity. 

 2. Mycotoxin Production by T. reesei 

Many fungi produce mycotoxins, and more produce other secondary metabolites 

that are inhibitory to various organisms but are typically less toxic. Mycotoxins are 

highly toxic secondary metabolites that are thought to provide the fungus with a 

competitive edge against other microorganisms, including other fungi. There is one 

report in the literature that T. reesei produces the mycotoxin trichodermin (Watts et al., 

1988). Trichodermin is a member of a group of 12,13-epoxytrichothecenes that are 

potent inhibitors of protein synthesis in eukaryotes, and is therefore highly toxic to 

animals, including humans, and to plants (Wei et al., 1974). According to Blumenthal 

(2004) trichodermin is a member of this family of potent food contamination toxins but it 

is much less toxic than some mycotoxins. 

Watts et al. (1988) reported that trichodermin and another secondary metabolite 

with antifungal activity designated as Tx were isolated from a mycelial extract of T. 

reesei strain P-12. They described the strain P-12 as a mutant of T. reesei strain 

QM9414 although the nature of the mutagenesis that resulted in their strain was not 

specified. As stated above, QM9414 is a radiation mutant of strain QM9123 (Morawetz 

et al., 1992) which is a radiation mutant of T. reesei QM6a (Mandels et al., 1971). In 

her paper on mycotoxin testing in food grade enzyme preparations produced by A. 

niger, A. oryzae, and T. reesei, Blumenthal (2004) questioned the identity of the strain 

used by Watts et al. (1988). She suggested that it was more likely a strain of T. 

harzianum (based upon a personal communication). However, Watts et al. (1988) 

specifically stated that they obtained the T. reesei strain QM9414 directly from the Army 
13

Laboratories in Natick, Massachusetts where the QM9414 strain was originally created. 

Thus, it would appear that there should be no question as to the proper identity of the 

strain as T. reesei. Rather than the strain used by Watts et al. (1988) being incorrectly 

identified, it may be possible that the unspecified mutagenesis that QM9414 was 

subjected to produce the mutant strain P-12 resulted in trichodermin production even 

though trichodermin is not typically produced by T. reesei. There are no other reports in 

the literature suggesting that T. reesei is capable of producing trichodermin. On the 

contrary, more recent papers have demonstrated that mycotoxin production is restricted 

to only certain species of the Trichoderma genus that are not closely related to T. 

reesei. 

In a review of peptaibiotics and mycotoxins from species in the genera 

Trichoderma and Hypocrea, Degenkolb et al. (2008b) stated that T. harzianum and T. 

viride, two biocontrol strains that are known to produce trichothecene toxins, have been 

re-identified as the closely related species T. brevicompactum and T. arundinaceum. 

Both of these species belong to the T. brevicompactum complex, a complex with 

members that have been proven to produce trichothecene-like mycotoxins such as 

trichodermin and harzianum A (Degenkolb et al., 2008a). Degenkolb et al. (2008b) 

specifically stated “only T. brevicompactum and three closely related species within the 

T. brevicompactum complex have been proven to produce trichothecenes-type 

mycotoxins (trichodermin, harzianum A)”. They further stated that two studies have 

shown that trichothecenes were not produced by any species outside the T. 

brevicompactum complex. These more current papers suggest that T. reesei is not 

capable of producing the mycotoxin trichodermin. 

According to Nevalanein et al. (1994) industrial strains of T. reesei are routinely 

checked by enzyme producers to confirm the absence of antibiotic activity and toxins 

including aflatoxin B, ochratoxin A, sterigmatocystin, T-2 toxin, and zearalenone 

according to the recommendations of the Joint FAO/WHO Expert Committee on Food 

Additives (JEFCA, 1981). Given that T. reesei has a long history of safe use in the 

production of food enzymes where there is a need to routinely check for the absence of 

toxins and harmful metabolites, T. reesei strains utilized for production of enzymes used 

in industrial applications likely also do not produce these compounds under the growth 

conditions used in submerged standard industrial enzyme fermentation. 

 3. Production of Secondary Metabolites by T. reesei 

 Secondary metabolites, like mycotoxins, may also provide the fungus with 

competitive mechanisms against other microorganisms. Martinez et al. (2008) stated 

that T. reesei has numerous genes that code for biosynthetic pathways of secondary 

metabolites, and the authors speculated that these may help it survive in its natural soil 

habitat. T. reesei has been shown to produce a secondary metabolite known as 

paracelsin which is a compound known as a peptaibol (Brückner and Graf, 1983). 

Peptaibols are small (up to 20 amino acids) linear peptides of 1000-2000 Daltons 
14

characterized by a high content of the non-proteinogenic amino acid α-aminoisobutyric 

acid (Aib), with an N-terminus that is typically acetylated, and a C-terminus that is linked 

to an amino alcohol which is usually phenylalaninol, or sometimes valinol, leucinol, 

isoleucinol, or tryptophanol (Szekeres et al., 2005). The term peptaibol was formed 

from the names of its three characteristic components, peptide, Aib, and amino alcohol. 

Peptaibiotics (peptide antibiotics) are similar secondary metabolites but the N-terminus 

is rarely acetylated (Szekeres et al., 2005). Peptaibols are associated with a wide 

variety of biological activities as they modify membranes and cause pore formation, 

resulting in leakage from the cell. They can have antifungal, antibacterial, sometimes 

antiviral, antiparasitic, and neurotoxic activity, and many fungal strains producing 

peptaibols are used as biological control agents against plant pathogens (Szekeres et 

al., 2005). Part of their role as biocontrol agents is that they induce plant defense 

mechanisms. They are also being investigated as therapeutic agents because of their 

anti-cancer activity (Mukherjee et al., 2010). 

 It is known that peptaibols are synthesized by large non-ribosomal peptide 

synthetases by a gene referred to as Tex1, and two other genes that code for different 

size peptaibols bearing high homology to Tex1, referred to as Tex2 and Tex3 (Kubicek 

et al., 2007; Mukherjee et al., 2010). However, peptaibol formation by Trichoderma is 

still apparently poorly understood. According to Kubicek et al. (2007) in a review article 

on peptaibols in Trichoderma, there are numerous data gaps regarding how peptaibols 

are actually produced. Kubicek et al. (2007) point out that in most studies investigating 

peptaibol formation, surface cultures were used, but with the same fungus under 

submerged conditions, peptaibols were not formed. There were a few exceptions to 

this, however, as peptaibol production in submerged culture was observed when an 

insoluble component, for instance, a carbon source such as cellulose, was added to the 

culture medium (Brewer et al., 1987). T. viride was shown to produce the peptaibol 

alamethicin in culture when the growth medium was supplemented with one of several 

insoluble components such as dextrin, pharmamedia, or fish meal. Yields of the 

peptaibol were greatly reduced in the absence of these solids (Brewer et al., 1987). 

These authors reported that a number of insoluble polysaccharides added to the culture 

medium increased the production of the peptaibol alamethicin, and that methylcellulose 

resulted in the largest increase. The production of the compound paracelsin by T. 

reesei as originally discovered by Brückner and Graf (1983) was accomplished by the 

solid phase fermentation of the fungus on bagasse and other cellulose-containing 

substrates. Schirmböck et al. (1994) also got peptaibol production with addition of a 

solid component to a T. harzianum culture. However, in this study, the solid surface 

was cells walls of the fungus Botyrtis cinerea. Further investigation revealed it was the 

presence of the cell wall material from the other fungus that triggered both peptaibol 

production and production of hydrolytic enzymes including chitinase, glucanase, and 

protease to degrade the fungal cell walls. Song et al. (2007) used solid-state 

fermentation (SSF) to optimize production of the peptaibols known as trichokonins from 

T. koningii. 
15

 Kubicek et al.’s (2007) summary of the literature also reported that peptaibol 

production in Trichoderma occurred in older cultures, usually more than 15 days old. 

They speculated that with older cultures sporulation was likely occurring. However, they 

stated that there was no definitive link between conidiation and peptaibol production. 

Later that year, researchers in Kubicek’s laboratory published a paper that 

demonstrated that the formation of the peptaibol atroviridin by Hypocrea atroviridis was 

conidiation-associated, and was regulated by both blue light and by a protein known as 

GNA3 (Komoń-Zelazowska et al., 2007b). It was shown that light triggered peptaibol 

formation, but it was only because the light triggered conidiation. No atroviridin was 

produced during vegetative growth of the fungus. 

 Stress conditions have also been shown to result in peptaibol production by 

Trichoderma. An earlier study by Vizcaino et al. (2006) showed that the T. harzianum 

peptaibol synthetase transcript could only be detected under nitrogen starvation. To 

date more than 900 peptaibol sequences have been published (http://www. 

cryst.bbk.ac.uk/ peptaibol/sequence.htm). However, Mukherjee et al. (2010) stated that 

despite the knowledge of the sequences, the genetics of peptaibol formation are not 

well-understood. 

Peptaibols have been shown to be produced in very high amounts in cultures of 

Trichoderma strains in some studies. Ooka et al. (1966) showed that T. viride produced 

a peptaibol in the extremely high concentration of 20 g/L over the course of 70 - 90 

hours (< 3 - < 4 days) with shake-flask culture of a broth supplemented with 4% soluble 

starch and 1% Polypepton at 30°C. Meyer and Reusser (1967) used submerged 

culture supplemented with molasses, dextrin, fish meal, and Pharmamedia to get 

production of 8 - 10 mg/L of a peptaibol from a strain of T. viride grown at 25°C for 4 - 5 

days. Song et al. (2007) obtained the maximum production of trichokonin VI of 4.07 

mg/g dry substrate under optimized conditions in solid-state fermentation. 

 Several different paracelsins have been discovered since the term was first 

used by Brückner and Graf (1983) for the toxic secondary metabolite produced by T. 

reesei. Paracelsins have 20 amino acids (Brückner and Graf, 1983), and are 

characterized by the presence of phenylalaninol as the C-terminal amino alcohol, and 

by the specific amino acid content and sequence (Ritieni et al., 1995). The structures of 

paracelsin A, B, C, and D (Brückner et al., 1984), which were isolated from both the 

fungal mycelium and the culture broth of T. reesei QM9414, were elucidated by Fast 

Atom Bombardment Mass Spectroscopy (fabms) by Przybylski et al. (1984). These four 

paracelsins, A, B, C, and D, were individual components in a microheterogeneous 

paracelsin mixture. Przybylski et al. (1984) stated that a characteristic of peptaibols is 

their microheterogeneity which is a result of their non-ribosomal biosysthesis 

mechanism. Paracelsin E was later isolated from T. saturnisporum which also 

produced paracelsin A, B, C, and D (Ritieni et al., 1995). 

Brückner and Graf (1983) demonstrated that paracelsin extracted from T. reesei 
16

QM9414 had antibacterial action against Bacillus subtilis, Micrococcus luteus, 

Staphylococcus aureus, Streptococcus faecalis, Streptococcus lactis, and 

Streptococcus thermophilus. It was not inhibitory to Candida albicans, Escherichia coli, 

Porteus vulgaris, Serratia marcescens, Salmonella typhimurium, Pseudomonas 

aeruginosa, or Enterobacter aerogenes. Paracelsin was shown to have membranemodifying properties and antibiotic activity, particularly against Gram-positive bacteria. 

In a study on peptaibols produced by 12 different species of Trichoderma, most 

of which had potential as biocontrol agents, a strain of T. reesei was shown to produce 

paracelsin (Solfrizzo et al., 1994). Paracelsin production was common among the 

Trichoderma species studied as it was also observed in T. citrinoviride, in one of two 

strains of T. hamatum, in T. inhamatum, T. koningii, T. longibrachiatum, T. 

parceramosum, T. polysporum, and T. pseudokoningii, in both strains of T. 

saturnisporum, and in T. viride. The only species in which paracelsin production was 

not observed was in the two strains of T. harzianum. 

 

 Paracelsin has been shown to have toxicity toward mammalian cells. Brückner 

et al. (1984) reported that paracelsin exhibited hemolytic activity on human erythrocytes. 

They stated that erythrocyte hemolysis is a common characteristic of other peptaibols 

such as alamethicin, suzukacillin, and trichotoxin. The in vitro hemolytic activity of 

paracelsin was reported as C50 = 3.7 x 10-5 mol/liter (where C50 is the concentration that 

causes 50% hemolysis). Paracelsin exhibited lesser hemolytic activity than trichotoxin 

(C50 = 2.1 x 10-5 mol/liter) and alamethicin F-50 (C50 = 1.6 x 10-5 mol/liter). Brückner et 

al. (1984), when discussing the hemolytic activity of paracelsin, also stated that “in vivo 

hemolysis is also suspected to be the reason why the lethal dose is about 5 mg/kg mice 

when paracelsin is administered intraperitoneally”. However, the authors do not present 

any data for determination of this lethal dose, nor do they cite a reference for this study. 

Therefore, it is unknown whether this value is from unpublished data from the authors or 

whether these are data from another study that cannot be located in the literature. 

Abu Raya et al. (1993) reported that paracelsin showed toxicity to PC12 cells (a 

cell line derived from a pheochromocytoma of the rat adrenal medulla) with a CC50 

(cytotoxicity concentration of 50%) of 21.8 μM. Grigoriev et al. (2003) showed that 

paracelsin A was toxic to mice at an intraperitoneal dosage of 20 mg/kg body weight. In 

this study, all of the highly membrane active peptaibols which included alamethicin F30, paracelsin A, chrysospermin C, and the lipopeptaibol texenomycin A were toxic to 

mice. Long lasting hypothermia in mice, which was an indicator of neuroleptic activity 

(affecting the brain, especially by reducing the intensity of nerve function, e.g., 

tranquilizing) was not observed in this study because the mice died shortly after 

administration of the compounds (Grigoriev et al., 2003). 

The toxicity of paracelsin reported in the two studies above seems quite high, 

particularly in comparison to the reported toxicity of trichodermin, an actual mycotoxin 

with high toxicity to both animals and plants (Wei et al., 1974). Blumenthal (2004) 
17

stated that trichodermin had an LD50 of 500 mg/kg of body weight with intraperitoneal or 

subcutaneous administration in mice according to the Registry of Toxic Effects of 

Chemical Substances. This Registry has other toxicity values for trichodermin in 

addition to this one reported value. The LD50‘s of trichodermin were 500 and 354 mg/kg 

in mice with intraperitoneal injection, and 500 mg/kg in mice with subcutaneous 

injection. LD50 values were approximately two orders of magnitude lower with oral 

administration of trichodermin. The Registry of Toxic Effects of Chemical Substances 

reported an LD50 of 6.2 mg/kg with oral administration of trichodermin to mice, and an 

LD50 of 7.5 mg/kg with oral administration of trichodermin to rats. 

In addition to paracelsin, other secondary metabolites have been reported from 

T. reesei. Trichodermatides, which are polyketide compounds that exhibit weak 

cytotoxicity against the A 375-S2 melanoma cell line, were reported by Sun et al. (2006) 

and Sun et al. (2008) as being from the marine fungus T. reesei. As stated previously, 

these two papers do not specify how the particular strain of Trichoderma was identified. 

T. reesei is not known as a marine fungus, so it is unknown whether the fungus studied 

in these two papers was actually T. reesei. There apparently are, however, marine 

strains of T. longibrachiatum (Mohamed-Benkada et al., 2006; Ruiz et al., 2007) and T. 

koningii (Landreau et al., 2002), the former of which was isolated from blue mussel and 

the latter from a shellfish (cockle) farm. 

 4. Test Data on Toxin Production 

A number of different studies have been conducted assessing the potential for T. 

reesei to produce toxins during submerged fermentation for production of enzymes for 

food, pharmaceutical, or industrial uses. In addition to the pathogenicity testing of the T. 

reesei strain used to produce the enzyme Cellulast as discussed above, the enzyme 

product itself has also been tested for toxicity (Hjortkjaer et al., 1986). The test material, 

referred to as the “tox-batch” was tested for general oral toxicity, effects on 

reproduction, mutagenic potential, inhalation toxicity, irritation to skin and to eyes, and 

for skin sensitization. Acute oral toxicity studies were conducted in mice, rats, and 

dogs. No toxicity was observed in any species and the acute oral toxicity was > 8 g/kg 

in rats, >16 g/kg in mice, and >5 g/kg in dogs (the highest dose tested in each species). 

Subchronic toxicity studies, which consisted of feeding the tox batch for 91 days at 0, 1, 

2, or 5%, were conducted in rats and dogs. There was no evidence of systemic effects 

in dogs up to 3.0 g/kg of bodyweight (bw) per day, the highest dose tested. In the 91-

day subchronic toxicity study in rats, the highest dietary level of the Celluclast enzyme 

product (tox-batch) of 5% depressed body weights in male rats but not female rats. 

Rats tolerated the tox-batch at the 1% or 2% dietary level (~1000 mg/kg/bw). 

Additional toxicity studies have been conducted on other enzymes produced by 

T. reesei. In GRAS petition No. 315, a 91-day subchronic feeding study with a cellulase 

was conducted in CD rats to assess the potential toxicity of the enzyme product. No 

mortalities were recorded throughout the entire investigation. There were no treatment-
18

related adverse effects on any of the parameters measured (systemic toxicity, food 

consumption, opthalmology, clinical chemistry, body weight, organ weight, urine 

analysis, hematology, necropsy, and histopathology). It was concluded that the 

cellulase from T. reesei did not result in toxicity up to and including a dose level of 5% 

w/w in the diet. A No Observable Effect Level (NOEL) was established at 5% in the diet 

corresponding to 3.35 and 4.05 g cellulase/kg bw/day for male and female rats, 

respectively (GRN No. 315). 

 Data from a number of other studies are also presented in this GRAS petition 

(GRN No. 315) for different enzymes from the same T. reesei strain including a high pI 

xylanase, a low pI xylanase, endoglucanase III, endoglucanase I, and protease. A 91-

day subchronic feeding study in CD rats at concentrations up to 50,000 mg/kg of a high 

pI xylanase reported two deaths, but they were not attributed to the treatment. No other 

treatment-related adverse effects were observed on any of the parameters measured 

(organ weight, urine analysis, hematology, necropsy, and histopathology) with the high 

pI xylanase indicating a lack of toxicity of the enzyme. Likewise, a 91-day subchronic 

feeding study was conducted with a low pI xylanase produced by T. reesei with feeding 

concentrations up to 3000 mg/kg/day. There were no deaths and no adverse treatmentrelated effects on any of the same parameters measured as listed previously. The 

NOEL was established at 3000 mg/kg for the low pI xylanase. 

 Also presented in GRAS Notice 315 is data from a 28-day oral toxicity study in 

rats for a T. reesei endoglucanse III administered at concentrations up to 1000 

mg/kg/day for four weeks. No mortalities were observed in the Charles River rats 

during the study. There were no treatment-related adverse effects either, which 

established a NOEL of 1000 mg/kg/day (GRN No. 315). For another T. reesei enzyme, 

endoglucanase I, there was a 14-day oral feeding study and a 91-day subchronic 

feeding study conducted. There were no adverse effects reported for any parameter 

monitored in the 14-day oral study in Charles River CrlCD®BR rats dosed with up to 

1000 mg/kg/day by oral gavage using a dosage volume of 10 ml/kg bw. Likewise, 

there was no toxicity observed in the 91-day subchronic feeding test. There were no 

mortalities, and no adverse treatment-related effects were observed on any of the 

parameters measured (organ weight, urine analysis, hematology, necropsy, and 

histopathology). At study termination, there were no differences in the behavioral test 

(open field) and functional tests (gripping reflex, startle reflex) between the control and 

treatment groups. A NOEL was established for this enzyme of 1000 mg/kg/day. 

 A protease produced by T. reesei was also tested for toxicity with a 91-day 

subchronic feeding test (GRN No. 315). There were no treatment-related deaths or 

adverse effects observed. A NOEL of 31.25 mg total protein/kg bw/day was established 

(the highest dosage studied). 

 Data from an acute toxicity test for another T. reesei enzyme, glucoamylase, are 

also presented in GRAS petition 315. Under the conditions of this study where the rats 
19

received a single oral dose followed by a 14-day period of observation, the oral LD50

was > 1996 mg total protein/kg bw which was the highest possible dose volume of 10 

ml/kg. This same enzyme was also tested in a 91-day subchronic feeding test in SD 

rats. There were no overt signs of systemic toxicity or adverse clinical observations 

attributable to treatment (GRN No. 315). 

 5. Allergic Reactions/Sensitization to T. reesei and its Enzymes

 Although fungi in general can be allergens, there are no reports in the literature 

specifically suggesting that T. reesei causes allergenicity in humans but there are 

reports for other Trichoderma species. There are two reported cases of hypersensitivity 

pneumonitis, also known as extrinsic allergic alveolitis, caused by T. koningii and by T. 

viride (Halpin et al., 1994; Enriquez-Matas et al., 2009). In addition, there is one report 

of T. longibrachiatum causing allergic fungal sinusitis (Tang et al., 2003). 

 Enzymes themselves are known allergens. However, there are no allergenicity 

issues specifically attributable to enzymes produced by T. reesei vs. enzymes produced 

by other fungi. 

 

 6. Human Health Hazard Conclusions 

 There is only one potential concern for human health hazards associated with T. 

reesei and that is paracelsin production. There is no concern for potential pathogenicity 

of T. reesei. Test data on various industrial strains typically do not show adverse 

effects, except in one case where pathogenicity to mice was demonstrated, but only 

under the conditions where the mice were intentionally immunosuppressed and then 

subjected to high inoculation rates. Toxicity testing on a number of enzymes produced 

by T. reesei indicates that the fungus does not produce toxins under the conditions used 

in submerged standard industrial fermentation for enzyme production. There is, 

however, concern for the production of the secondary metabolite paracelsin if T. reesei 

was to be grown under certain other conditions. Paracelsin has been shown to be 

capable of hemolysis of human erthyrocytes, and has been reported to be toxic to mice 

at a concentration of 20 mg/kg (Grigoriev et al., 2003) which is a lower amount than that 

of an actual mycotoxin known as trichodermin that is produced by some Trichoderma 

spp. 

 Paracelsin production is not expected to occur under the conditions of 

submerged standard industrial fermentation for enzyme production. However, 

according to information available in the literature, extended fermentations, or 

fermentations that contain insoluble carbon sources such as cellulose may result in 

paracelsin production. Therefore, the potential for paracelsin production during 

fermentation of plant material in a cellulosic biofuel facility, and the potential 

environmental introduction of paracelsin resulting from the use of T. reesei in cellulosic 

material degradation warrants further investigation. Even before interest in using T. 
20

reesei for biofuels, Brückner et al. (1984) came to a similar conclusion years ago with 

other uses of Trichoderma, and it was Brückner and Graf (1983) that first isolated 

paracelsin. Brückner et al. (1984) stated “With regard to the most intensive efforts for a 

biotechnological application of Trichoderma – both in submerged and solid-substrate 

fermentation – for the enzymatic conversion of cellulosic materials used for animal feed 

or human food and, moreover, the contribution of these microfungi and their relatives to 

the moldiness, spoilage and deterioration of organic matter, the excretion of paracelsin 

and related metabolites should be seriously taken into account. These mycotoxins 

exhibit a great variety of biological activity and are sometimes produced in 

extraordinarily high amounts”. 

 

B. Environmental Hazards 

 1. Hazards to Animals 

T. reesei is not pathogenic to animals, and there are no reports in the literature 

suggesting it can cause infection or disease in animals. In a summary of the literature 

Klein and Eveleigh (1998) stated that some of the secondary metabolites produced by 

some Trichoderma spp. such as glioviridin (a diketopiperazine), sesquiterpenoids, 

trichothecenes, cyclic peptides, and isocyanide-containing metabolites (trichoviridin) 

may be inhibitory to organisms other than microorganisms. Some of these compounds 

may result in ill-thrift of sheep due to their inhibitory action on cellulolytic rumen 

microflora (Brewer et al., 1982). It is known that paracelsin, the peptaibol produced by 

T. reesei, is inhibitory to Gram-positive bacteria such as Bacillus subtilis, Micrococcus 

luteus, Staphylococcus aureus, Staphylococcus faecalis, Streptococcus lactis, and 

Streptococcus thermophilus (Brückner and Graf, 1983). Brückner et al. (1984), in a 

discussion of the potential effects of paracelsin, stated “the increase of the digestion of 

starch and cellulose in rumen liquor by the addition of low concentrations of paracelsin 

should be considered in the discussion of the association of metabolites from 

Trichoderma with the ill-thrift of pasture fed ruminants, and may be explained by the 

suppression of various feed rivals of the cellulolytic activ(e) microorganisms”. 

 Paracelsin has been shown to exhibit toxicity to mammalian cells. Data from 

mice and rat studies were previously presented in the human health effects section. 

The toxicity of paracelsin to aquatic species has also been directly tested. Solfrizzo et 

al. (1994) studied the toxicity of paracelsin and other compounds extracted from six 

Trichoderma species to Artemia salina (brine shrimp) larvae. Paracelsin was shown to 

have an LC50 (concentration that is lethal to 50% of the organisms) of 2.2 μM which was 

equivalent to approximately 4 μg/ml (Solfrizzo et al., 1994). The toxicity of the extracts 

of five of the six Trichoderma strains to A. salina could be attributed to the production of 

paracelsin by these strains, whereas in T. harzianum, toxicity was attributed to an 

unidentified peptaibol(s). 

Favilla et al. (2006) tested the toxicity of various secondary metabolites of 
21

biocontrol fungi to both Artemia salina and Daphnia magna (water flea). The three 

peptaibols tested included alamethicin, paracelsin, and antiamoebin, the first two of 

which are produced by some Trichoderma species. The paracelsin used in these 

studies was a mixture of paracelsin homologs (purity 88.4%) purchased from a chemical 

supply company. With 24 hr. exposure to A. salina, the LC50 of paracelsin was 21.26 

μM (equivalent to 40.84 μg/ml). The LC50 decreased with 36 hr. exposure to 9.66 μM 

(equivalent to 18.56 μg/ml). One of the most toxic fungal compounds was alamethicin, 

which is produced by some Trichoderma species including all the members of the 

Trichoderma brevicompactum clade that previously included strains of T. viride

(Degenkolb et al., 2008a). For comparison, the LC50 of alamethicin was 5.32 μM (10.43 

μg/ml) and 1.96 μM (3.84 μg/ml) for 24 and 36 hr. exposure, respectively, so it is 

approximately four to five times more toxic to brine shrimp than paracelsin. However, 

paracelsin was about twice as toxic to brine shrimp as gliotoxin, an actual mycotoxin 

produced by several fungi such as Aspergillus, Penicillium, and Trichoderma. In 

experiments with D. magna, the LC50 of paracelsin was 7.70 μM (equivalent to 14.79 

μg/ml) with a 24 hr. exposure, and 5.60 μM (equivalent to 10.76 μg/ml) with a 36 hr. 

exposure (Favilla et al., 2006). These LC50 values for D. magna are considered 

moderately toxic. Favilla et al. (2006) stated that the biological activity of peptaibols is a 

result of their ability to form pores in lipid membranes by forming gated ion channels. 

The pores lead to the loss of osmotic balance from leakage. 

 Poirier et al. (2007a) reported that peptaibols and peptaibol-producing 

Trichoderma were detected in marine sediments that exhibited a high toxicity to mussel 

larvae from a shellfish farming area in France. Peptaibols were also detected in the 

marine bivalves. Poirier et al. (2007b) conducted bioassays with oyster (Crassostrea 

gigas) embryos with alamethicin and different groups of peptaibols that were produced 

by a strain of T. longibrachiatum that was isolated from the mussels. They found that 

peptaibols of both long and short sequence disturbed embryogenesis of oysters at 

nanomolar concentrations. Toxicity of the peptaibols was between 1.3 and 8 times 

greater than the toxicity induced by copper which was used for reference, and the 

longer peptaibols were found to be more toxic than the shorter ones. The toxicity was 

suggested to be associated with the membrane-modifying properties of peptaibols 

wherein they interact with biological membranes and modify their permeability by 

forming voltage-dependent membrane ion channels through which cytoplasmic contents 

can leak out (Poirier et al., 2007b). 

 In an earlier study, Sallenave et al. (1999) examined the experimental 

bioaccumulation in filter-feeding mollusks of fungal “mycotoxins”, which were later 

referred to as peptaibols rather than mycotoxins by Poirier et al. (2007b). Sallenave et 

al. (1999) exposed mussels to culture filtrates of Trichoderma koningii that was 

previously isolated from cockles (Cerastiderma edule). The toxic metabolites 

bioaccumulated in the mussels, and the digestive gland of the mussels had the greatest 

toxicity indicating that contamination was from filtration. There are no reports in the 

literature of bioaccumulation of peptaibols by aquatic species in the environment. 
22

However, it was not until 2007 when peptaibols were actually detected in the 

environment in marine sediments and in shellfish (Poirier et al., 2007a). 

In a review of peptaibiotics and mycotoxins in the genera Trichoderma and 

Hypocrea, Degenkolb et al. (2008b) commented that the high toxicity reported for the 

peptaibols by Poirier et al. (2007b) (alamethicin in oysters) and Favilla et al. (2006) 

(alamethicin, paracelsin, and others in A. salina and D. magna) in aquatic invertebrate 

studies may be a result of contamination of the alamethicin used in these experiments. 

Degenkolb et al. (2008b) suggested that the alamethicin standard used in these studies 

(Sigma-Alrich A-4665) may have been contaminated with the trichothecene mycotoxin 

harzianum A which was shown to be the case in some of their own studies. Harzianum 

A has been shown to be highly toxic to A. salina. However, this possible explanation for 

the toxicity of alamethicin in these two studies has no relevance to the toxicity values of 

paracelsin to brine shrimp or D. magna presented by Favilla et al. (2006), nor on the 

toxicity values of paracelsin to brine shrimp reported by Solfrizzo et al. (1994). 

Landreau et al. (2002) speculated that the neurotoxicity observed with peptaibols 

from T. koningii was related to the amphiphilic nature of the molecules that form pores 

in phospholipid membranes. In addition to pore formation, peptaibols have been shown 

to uncouple oxidative phosphorylation of mitochondria, stimulate catechol secretions of 

adrenal cells, activate membrane-bound enzymes, and impart cytotoxicity, neurotoxicity, 

and neuroleptic effects (as summarized in Poirier et al., 2007b; Szekeres et al., 2005). 

 2. Hazards to Plants 

Trichoderma reesei is not a pathogen of plants. Although it is capable of 

degrading cellulose and hemicellulose due to the copious quantities of these enzymes it 

can produce, it is not a primary colonizer on plant tissue. Genetic studies have shown 

that it does not contain any genes for ligninases that are required for initial breakdown 

of plant material (Maheshwari, 2008). T. reesei is known as a wood rot fungus, but it 

apparently attacks decaying plant material, not live plants. This species does not 

appear to pose a hazard to plants. 

 3. Effects on Other Organisms 

As previously mentioned, peptaibols, including paracelsin, are toxic to Grampositive bacteria and to some fungi. Peptaibols are apparently not inhibitory to Gramnegative bacteria, and it assumed that this is a result of the differences in the cell walls 

of Gram-positive and Gram-negative bacteria. Paracelsin has also been shown to be 

inhibitory to the fungus Phoma destructiva (Grigoriev et al., 2003). They measured 

differences in membrane pore formation by a number of peptaibols produced by a 

number of different fungi including paracelsin isolated from an unspecified Trichoderma 

sp. The 19-20mer peptaibols such as alamethicin F-30, paracelsin A, and 

chrysospermin C all had very high membrane activity, and the larger peptaibols 
23

exhibited higher activity by several orders of magnitude compared with the smaller 

compounds, the 11-17mer peptaibols. Watts et al. (1988) reported that two metabolites 

produced by T. reesei, trichodermin, and another compound designated as Tx, were 

inhibitory to species of Aspergillus, Cladosporium, Fusarium, Rhizoctonia, and Pythium. 

However, as mentioned previously, the identity of that fungus as T. reesei has been 

questioned. Other studies have investigated the biocontrol potential of T. reesei. 

Harjono and Widyastuti (2001) found that high concentrations of an endochitinase 

produced by T. reesei lysed the hyphae of the plant pathogenic fungus Ganoderma 

philipii. Seidl et al. (2004) reported that the teleomorph of T. reesei, H. jecorina, was 

antagonistic to the plant pathogen Pythium ultimatum in plate confrontation tests. 

Apparently T. reesei can be antagonistic to other fungi, but it has not been developed 

commercially as a biocontrol agent because its antifungal activity is inferior to that of 

other strains of the genus. 

As previously mentioned, some species of Trichoderma, specifically T. 

aggressivum, T. pleuotrophilum, and T. fulvidum are pathogens of mushrooms, which of 

course, are fungi. However, T. reesei is not a pathogen of mushrooms. 

 4. Stability of Peptaibols in the Environment 

There is no information in the literature on the stability of the paracelsin molecule 

in the environment. There is little literature on the stability of other peptaibols either, but 

it is known that peptaibols form helical structures in phospholipid membranes. In a 

paper on the structure-functional relationship of model α-aminoisobutyric acid peptides, 

Higashimoto et al. (1999) stated that “ the presence of α-aminoisobutyric acid, because 

of its preference for certain dihedral angles most suitable for 310 – or α-helical 

structures, gives a particular stability to their conformations in membranes in a variety of 

environments”. 

Poirier et al. (2007a) were the first to detect the presence of peptaibols produced 

by Trichoderma in the environment, in marine sediments. Their isolation of peptaibols 

from the bivalve mussels cultivated in those sediments led them to state that theirs was 

“the first observation of contamination of the marine human-food chain by fungal 

metabolites”. The fact that they could still detect peptaibols in sediments samples that 

had been frozen for years led Poirier et al. (2007a) to suggest that peptaibols could be 

considered as stable markers for fungal presence in the marine environment. 

Song et al. (2006) described the stability of secondary metabolites produced by 

T. koningii called trichokonins, which they referred to as peptaibol-like antimicrobial 

compounds. These trichokonins were referred to as peptaibols, rather than just 

peptaibol-like compounds, in a review article by Samuels et al. (2006). They are also 

considered to be peptaibols by others as they are included in the peptaibol database 

(http://www.cryst.bbk.ac.uk/peptaibol/home.shtml). Trichokonins also contains the nonproteinogenic α-aminoisobutyric acid as do peptaibols. Song et al. (2006) reported that 
24

trichokonins were inhibitory to Gram-positive bacteria and a number of plant pathogenic 

fungi. To evaluate their stability to temperature, a solution of the antimicrobial 

compounds dissolved in buffer was heated for 30 minutes at 50, 70, 90, or 100C, and 

was also subjected to autoclaving. In addition, the solution was stored at 4C for 10 

days, at -20C for 30 days, and the frozen solution was then lyophilized. The 

trichokonins were found to be extremely heat resistant, with no loss of activity at any 

temperature tested, including autoclaving at 121C and 15 psi for 15 minutes. Cold 

storage and lyophilization did not affect their activity either. Their biological activity was 

retained over a pH range of 3.0 to 10.0 as well. The sensitivity of trichokonins to 

proteolytic enzymes such as trypsin, neutral protease, basic protease, and multiplex 

protease was also tested by Song et al. (2006). The trichokonins were insensitive to 

treatment with proteolytic enzymes, and the majority of their antimicrobial activity was 

retained. 

 5. Environmental Hazard Conclusions 

The issues for environmental hazards are similar to those for human health 

hazards. The primary hazard concern for T. reesei is for the production of paracelsin, 

the moderately toxic secondary metabolite. Under usual conditions of submerged 

culture for standard enzyme production, well-established commercial strains of this 

species do not seem to produce paracelsin. However, fungal secondary metabolites 

can be produced after extended fermentation, particularly when sporulation occurs after 

depletion of nutrients. It has also been demonstrated that these peptaibol compounds 

may be produced in the presence of an insoluble carbon source such cellulose. 

Therefore, its growth on plant material as proposed for on-site production of enzymes in 

cellulosic biofuel facilities by NREL (Wooley et al., 2009; Kazi et al., 2010) may 

potentially result in the production of significant quantities of paracelsin in the 

fermentation broth. Large quantities of paracelsin could be possible with extended 

fermentations and/or the absence of exponential growth (i.e., the stationary growth 

phase) in the presence of a surplus of carbon with a shortage of nitrogen, conditions 

that are known to stimulate the formation of secondary metabolites by fungi. 

 

 There are two issues warranting consideration with the use of T. reesei cells in 

cellulosic biofuel plants. One is the potential for cells of T. reesei to escape 

containment, and T. reesei is expected to survive in the environment if inadvertently 

released. However, the releases expected from closed system use are expected to be 

low. It is unknown whether T. reesei would produce any quantity of paracelsin in the 

environment that would be of any concern. However, there needs to be a consideration 

of the concentrations of paracelsin that could be released in wastewater from a 

cellulosic biofuel facility. It is unknown whether paracelsin, if it was formed, would be 

denatured, with a resulting loss of toxicity, at some point in the process stream, such as 

with high temperatures used for ethanol distillation. It is also not known whether 

paracelsin would be rapidly degraded during wastewater treatment, or whether it could 
25

possibly remain intact in the stable α-helical form known to occur in membranes. There 

are no data in the literature on the degradation of peptaibols in the environment, except 

for the one paper of Poirier et al. (2007a) that reported the persistence of peptaibols in 

marine sediments and stability of the compounds for years when in frozen sediments. 

Extraordinary stability was observed for the peptaibols known as trichokonins, as 

extremes in temperature, including autoclaving, and freezing followed by lyophilization, 

did not denature the compounds. The compounds also functioned over a wide pH 

range. In addition, they also were fairly resistant to attack by trypsin and proteases as 

judged by the retention of their antimicrobial activity. It is unknown if paracelsin would 

be similarly resistant to temperature extremes, pH levels, or to enzymatic degradation 

as are the trichokonin peptaibols. 

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 T. reesei. 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 collected 

from non-engineered microorganisms. These values are based on reasonable worstcase 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, one supervisory worker, all with three shifts 

per day. 
26

 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 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 106 to 1.8 x 107 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 T. reesei 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 fermentor off-gases. 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. 
27

Table 1. Estimated Numbers of Viable T. reesei Cells 

 Released During Production 

_____________________________________________________________________ 

 Uncontrolled/ Full 

Release Media Untreated Exemption Release 

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

AIR 

 Fermentor exhaust 2 x 105 < 2 x 105

 350 

 Rotary Drum Filter 7 x 105

 < 7 x 105 100 

 

WATER - to WWT 

 Fermentor cleaning 1.4 x 1013 1.4 x 107

 100 

LAND - to landfill 

 Filter cakes 7 x 1014 7 x 108

 100 

____________________________________________________________________ 

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

cell density in the fermentation broth of 107 cfu/ml for fungi, 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 

105 cfu/day each from treatment of fermentor and rotary drum exhausts. 

 1. Releases to Air 

 Specific data which indicate the survivability of T. reesei 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. 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 these sources, 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 < 1 

cfu/yr from both the fermentor off-gas and from the rotary drum filtration exhaust, and 

less than that for systems meeting the full exemption criteria. It should be noted that 
28

these estimates represent hypothetical exposures under reasonable worst case 

conditions. 

 2. Releases to Water 

 The concentrations of T. reesei 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 values (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 T. reesei 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 T. 

reesei in surface water for the uncontrolled/untreated and the full exemption scenarios 

are tabulated in Table 2. 

TABLE 2. T. reesei 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 104

 2.50 x 106 

 50th Percentile 768 68.13 1.82 x 104 2.05 x 105

Full Exemption 

 10th Percentile 156 5.60 8.97 x 10-2 2.50 

 50th Percentile 768 68.13 1.82 x 10-2 2.05 x 10-1

_________________________________________________________________ 

*MLD = million liters per day 
29

C. Release and Exposure Summary 

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

suggest high levels of exposure of T. reesei to either workers or the public resulting 

from typical enzyme fermentation operations. 

 

 D. Survival of the Organism in the Environment 

 The species T. reesei is known only from the single original isolate QM6a from 

the Solomon Islands in the 1940s. Therefore, there is little information on its prevalence 

or behavior in the environment. However, microcosm studies conducted on T. reesei

suggests that it would survive in the environment. Providenti et al. (2004) studied the 

survival of a derivative of QM6a marked with a recombinant hygromycin-B (HygB) 

resistance gene, designated as QM6a#4, added to laboratory contained intact soil-core 

microcosms containing three different textured soils. In one of those soils, survival was 

also tested with or without a plant rhizosphere (bush lima beans, Phaseolus limensis). 

Soil cores were incubated at 22 ̊C and were sampled periodically over a 4-month 

period. Then the soil cores were subjected to a simulated winter treatment 

(approximately 2 weeks at 4 ̊C, 3 weeks at -20 ̊C, and 2 weeks at 4 ̊C), and then 

returned to 22 ̊C for another 2 weeks. The levels and viability of T. reesei were 

determined by quantitative polymerase chain reaction (PCR) of total soil DNA extracts 

and by dilution-plating of soil on a semiselective growth medium, respectively. QM6a#4 

was added at a rate of 2.0 ml of inocula (consisting of 0.35 g of wet fungal mycelia per 

ml that had been processed at high speed in a Waring blender) onto the top 1 cm of the 

soil core. In Soil 1 (sandy loam), QM6a#4 numbers decreased over the first 2 - 3 

months (40- to 170-fold for the soil with or without a plant rhizosphere, respectively), 

after which a steady state was reached. In Soil 2, (medium texture, soil only), the 

decrease in QM6a#4 was observed within the first month of incubation (80-fold) after 

which a steady state was reached. In Soil 3 (clay loam, soil only), no clear trend was 

apparent, but there was an approximate 4-fold decrease observed by day 114. 

After the cores were subjected to the simulated overwinter treatment, QM6a#4 

levels did not change in any of the three soils without the plant rhizosphere. In the one 

soil with the bean rhizosphere (Soil 1), there was a statistically significant approximately 

3-fold increase in QM6a#4 levels, reflecting a “bloom” associated most likely with an 

increase in nutrients from the decaying dead plant roots after thawing of the cores. In 

all three soils, QM6a#4 remained viable and culturable, even after the simulated winter 

treatment. 

 From this data given in Providenti et al. (2004) above, it appears that even 

though T. reesei was originally isolated from a tropical climatic region, it is able to 

persist in soils for extended periods of time. T. reesei survived in soil even after colder 

temperatures of a simulated overwintering treatment of 4 ̊C for 2 weeks, -20 ̊C for 3 

weeks, followed by 4 ̊C for 2 weeks. In addition to mere persistence, fungal growth 
30

actually occurred after the winter treatment in the soil with the bean rhizosphere, 

presumably due to the nutrient release from the dead roots. 

 

V. INTEGRATION OF RISK 

 The fungus T. reesei has a long history of safe use in submerged standard 

industrial fermentation for enzyme production with food, feed, pharmaceuticals and 

industrial uses. It is not pathogenic to humans, animals, or to plants. The fungus has 

been shown to be nonpathogenic to healthy rats in a number of acute and chronic 

pathogenicity tests. The enzyme fermentation industry routinely tests for toxin 

production in their enzyme products, and T. reesei has not been associated with 

mycotoxin production during typical enzyme fermentation conditions. Acute and chronic 

toxicity tests on a number of enzymes for which GRAS petitions were made to the FDA 

have demonstrated the lack of toxicity of T. reesei enzymes such as cellulase, xylanase, 

endoglucanase, glucanase, protease, and pectin lyase. The use of T. reesei in 

submerged standard industrial fermentation poses low risk. T. reesei has a long history 

of safe use in enzyme production and is a good candidate for the list of eligible recipient 

microorganisms in the Tiered Exemptions if used for production of enzymes under the 

conditions of submerged standard industrial fermentation. 

 However, T. reesei is not an appropriate recipient microorganism for the 5(h)4 

Tiered Exemptions for any other closed system use or under conditions unlike those 

used in standard submerged enzyme fermentation. T. reesei is known to produce a 

secondary metabolite known as paracelsin in submerged fermentation in the presence 

of insoluble substrates such as cellulose. Cellulose is, of course, the main component 

of plant material, and in some form or other, the substrate of choice for biofuel 

production. Cellulosic biofuel facilities pre-treat various types of plant material such as 

sugarcane, switchgrass, corn stover, wood chips, etc. with acid or other chemicals to 

initiate breakdown of the lignin in the plant material. Then, the fungus might actually be 

grown on the plant hydrolysate in the seed fermentor, which would then be transferred 

to a cellulase fermentor, for on-site enzyme production. One approach would be to 

subsequently transfer both the broth containing high levels of cellulases and live cells to 

the saccharification tank wherein the cellulases would breakdown the cellulose in the 

pretreated plant material into sugars which could then be fermented into ethanol. 

 Given this knowledge that an insoluble substrate such as cellulose could 

stimulate peptaibol synthesis, there are a number of different scenarios to consider. 

The first of which is whether paracelsin would actually be produced in a cellulosic 

biofuel facility in the plant biomass breakdown step, and if so, at what levels. It is 

possible that paracelsin would not be produced in submerged fermentation even in the 

presence of plant material, particularly if other conditions known to be conducive to 

peptaibol formation such as the use of older cultures, exposure to light, exposure to 

stress such as nitrogen limitation, and sporulation are all avoided. It is known that, in 
31

general, that production of secondary metabolites in fungal liquid culture usually occurs 

late in the growth stage, i.e., the stationary phase, after exponential growth has ceased 

and when there is a nitrogen limitation in the presence of excess carbon substrates. 

These conditions are known to cause fungal metabolism to shift into production of 

secondary metabolites. 

 If in fact paracelsin was produced in the plant biomass breakdown tank, the 

ethanolic fermentation step following degradation of the plant material into sugars could 

be inhibited because paracelsin is inhibitory to the growth of other fungi, and a yeast (a 

fungus) is typically used for the ethanolic conversion step. However, a particular yeast, 

Candida, studied by Brückner and Graf (1983) was not inhibited by paracelsin, so it is 

not known whether the particular yeasts used for ethanol production would be inhibited. 

If paracelsin was produced at high concentrations with the growth of T. reesei on the 

plant material rather than using purified enzymes void of the fungus, the manufacturer 

may find that efficient ethanol fermentation was not possible. However, microorganisms 

other than yeast, such as the Gram-negative bacteria Escherichia coli or Zymonomas 

mobilis, could alternatively be used as the ethanologen. Gram-negative 

microorganisms would not be inhibited by paracelsin, so paracelsin production could 

possibly go undetected. 

 Another scenario for consideration is that the production of paracelsin in the 

fermentation tanks used for plant material breakdown may be inconsequential if at some 

other point in the ethanol production process the compound is denatured. It is possible 

that the high temperatures (78°C) used for ethanol distillation could break down the 

peptide. However, other peptaibols have been shown to retain their biological activity 

after high temperature treatment. It is unknown if paracelsin would be denatured or 

whether it would retain its conformation and biological activity. Another possibility is that 

if paracelsin is not degraded during the latter stages of the ethanol production process, 

it could be broken down during wastewater treatment of the spent fermentation broth, 

and thus, would not enter the environment. However, there is no information available 

in the literature that addresses the potential breakdown of paracelsin in a wastewater 

treatment system, nor is there information on the breakdown or persistence of 

paracelsin in the environment. There is some indication that other peptaibols appear to 

be relatively stable due to formation of α-helical structures resulting from the presence 

of the α-aminoisobutyric acid which favors that structure. There is one article in the 

literature that detected peptaibols in marine sediments that were causing toxicity to 

shellfish. The peptaibols were apparently rather persistent in the environment, and the 

authors suggested that peptaibols could be used as stable markers for the presence of 

Trichoderma fungi in the marine environment (Poirier et al., 2007a). There is also the 

one article on the stability of peptaibol-like compounds produced by Trichoderma 

koningii called trichokonins that also contain high proportions of α-aminoisobutyric acid 

and have antimicrobial properties like other peptaibols. These trichokonins, which now 

are recognized as peptaibols, were found to be stable at high temperatures, up to 

100°C, and even after autoclaving. They retained their biological activity after low 
32

temperature treatment as well, including freezing followed by lyophilization, and over a 

pH range of 3.0 to 10.0. They were also found to be resistant to enzymes such as 

trypsin and other proteases that normally break down proteinaceous materials (Song et 

al., 2006). 

 In the absence of any data or literature specifically on paracelsin and its 

degradation or persistence in the environment, the consequences of paracelsin 

exposure to environmental receptors must be considered. At the far extreme, it is 

possible that large quantities of paracelsin could be produced with the growth of T. 

reesei on plant material, and that paracelsin would not be denatured/inactivated at any 

other point in the ethanolic fermentation process stream. Under this scenario, 

paracelsin could adversely affect the functioning of wastewater treatment by inhibition of 

growth of Gram-positive bacteria in the POTW or on-site treatment facility with disposal 

of liquid wastes. There is no information available in the literature on whether 

paracelsin would be rapidly broken down in the wastewater treatment system, or 

whether the molecule is resistant to proteases or other enzymes present in wastewater. 

If the paracelsin remains intact through wastewater treatment, then the potential 

consequences of exposure of paracelsin to the environment must be considered. 

Recommendations 

T. reesei (QM6a and its derivatives) is recommended as an eligible recipient 

microorganism for the 5(h)4 Tiered Exemption when used with submerged standard 

industrial fermentation conditions for enzyme production where no solid material is 

present in the fermentation broth. However, in the absence of data on potential 

production of paracelsin, T. reesei is not recommended for an exemption from review 

when used in closed systems under conditions unlike those encountered in submerged 

standard enzyme fermentation, such as the growth directly on plant material. 

 
33

VI. REFERENCES 

Abu-Raya., S., V. Trembovier, E. Shohami, and P. Lazarovici. 1993. Cytolysins 

increase intracellular calcium and induce eicosanoids release by pheochromocytoma 

PC12 cell cultures. Natural Toxins 1:263-270. 

Antal, Z., J. Varfa, L. Kredics, A. Szekeres, L. Hatvani, L. Manczinger, C. Vágvölgyi, and 

E. Nagy. 2006. Intraspecific mitochondrial DNA polymorphism within the emerging 

filamentous fungal pathogen Trichoderma longibrachiatum. J. Medical Microbiol. 55:31-

35. 

Askolin, S., T. Nakari-Setala, and M. Tenkanen. 2001. Overproduction, purification and 

characterization of the Trichoderma reesei hydrophobin HFBI. Appl. Microbiol. 

Biotechnol. 57:124-130. 

Atanasova, L., W.M. Jaklitsch, M. Komoń-Zelazowska, C.P. Kubicek, and I.S. 

Druzhinina. 2010. Clonal species T. parareesei sp. nov. likely resembles the ancestor 

of the cellulase producer Hypocrea jecorina/T. reesei. Appl. Environ. Microbiol. 

76:7259-7267. 

Avcin, J. 2011. Engineering Assessment for Trichoderma reesei for the 5(h)4 Tier 

Exemptions. Unpublished. U. S. Environmental Protection Agency, Washington, DC. 

Benitez, T., A.M. Rincon, M.C. Limon, and A.C. Codon. 2004. Biocontrol mechanisms 

of Trichoderma strains. Int. Microbiol. 7(4):249-260. 

Blumenthal, C. 2004. Production of toxic metabolites of Aspergillis niger, Aspergillis 

oryzae, and Trichoderma reesei: Justification of mycotoxin testing in food grade 

enzyme preparations derived from the three fungi. Reg. Tox. Pharmacol. 39:214-228. 

Bokari, F. 2009. Efficacy of some Trichoderma species in the control of Rotylenchulus 

reniformis and Meloidogyne javanica. Archives Phytopathology and Plant Protection 

42:361-369. 

Brewer, D., A. Feicht, A. Taylor, J. W. Keeping, A. A. Taha, and V. Thaller. 1982. 

Ovine ill-thrift in Nova Scotia. 9. Production of experimental quantities of isocyanide 

metabolites of Trichoderma hamatum. Can. J. Microbiol. 28:1252-1260. 

Brewer, D, F.G. Mason, and A. Taylor. 1987. The production of alamethicins by 

Trichoderma sp. Can. J. Microbiol. 33:619-625. 

Brückner, H. and H. Graf. 1983. Paracelsin, a peptide antibiotic containing αaminoisobutyric acid, isolated from Trichoderma reesei Simmons. Experientia 39:528-

530. 
34

Brückner, H., H. Graf, and M. Bokel. 1984. Paracelsin; characterisation by NMR 

spectroscopy and circular dichroism, and hemolytic properties of a peptaibol antibiotic 

from the cellulolytical active mold Trichoderma reesei . Part B. Experientia 40:1189-96. 

Chemical Engineering Branch. 2011. Generic scenario for fungal releases from an 

enzyme fermentation facility. Unpublished. U. S. Environmental Protection Agency, 

Washington, DC. 

Degenkolb, T., T. Grafenhan, A. Berg, H.I. Nirenbrg, W. Gams, and H. Brückner. 2006. 

Peptabiomics: Screening for polypeptide antibiotics (peptaibiotics) from plant-protective 

Trichoderma species. Chemistry & Biodiversity 3:593-610. 

Degenkolb, T., R. Dieckmann, K.F. Nielsen, T. Grafenhan, C. Thies, D. Zafari, P. 

Chaverri, A. Ismaiel, H. Brückner, H. von Dorhe, U. Thrane, O. Petrini, and G.J. 

Samuels. 2008a. The Trichoderma brevicompactum clade: a separate lineage with 

new species, new peptaibols, and mycotoxins. Mycol. Progress 7:177-219. 

Degenkolb, T., H. von Dorhen, K.F. Nielsen, G.J. Samuels, and H. Brückner. 2008b. 

Recent advances and future prospects in peptaibiotics, hydrophobin, and mycotoxin 

research, and their importance for chemotaxonomy of Trichoderma and Hypocrea. 

Chem. Biodiversity 5:671-680. 

Druzhinina I., M. Komoń-Zelazowska, L. Atanasova, V. Seidl, C. Kubicek. 2010. 

Evolution and ecophysiology of the industrial producer Hypocrea jecorina (anamorph 

Trichoderma reesei) and a new sympatric agamospecies related to it. 

PLoS ONE 5(2):e9191 

Druzhinina, I.S., M. Komoń-Zelazowska, L. Kredics, L. Hatvani, Z. Antal, T. Belayneh, 

and C.P. Kubicek. 2008. Alternative reproductive strategies of Hypocrea orientalis and 

genetically close but clonal Trichoderma longibrachiatum, both capable of causing 

invasive mycoses of humans. Microbiol. 154:3447-59. 

Druzhinina I. S., A.G. Kopchinskiy, M. Komoń, J. Bissett, G. Szakacs, and C. P. 

Kubicek. 2005. An oligonucleotide barcode for species identification in Trichoderma

and Hypocrea. Fungal Genet. Biol. 42: 813–828. 

Enriquez-Matas, A., S. Quirce, N. Cubero, J. Sastre, and R. Melchor. 2009. 

Hypersensitivity pneumonitis caused by Trichoderma viride. Archivos de 

Bronconeumologia 45:304-305. 

ETA. 2010. Letter from the Enzyme Technical Association to EPA, Re: Response to 

request for additional information regarding the application to list Trichoderma reesei as 
35

a recipient microorganism eligible for exemption under 40 CFR Part 725, Subpart G; 

relating to the potential production of paracelsin by the organism during enzyme 

manufacture. Letter from Anthony T. Pavel to Greg Schweer, August 24, 2010. 

Eveleigh, D. E. 1985. Trichoderma. In: A.L. Demain and N.A. Solomon (eds.). Biology 

of Industrial Microorganisms. Chapter 16, pp. 487-509. Benjamin/Cummings 

Publishing Co., Menlo Park, CA. 

Favilla, M., L. Macchia, A. Gallo, and C. Altomare. 2006. Toxicity assessment of 

metabolites of fungal biocontrol agents using two different (Artemia salina and Daphnia 

magna) invertebrate assays. Food Chem. Toxicol. 44:1922-31. 

Gams, W. and J. Bissett. 1998. Morphology and identification of Trichoderma. In: 

C.P. Kubicek and G.E. Harman (eds.). Trichoderma and Gliocladium. Volume 1. Basic 

Biology, Taxonomy, and Genetics. Taylor and Frances Inc. Bristol, PA, pp. 3-34. 

Grigoriev, P.A., B. Schlegel, M. Kronen, A. Berg, A. Harti, and U. Grafe. 2003. 

Differences in membrane pore formation by peptaibols. J. Peptide Sci. 9(11-12):763-

68. Published online 5 Nov 2003, Wiley Interscience. 

GRN No. 32. Röhm Enzyme GmbH Kirschenallee 45; D-64293 Darmstadt, GERMANY. 

Pectin lyase derived from Trichoderma reesei carrying a gene encoding pectin lyase 

from Aspergillus niger. Sept, 29, 1999. 

GRN No. 315. Genencor, a Danisco Division, Palo Alto, CA. 2009. Transglucosidase 

enzyme preparation derived from Trichoderma reesei expressing the transglucosidase 

gene from Aspergillus niger. Dec. 4, 2009. 

GRN No. 333. Genencor, a Danisco Division, Palo Alto, CA. 2010. Acid fungal 

protease enzyme preparation from Trichoderma reesei expressing the gene encoding 

acid fungal protease from T. reesei. March 26, 2010. 

Halpin, D.M., B.J. Graneek, M. Turner-Warwick, and A.J. Taylor Newman. 1994. 

Extrinsic allergic alveolitis and asthma in a sawmill worker: case report and review of 

the literature. Occup. Environ. Med. 51:160-164. 

Haran, S., H. Schickler, and I. Chet. 1996. Molecular mechanisms of lytic enzymes 

involved in the biocontrol activity of Trichoderma harzianum. Microbiol. 142:2321-31. 

Harjono and S.M. Widyastuti. 2001. Antifungal activity of purified endochitinase 

produced by biocontrol agent Trichoderma reesei against Ganoderma philippii. 

Pakistan J. Biological Sciences 4:1232-34. 

Harman, G.E., C.R. Howell, A. Viterbo, I. Chet, and M. Lorito. 2004. Trichoderma 
36

species -- opportunistic, avirulent plant symbionts. Microbiol. 2:43-56. 

Higashimoto, V., H. Kodama, M. Jelokhani-Niaraki, F. Kato, and M. Kondo. 1999. 

Structure-function relationship of model Aib-containing Peptides as ion transfer 

intermembrane templates. J. Biochem. 125:705-712. 

Hjortkjaer, R.K., V. Billie-Hansen, K.P. Hazelden, M. McConville, D.B. McGregor, E. 

Chapman, J.R. Gardner, and R. Ashby. 1986. Safety evaluation of Celluclast®, an acid 

cellulase derived from Trichoderma reesei. Fd. Chem. Toxic. 24:55-63. 

Hou, C.T., A. Ciegler, and C.W. Hesseltine. 1972. New mycotoxin, trichotoxin A, from 

Trichoderma viride isolated from southern leaf blight-infected corn. Appl. Microbiol. 

23:183-185. 

Jaklitsch, W. 2009. European species of Hypocrea Part I. The green-spored species. 

Studies in Mycology 63:1-91 (http:www.studiesinmycology.org) doi:10.3114/ 

sim.2009.63.01. 

JEFCA. 1981. Joint FAO/WHO Expert Committee on Food Additives. Specifications for 

identity and purity of carrier solvents, emulsifiers and stabilizers, enzyme preparations, 

flavoring agents, food colours, sweetening agents and other food additives, FAO Food 

and Nutrition Paper 19, p. 214.

Jones-Brown, Y. 2011. TSCA uses and the history of safe use of Trichoderma reesei 

for the 5(h)4 Tier Exemptions. Unpublished. U. S. Environmental Protection Agency, 

Washington, DC. 

Kar, S. A. Mandal, P.K. das Mohapatra, K.C. Mondal, and B.R. Pati. 2006. Production 

of cellulase-free xylanase by Trichoderma reesei SAF3. Brazilian J. Microbiol. 37:462-

464. 

Kazi, F.K., J. Fortman, R. Anex, G. Kothandaraman, D. Hsu, A. Aden, and A. Dutta. 

2010. Techno-Economic Analysis of Biochemical Scenarios for Production of Cellulosic 

Ethanol. Technical Report NREL/TP-6A2-46588, June 2010. 

Klein, D. and D.E. Eveleigh. 1998. Ecology of Trichoderma. In: C.P. Kubicek and 

G.E. Harman (eds.). Trichoderma and Gliocladium. Volume 1. Basic Biology, 

Taxonomy, and Genetics. Taylor and Frances Inc. Bristol, PA, pp. 57-74. 

Komoń-Zelazowska, M., J. Bissett, D. Zafari, L. Hatvani, L. Manczinger, S. Woo, M. 

Lorito, L. Kredics, C.P. Kubicek, and I.S. Druzhinina. 2007a. Genetically closely related 

but phenotypically divergent Trichoderma species cause green mold disease in oyster 

mushroom farms worldwide. Appl. Environ. Microbiol. 73:7415-26. 
37

Komoń-Zelazowska, M., T. Neuhof, R. Dieckmann, H. von Döhren, A. Herrera-Estrella, 

C.P. Kubicek, and I.S. Druzhinina. 2007b. Formation of atroviridin by Hypocrea 

atroviridis is conidiation associated and positively regulated by blue light and the G 

protein GNA3. Eukaryotic Cell 6:2332-2342. 

Kredics, L., Z. Antal, A. Szekeres, L. Hatvani, L. Manczinger, C. Vágvölgyi, and 

E. Nagy. 2005. Extracellular proteases of Trichoderma species. A Review. Acta 

Microbiol. Immunol. Hung. 52:169-84. 

Kubicek, C.P. and G.E. Harman. 1988. Preface. In: C.P. Kubicek and G.E. Harman 

(eds.). Trichoderma and Gliocladium. Volume 1. Basic Biology, Taxonomy, and 

Genetics. Taylor and Frances Inc. Bristol, PA. pp. ix. 

Kubicek, C.P., M. Komoń-Zelazowska, E. Sándor, and I.S. Druzhinina. 2007. Facts 

and challenges in the understanding of the biosynthesis of peptaibols by Trichoderma. 

Chemistry & Biodiversity – Vol. 4, pp.1068-1082. 

Kuhls, K. E. Lieckfeldt, G.J, Samuels, W. Kovacs, W. Meyers, O. Petrini, W. Gams, T. 

Borner, and C.P. Kubicek. 1996. Molecular evidence that the asexual fungus 

Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina. Proc. 

Natl. Acad. Sci. 93:7755-7760. 

Kuhls, K., E. Lieckfeldt, T. Börner, and E. Guého. 1999. Molecular re-identification of 

human pathogenic Trichoderma isolates as Trichoderma longibrachiatum and 

Trichoderma citrinoviride. Medical Mycology 37:25-33. 

Landreau, A., Y.F. Pouchus, C. Sallenave-Namont, J.-F. Biard, M.-C. Boumard, T.R. du 

Pont, F. Mondeguer, C. Goulard, and J.-F. Verbist. 2002. Combined use of LC/MS and 

a biological test for rapid identification of marine mycotoxins produced by Trichoderma 

koningii. J. Microbiol. Methods 48:181-194. 

Lieckfeldt, E., C. Kullnig, G.J. Samuels, and C.P. Kubicek. 2000. Sexually competent, 

sucrose- and nitrate-assimilating strains of Hypocrea jecorina (Trichoderma reesei) from 

South American soils. Mycologia 92:374-380. 

Lübeck, M., S.K. Poulsen, P.S. Lübeck, D.F. Jensk, and U. Thrane. 2000. Identification 

of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR 

fingerprinting and UP-PCR cross hybridization. FEMS Microbiology Letters 185:129-

134. 

Lynch, D. 2011. Exposure assessment of Trichoderma reesei for the 5(h)4 Tier 

Exemptions. Unpublished. U. S. Environmental Protection Agency, Washington, DC. 
38

Maheshwari, R. 2008. Genome sequence of a fungus for the biofuel industry. Current 

Sci. 95(2):161-162. 

Malmos, H. 1977. Enzyme applications in food, pharmaceutical, and other industries: 

Industrial applications of cellulase, American Institute Chem. Engineers Symposium 

Series 74:93-99. 

Mandels, M. and E.T. Reese. 1957. Induction of cellulase in Trichoderma viride as 

influenced by carbon sources and metals. J. Bacteriol. 73:269-278. 

Mandels, M. J. Weber, and R. Parizek. 1971. Enhanced cellulase production by a 

mutant of Trichoderma reesei. Appl. Microbiol. 21:152-154. 

Martinez, D., R.M. Berka, B. Henrissat, M. Saloheimo, M. Arvas, S.E. Baker, J. 

Chapman, O. Chertkov, P.M. Coutinho, D. Cullen, E.G.J. Danchin, I.V. Grigoriev, P. 

Harris, M. Jackson, C.P. Kubicek, C.S. Han, I. Ho, and L.F. Larrondo, A.L. Leon, J.K. 

Magnuson, S. Merino, M. Misra, B. Nelson, N. Putnam, B. Robbertse, A.A.Salamov, M. 

Schmoll, A. Terry, N. Thayer, A. Westerholm-Parvinen, C.L.Schoch, J. Yao, R. 

Barabote, M.A. Nelson, C.Detter, D. Bruce, C.R. Kuske, G. Xie, P. Richardson, D.S. 

Rokhsar, S.M. Lucas, E.M. Rubin, N. Dunn-Coleman, M. Ward, T.S. Brettin. 2008. 

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei

(syn. Hypocrea jecorina). Nature Biotechnol. 26:553-560. 

McClung, G. 2011. Ecological hazard assessment of Trichoderma reesei for the 5(h)4 

Tier Exemptions. Unpublished. U. S. Environmental Protection Agency, Washington, 

DC. 

Meyer, C.E. and F. Reusser. 1967. A polypeptide antibacterial agent isolated from 

Trichoderma viride. Specialia 15:85-86. 

Mohamed-Benkada, M., M. Montaqu, J.F. Baird, F. Mondequer, P. Verite, M. 

Dalgalarrondo, J. Bissett, and Y.F. Pouchus. 2006. New short peptaibols from a 

marine Trichoderma strain. Rapid Commun. Mass Spectrom. 20(8);1176-1180. 

Montenecourt, B.S. and D.E. Eveleigh. 1977. Preparation of mutants of Trichoderma 

reesei with enhanced cellulase production. Appl. Environ. Microbiol. 34:777-782. 

Morawetz, R. F. Granz, R. Messner, and C.P. Kubicek. 1992. Presence, transcription 

and translation of cellobiohydrolase genes in several Trichoderma species. Current 

Genetics 21:31-6. 

Mukherjee, P., A. West, N. Ruiz, A. Keighttley, M.E. Moran-Diez, K. McCluskey, Y.F. 

Pouchus, and C.M. Kenerley. 2010. Two classes of new peptaibols are synthesized by 

a single non-ribosomal peptide synthetase of Trichoderma virens. J. Biological 
39

Chemistry 286:4544-4554. Published on-line Dec 1, 2010 

(http://www.jbc.org/cgi/doi/10.1074/jbc.M110.159723). 

 

Nevalainen, H., P. Suominen, and K. Taimisto. 1994. On the safety of Trichoderma 

reesei. J. Biotechnol. 37:193-200. 

Olempska-Beer, Z., R.I. Merker, M.D. Ditto and M.J. DiNovi. 2006. Food-processing 

enzymes from recombinant microorganisms – a review. Regulatory Toxicology 

Pharmacology 45:144-158. 

 

Ooka, T., Y. Shimojima, T. Akimoto, I. Takeda, S. Senoh, and J. Abe. 1966. A new 

antibacterial peptide “Suzukacillin”. Agr. Biol. Chem. 30:700-702. 

Pfaller, M. and D.J. Diekema. 2010. Epidemiology of invasive mycoses in North 

America. Critical Rev. Microbiol. 36:1-53. 

Poirier, L., M. Montagu, A. Landreau, M. Mohamed-Benkada, O. Grovel, C. SallenaveNamont, J.-F. Biard, C. Amiard-Triquet, J.-C. Amiard, and Y.F. Pouchus. 2007a. 

Peptaibols: Stable markers of fungal development in the marine environment. Chem. & 

Biodiversity 4:1116-1128. 

Poirier, L., F. Quiniou, N. Ruiz, M. Montagu, J.-C. Amiard, and Y.F. Poucheus. 2007b. 

Toxicity assessment of peptaibols and contaminated sediments on Crassostrea gigas 

embryos. Aquatic Toxicology 83:254-62. 

Providenti, M.A., S.I. Mautner, O. Chaudhry, M. Bombardier, R. Scroggins, E. 

Gregorich, and M.L. Smith. 2004. Determining the environmental fate of a filamentous 

fungus, Trichoderma reesei, in laboratory-contained intact soil-core microcosms using 

competitive PCR and viability plating. Can. J. Microbiol. 50:623-631. 

Przybylski, M., I. Dietrich, I. Manz, and H. Brückner. 1984. Elucidation of structure 

microheterogeneity of the polypeptide antibiotics paracelsin and trichotoxin A-50 by fast 

atom bombardment mass spectrometry in combination with selective in situ hydrolysis. 

Biomed. Mass Spectrom. 11: 569–582. 

Ritieni, A, V. Fogliano, D. Nanno, G. Randazzo, C. Altomare, G. Perrone, A. Bottalico, 

L. Maddau, and F. Marras. 1995. Paracelsin E, a new peptaibol from Trichoderma 

saturnisporum. J. Nat. Prod. 58(11):1745-8. 

Roodsari, F.N., D. Zafari, S.A. Khodaparast, and H. Rouhani. 2007. Introducing some 

new species of Trichoderma for Iran. Rostaniha 8:14-18. 

Ruiz, N. G. Wielgosz-Collin, L. Poirier, O. Grovel, K.E. Petit, M. Mohamed-Benkada, 

T.R. du Pont, J. Bissett, P. Verite, G. Barnathan, and Y.F. Pouchus. 2007. New 
40

trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma 

longibrachiatum. Peptides 28: 1351-1358. 

Sallenave, C., Y.F. Pouchus, M. Bardouil, P, Lassus, M.-F. Roquebert, and J.-F. 

Verbist. 1999. Bioaccumulation of mycotoxins by shellfish: Contamination of mussels 

by metabolites of a Trichoderma koningii strain isolated in the marine environment. 

Toxicon 37:77-83. 

Samuels, G., S.L. Dodd, W. Gams, L.A. Castlebury, and O. Petrini. 2002. Trichoderma 

species associated with the green mold epidemic of commercially grown Agaricus 

bisporus. Mycologia 94:146-170. 

Samuels, G., S.L. Dodd, B.-S. Lu, O. Petrini, H.-J. Schroers, and I.S. Druzhinina. 2006. 

The Trichoderma koningii aggregate species. Studies in Mycology 56:67-133. 

Samuels, G.J., O. Petrini, K. Kuhls, E. Lieckfeldt, and C.P. Kubicek. 1998. The 

Hypocrea schweinitzii complex and Trichoderma sect. longibrachiatum. Studies in 

Mycology 41:1-54. 

Schirmböck, M. Lorito, Y.-L. Wang, C.K. Hayes, I. Arisan-Atac, F. Scala, G.E. Harman, 

and C.P. Kubicek. 1994. Parallel formation and synergism of hydrolytic enzymes and 

peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of 

Trichoderma harzianum against pathogenic fungi. Appl. Environ. Microbiol. 60:4364-

4370. 

Segal, M. 2011. Taxonomy of Trichoderma reesei for the 5(h)4 Tier 

Exemptions. Unpublished. U.S. Environmental Protection Agency, Washington, DC. 

Seidl, V., M. Schmoll, B. Scherm, V. Balmas, B. Seiboth, Q. Migheli, and C.P. Kubicek. 

2004. Use of Hypocrea jecorina (anamorph Trichoderma reesei) as a model system for 

Trichoderma biocontrol of Pythium blight identifies new targets for genetic strain 

improvement. J. Zhejiang University 30(4):404. 

Seidl, V., C. Seibel, C. Kubicek, and M. Schmoll. 2009. Sexual development in the 

industrial workhorse Trichoderma reesei. Proc Natl. Acad. Sci. 106:13909-13914. 

Simmons, E.G. 1977. Classification of some cellulase-producing Trichoderma species, 

p. 618. Abstract of the Second International Mycological Congress, Tampa, FL. 

Solfrizzo, M., C. Altomare, A. Visconti, A. Bottalico, and G. Perrone. 1994. Detection of 

peptaibols and their hydrolysis products in cultures of Trichoderma species. Nat. Toxins 

2(6):360-5. 

Song, X.Y., Q.-T. Shen, S.-T. Xie, X.-L. Chen, C.-Y. Sun, and Y.-Z. Zhang. 2006. 
41

Broad spectrum antimicrobial activity and high stability of trichokonins from Trichoderma 

koningii SMF2 against plant pathogens. FEMS Microbiol. Letters260:119-125. 

Song, X.Y., S.-T. Xie, X.-L. Chen, C.-Y. Sun, and Y.-Z. Zhang. 2007. Solid-state 

fermentation for trichokonins production from Trichoderma koningii SMF2 and 

preparative purification of trichokonin VI by a simple protocol. J. Biotech. 131:209-215. 

Sun,Y., L. Tian, Y.-F. Huang, Y. Sha, and Y.-H. Pei. 2006. A new cyclotetrapeptide 

from marine fungus Trichoderma reesei. Pharmazie 61:809-810. 

 

Sun, Y., L. Tian, J. Huang, H.-Y. Ma, Z. Zheng, A.-L. Lv, K. Yasukawaw, and Y.-H. Pei. 

2008. Trichodermatides A-D, novel polyketides from the marine-derived fungus 

Trichoderma reesei. Organic Letters 10:393-396. 

 

Szekeres, A., B. Leitgeb, L. Kredics, A. Antal, L. Hatvani, L. Manczinger, and C. 

Vagvolgyi. 2005. Peptaibols and related peptaibiotics of Trichoderma. A review. Acta 

Microbiol. Immunol. Hung. 52(2):137-68. 

Tang, P., S. mohan, L. Sigler, I. Witterick, R. Summerbell, I. Campbell, and T. Mazzulli. 

2003. Allergic fungal sinusitis associated with Trichoderma longibrachiatum. J. Clin. 

Microbiol. 41:5333-36. 

Taylor, J.W., D.J. Jacobson, S. Kroken, T. Kasuga, D.M. Geiser, D.S. Hibbett, and M.C. 

Fisher. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal 

Genetics and Biology 31:21-32. 

Toyoma, N. 1969. Applications of cellulase in Japan. In: R.F. Gould (ed.). Cellulases 

and Their Application. Advances in Chemistry Series 95, American Chemical Society, 

Washington, DC, pp. 359-390. 

Turner, D.B. 1970. Workbook of Atmospheric Dispersion Estimates. PHS Publication 

No. 999-AP-26. U.S. Department of Health, Education, and Welfare, National Air 

Pollution Control Administration, Cincinnati, OH. 

Verma, M., S.K. Brar, R.D. Tyagi, R.Y. Surampalli, and J.R. Valero. 2007. Antagonistic 

fungi, Trichoderma spp.: Panoply of biological control. Biochem. Engineering J. 37:1-

20. 

Vizcaino, J.A., R.E. Cardoza, L. Dubost, B. Bodo, S. Gutierrez, and E. Monte. 2006. 

Detection of peptaibols and partial cloning of a putative peptaibol synthetases gene 

from Trichoderma harzianum CECT 2413. Folia Microbiol. 51:114-120. 

Ward, R. 2011. Human health effects of Trichoderma reesei for the 5(h)4 Tier 

Exemptions. Unpublished. U.S. Environmental Protection Agency, Washington, DC. 
42

Watts, R., J. Dahiya, K. Chaudhary, and P. Tauro. 1988. Isolation and characterization 

of a new fungal metabolite of Trichoderma reesei. Plant and Soil 107:81-84. 

Wei, C.-M., B.S. Hansen, M.H. Vaughan Jr., and C.S. McLaughlin. 1974. Mechanisms 

of action of the mycotoxins trichodermin, a 12,13-epoxytrichothecene. Proc. Natl. Acad. 

Sci. 71:713-717. 

Woo, S.L., F. Scal, M. Ruocco, and M. Lorito. 2006. The molecular biology of the 

interactions between Trichoderma spp., phytopathogenic fungi, and plants. 

Phytopatholgy 96:181-5. 

Wooley, R., M. Ruth, J. Sheehan, K. Ibsen, H. Majdeski, and A. Galvez. 1999. 

Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-current 

Dilute Acid Prehydrolysis and Enzymatic Hydrolysis Current and Future Scenarios. 

Report No. NREL/TP-580-26157. National Renewable Energy Laboratory, Golden, CO. 

Wösten, H.A. 2001. Hydrophobins: Multipurpose proteins. Ann. Rev. Microbiol. 55: 

625-646.