Pichia anomala J121: a 30-year overnight near success biopreservation story

ORIGINAL PAPER

Pichia anomala J121: a 30-year overnight near successbiopreservation story

Johan Schnurer • Anders Jonsson

Received: 3 September 2010 / Accepted: 9 September 2010 / Published online: 25 September 2010

� Springer Science+Business Media B.V. 2010

Abstract Thirty years ago, the ascomycetous yeast

Pichia anomala strain J121 was isolated from moist

wheat grain stored under conditions of restricted air

access. Early observations indicated that an inverse

relationship existed between mould and P. anomala

colony forming units in grain. This yeast strain was

later found to have strong antifungal properties in

laboratory, pilot and farm studies with high-moisture

wheat under malfunctioning airtight storage. P. ano-

mala had the highest inhibitory activity of 60 yeast

species evaluated against the mould Penicillium

roqueforti. It also demonstrated strong inhibitory

effects against certain Gram-negative bacteria. P. ano-

mala J121 possesses a number of physiological

characteristics, i.e. capacity to grow under low pH,

low water activity and low oxygen tension and ability

to use a wide range of carbon and nitrogen sources,

enabling it to act as an efficient biopreservative agent.

The biocontrol effect in grain was enhanced by

addition of glucose, mainly through formation of the

volatile antimicrobial ethyl acetate. Animal feeding

trials with P. anomala J121 inoculated grains, fed to

chickens and beef cattle, demonstrated that mould

control observed in vitro in small scale laboratory

experiments could be extended to large scale farm

trials. In addition, no adverse effects on animal weight

gain, feed conversion, health or behaviour were

observed. We have now studied P. anomala J121

biology, ecology and grain preservation ability for

30 years. Over this period, more than 40 scientific

publications and five PhD theses have been written on

different aspects of this yeast strain, extending from

fundamental research on metabolism, genetics and

molecular biology, all the way to practical farm-scale

level. In spite of the well documented biopreservative

ability of the yeast, it has to date been very difficult to

create the right constellation of technical, agricultural

and biotechnical industries necessary to reach a

commercial launch of a P. anomala J121 based

biopreservation system. Additionally, the complica-

tions caused by a complex EU regulatory system

remain a significant barrier to practical applications.

Keywords Pichia anomala � Hansenula �Wickerhamomyces � Application � Feed trials �Grain storage

General background

A world population increasing from 6.3 billion to

9–10 billion in 2050 will require 70% more food

J. Schnurer (&)

Department of Microbiology, Swedish University

of Agricultural Sciences (SLU), P.O. Box 7025,

750 07 Uppsala, Sweden

e-mail: [email protected]

A. Jonsson

Department of Soil and Environment, Swedish University

of Agricultural Sciences (SLU), Box 234,

532 23 Skara, Sweden

123

Antonie van Leeuwenhoek (2011) 99:5–12

DOI 10.1007/s10482-010-9509-2

(FAO 2009). Worldwide demand for renewable

energy is also increasing drastically, driven by

environmental, economic and strategic concerns.

The global agricultural sector, simultaneously faced

with adverse climate changes and land loss/soil

destruction, thus has to produce increasing amounts

of food and animal feed, as well as bioenergy, while

at the same time minimising waste and food/feed

spoilage. Cereal grains, in addition to being the basis

for human nutrition, are a main component of animal

feed in large parts of the world. For example, In

Sweden, about 60% of grain produced is used as

animal feed (SJV 2007). The microbial population on

cereal grain may interfere with feed hygiene, storage

stability, bioavailability of minerals and protein and

may reduce palatability of the feed (Magan et al.

2003; Olstorpe 2008). In Sweden, the average grain

moisture content at harvest is approximately 21% and

to avoid fungal spoilage, cereal grains are normally

hot-air dried to 14% moisture at a high energy and

economic cost (Jonsson and Pettersson 2000). Grain

drying using fossil fuels and electricity consumes

approximately 60% of the energy used during total

plant husbandry operations (Pick et al. 1989). Feed

grains are later re-moistened before feeding to

animals, i.e. a futile energy cycle of removing and

adding water is created.

With increasing energy prices and climate change

concerns, the interest in finding alternative preserva-

tion methods, not reliant on fossil fuels, has

increased. Cereal grain can be stored moist without

drying in certain storage system, but the feed hygiene

can easily be impaired due to growth of deteriorative

and hazardous microorganisms. Airtight storage, in

combination with biopreservation (the use of bene-

ficial micro-organisms), is one way to prevent growth

of deleterious moulds and bacteria. Airtight storage

of high moisture feed grain requires only about 2% of

the energy consumed in conventional high-tempera-

ture drying (Pick et al. 1989). The yeast Pichia

anomala (P. anomala) improves stability of grain

with intermediate moisture in airtight storage sys-

tems, decreasing sensitivity to oxygen leakage (Dru-

vefors 2004). Grain can also be preserved by

fermentation: crimped, high moisture grains tightly

packed into bunkers or large plastic tubes establish

anaerobiosis conducive to indigenous lactic acid

bacterial fermentation (Olstorpe 2008). Fermentation

requires moisture contents high enough to allow

growth of lactic acid bacteria (30–45%). If grain is

harvested at too low moisture contents for lactic acid

bacteria, but too high for complete dry preservation,

incomplete fermentation may lead to spoilage (Ols-

torpe et al. 2010a). Starter cultures are needed to

preserve grains and silage over a range of moisture

contents in airtight storage systems. A starter culture

for grain/silage may comprise a combination of

several microorganisms, most likely yeasts together

with lactic acid bacteria. For high moisture content

feeds, lactic acid bacteria that decrease pH and

produce organic acids and other antifungal com-

pounds have been identified (Broberg et al. 2007),

while yeasts may better control mould growth at

lower moisture contents (Passoth et al. 2006).

Discovery of P. anomala strain J121

In the early 1980 colleagues at the Department of

Microbiology, SLU, Uppsala and the Institute of

Agricultural Engineering (JTI), Uppsala ran a series

of experiments on airtight storage of moist feed

grains in large-scale experimental silos (Ekstrom and

Lindgren 1987). The changes in grain microbiota

were followed after addition of carbon dioxide,

propionic acid or lactic acid bacteria. In one of the

first pilot scale experiments, the grain in some of

these silos was found to be completely dominated by

a yeast. This yeast was later identified by one of the

authors (Jonsson, at the time a PhD student), as

Hansenula anomala. The identification was based on

the yeast taxonomy of that period, relying on the key

phenotypic characters: fermentative, nitrate assimi-

lating yeast with hat-shaped ascospores, although

DNA based techniques had started to appear (Kurtz-

man 1984). During subsequent experiments, pub-

lished mainly in Swedish, it was realised that this

yeast possessed the ability to outnumber moulds

during grain storage (Ekstrom and Lindgren 1987;

Kaspersson et al. 1988).

Pichia anomala J121 mould inhibition in vitro,

in test-tubes and in pilot scale silos

For several years, the yeast isolate was kept in the

culture collection of the Department of Microbiology,

6 Antonie van Leeuwenhoek (2011) 99:5–12

123

SLU, Uppsala, until used in a published MSc project

on in vitro interactions between grain storage moulds

and P. anomala (Hansen) Kurtzman strain J121

(Bjornberg and Schnurer 1993). This study found that

P. anomala inhibited both Aspergillus candidus and

Penicillium roqueforti in nutrient rich, as well as in

nutrient poor agar, and over a range of water activities.

The inhibitory effect was observed as suppression of

mould colony forming units and importantly, also as a

reduction in mould hyphal length (direct microscopic

measurements). An additional and very important

finding was that P. anomala was completely inhibited

by 10 ppm cycloheximide, a concentration not effect-

ing mould spore germination and hyphal growth. This

finding permitted quantification of mould inhibition in

cereal grain, without observing false positive effects

caused by yeast inhibition of mould growth on agar

plates used for assays.

Promising results on mould inhibition in vitro led

to further studies in model grain storage systems,

comparing the inhibitory effects of P. anomala, the

fruit biocontrol yeast Meyerozyma guillermondii

(Pichia guiliermondii) and bakers’ yeast Saccharo-

myces cerevisiae on growth of P. roqueforti in moist

wheat grains in 18 g test tube systems (Petersson and

Schnurer 1995). The mould P. roqueforti, strains of

which are used in cheese production, is a very robust

and highly competitive silage and grain spoilage

fungus. In test-tube models of silo grain storage,

S. cerevisiae had no inhibitory effects, Pichia guiller-

mondii gave only marginal effects, whilst P. anomala

(at inocula levels exceeding 105 CFU g-1) com-

pletely inhibited growth of P. roqueforti. In theory, it

is conceivable that the stress imposed on moulds by

sub-inhibitory levels of yeast may stimulate myco-

toxin production. At least for ochratoxin A formation

from Pencillium verrucosum, this turned out not to be

true. In fact, we showed that P. anomala reduced

ochratoxin A formation in a dose-dependent manner

in both agar and wheat (Pettersson et al. 1998).

Mycotoxin formation was also more sensitive to

presence of the yeast than mould growth, i.e. the

opposite of what might have been expected/feared.

The mould inhibitory effect of P. anomala during

storage of moist grain kernels under restricted air

supply was applicable to a large number of wheat,

rye, oat and barley cultivars, suggesting broad

potential of this yeast as a feed grain biopreservative

agent (Petersson and Schnurer 1998). Using finger-

printing techniques based on polymorphic DNA,

we further investigated the inhibitory effects of

P. anomala on two other Penicillia: Penicillium

carneum and Penicillium paneum. These were found

to be similarly sensitive as P. roqueforti to inhibition

by the yeast, either when growing individually or

when all three organisms were inoculated (Boysen

et al. 2000).

We subsequently scaled up experiments from 18 g

wheat in test tubes to 160 kg wheat in plastic barrel

silos stored outdoor for 11 months, including a winter

period with temperatures below -15�C (Petersson

et al. 1999). Carbon dioxide levels increased rapidly

after closure reaching [70%, while oxygen concen-

tration fell below 1%. Inoculated P. anomala sur-

vived well during storage and grew slowly to around

107 CFU g-1 grain (after 11 months storage).

P. roqueforti growth was clearly restricted under

such conditions, and the aerobic stability after ‘‘feed-

out’’ was also prolonged for yeast inoculated grain.

Low numbers of naturally occurring P. anomala cells

were detected in non-inoculated barrels. The natural

occurrence of P. anomala in moist grain under air-

restricted storage has important implications for

regulatory considerations of the approval of the yeast

as a biopreservative agent.

Pilot scale experiments were repeated some years

later in a 14 month study with silo barrels of different

air permeability, i.e. extending storage from one

harvest to beyond the next harvest (Druvefors et al.

2002). It was found that oxygen levels reduced below

the detection limit after 1 day, while carbon dioxide

levels increased to 80–90% within 1 month. The

inoculated P. roqueforti did not grow in wheat treated

with P. anomala, regardless of silo permeability, but

increased to 105 CFU g-1 grain after 14 months

storage in non-treated grain. Naturally occurring

P. anomala in the non-treated grain increased from

102 to about 105 CFU g-1 during the first month and

subsequently reached the same level as in treated

silos, i.e. about 107 CFU after 9 months (Druvefors

et al. 2002). These results indicated the importance of

dynamic sampling over a time period, versus using

end-point sampling only. In the latter cases, different

final mould counts observed in yeast-inoculated and

control grain would be difficult to explain from

P. anomala end-point values only.

Antonie van Leeuwenhoek (2011) 99:5–12 7

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Pichia anomala J121 physiology

A study of the physiological characteristics of

P. anomala J121 revealed an extremely robust and

versatile organism (Fredlund et al. 2002). Strain J121

grows under strictly anaerobic conditions (if supplied

with ergosterol and unsaturated fatty acids), at

temperatures between 3 and 37�C, at pH values

between 2.0 and 12.4, at water activities down to 0.92

(NaCl), and even down to as low as 0.85 in glycerol

adjusted media. P. anomala J121 can assimilate a

wide range of carbon and nitrogen sources, including

starch, pectin, nitrate and urea. The strain is also a

strong producer of the phosphate releasing enzyme

phytase (Olstorpe et al. 2009)

In light of the very competitive nature of P. anomala

J121, as well as its ability to grow across an extremely

wide range of environmental conditions, prompted a

study of antimicrobial compounds inhibitory to this

yeast. We evaluated the sensitivity of P. anomala J121

to clinically used antifungal compounds and found low

minimum inhibitory concentrations (0.1–5 lg ml-1)

for amphothericin B, ketoconazole, miconazole and

nystatin, as well as for the crop protectants benomyl

and carbendazime (Fredlund et al. 2002). On the other

hand, the strain could grow at high levels of food and

feed preservatives, e.g. at 5000 lg ml-1 propionic

acid at pH 3.6, and thus qualifies as a preservative

resistant yeast (Lind et al. 2005).

Antimicrobial activity of P. anomala J121

Pichia anomala J121 displays strong biocontrol

activity against a variety of moulds when grown on

different cereal grains in both lab- and medium-scale

pilot silo experiments (Petersson 1998; Druvefors

2004). Recently, we also discovered a strong inhib-

itory activity of P. anomala J121 against Gram-

negative bacteria of the Enterobacteriacae group

growing on cereal grains. These observations were

made in large scale farm trials (5 log unit reduction

versus control, Olstorpe et al. 2010), and in controlled

laboratory studies with a number of bacterial species

(Olstorpe et al., unpublished data).

Druvefors and Schnurer (2005) found that P.

anomala was the best yeast among 60 different yeast

species tested with regard to inhibition of Penicillium

roqueforti growth in test tube versions of airtight

grain silos. Several yeasts grew to the same levels as

P. anomala, but failed to show mould inhibitory

effects. This argues against competition for space

and nutrients as the main antifungal mechanism of

P. anomala. Moreover, addition of nutrients in lab-

scale biocontrol experiments did not impair the

biocontrol activity of P. anomala (Druvefors et al.

2005). Addition of glucose even increased the anti-

fungal effect of P. anomala J121, without substan-

tially changing yeast biomass. Glucose did not inhibit

the test fungus (Penicillium roqueforti) when added to

cultures without yeasts. This suggested that a product

from glucose metabolism, e.g. ethanol or ethyl

acetate, was responsible for mould inhibition (Fredl-

und 2004; Fredlund et al. 2004a; Druvefors et al.

2005). When the haploid P. anomala strain CBS 1984

was cultivated at a water activity (aw) of 0.95, it

produced less ethyl acetate than at aw 0.98 and had

a diminished capacity to inhibit Penicillium roque-

forti (Fredlund et al. 2004b). Further investigations

confirmed that a decrease in mould growth was

accompanied by an increase in ethyl acetate concen-

tration, which was shown to have antifungal activity

(Fredlund 2004; Druvefors et al. 2005). P. anomala

killer toxins may also have an inhibitory effect on

detrimental moulds and yeasts (Walker et al. 1995).

Together, these findings suggest that the antifungal

activity of P. anomala in many cases may be due to a

combination of factors. The biocontrol activity

against moulds in moist grain stored under air

restrictions is most likely caused by a competition

for oxygen and the formation of products of glucose

metabolism, mainly ethyl acetate, as well as the

production of high carbon dioxide levels (Druvefors

et al. 2002; Druvefors 2004; Fredlund 2004; Druvef-

ors et al. 2005). However, it is difficult to design

experiments that conclusively prove that competition

for nutrients, e.g. some specific micronutrient, does

not contribute to inhibition of fungal growth.

Animal feeding trials with P. anomala J121

We have conducted several feeding trials with moist

wheat inoculated with P. anomala. Some of these

studies have been done together with industry and

experimental details are thus not openly available.

Nevertheless, a brief summary of some unpublished

results can be given here. In a carefully controlled


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feeding experiment with broiler chickens, six groups

of 30 animals were fed either a standard diet with

dried wheat or with moist wheat (water activity 0.95).

The moist wheat had been inoculated directly after

harvest with different levels of P. anomala J121 cells

and stored for 2 months under airtight conditions

before the feeding started. The treatments were

inoculation with 0, 102, 104, 106 and 108 P. anomala

cells per gram grain. The weights of the chickens

were followed over 36 days and feed conversion

ratios were calculated. In summary, no negative

effects on animal health, growth rate or feed conver-

sion was observed even at the highest P. anomala

level, and there was a small tendency of increased

animal growth with increasing yeast inoculation level

(Persson and Schnurer, unpublished data).

This study was followed by a larger scale experi-

ment at two commercial broiler chicken farms in

Southern Sweden. Here, several hundred tons of wheat

grains were inoculated with 106 P. anomala J121 cells

per gram using an acid applicator pump. The wheat was

stored in two different types of airtight grain silo

storage systems (steel silo and flexible rubber silos).

The inoculated grain was feed to more than 500,000

broiler chickens handled in normal commercial oper-

ations. Similar to the controlled experiment described

above, normal production results were observed at both

farms (Rejholt and Schnurer, unpublished results).

This substantial farm scale experiment provided sev-

eral practical insights with regard to logistic problems

when having to rely on using fresh yeast inocula,

difficulties with large scale applications, dependency

on weather conditions, etc.

The final example concerns the feeding of P. ano-

mala inoculated moist crimped barley grain to beef

cattle (Charolais breed) at a commercial farm on the

Swedish island of Gotland (Olstorpe et al. 2010b).

Immediately after harvest at 16–18% moisture (water

activity 0.83–0.85), the barley grain was inoculated

with 106 P. anomala cells per gram. The grain was

stored in three very large plastic tubes of 16 tons

barley each, with a control of three 16 tons tubes with

non-inoculated moist crimped barley grain. The

inoculated and control barley were fed to groups of

42 and 35 Charolais bulls, respectively. The weight

gain of the two groups of cattle were similar, with a

slight tendency for increased daily weight gain in

cattle fed P. anomala inoculated grain. No negative

effects of the P. anomala inoculation on animal

health status and behaviour were observed during the

trial by the farmer or by the veterinarian responsible

for animal welfare. In this large scale farm trial,

addition of the biocontrol yeast P. anomala slightly

increased amount of essential amino acids, reduced

grain phytate content, and diminished numbers of

undesirable moulds by 2 log units and Enterobacte-

riacae by 5 log units (Olstorpe et al. 2010b).

In conclusion, animal feeding trials with chickens

and beef cattle have demonstrated that the hygienic

improvements and mould control observed with

P. anomala in vitro and in small scale grain labora-

tory experiments can be extended to large scale farm

trials. In addition, no adverse effects on animal

weight gain, feed conversion, health or behaviour

were observed in any of the studies.

Barriers to practical applications

Given the accumulated results presented above, one

would have expected to see the appearance of

P. anomala products for biopreservation of cereal

grains on the market. In reality, even if the biopre-

servative effect is now very well documented, several

hurdles exist on the way to agricultural applications.

Some of these barriers relate to technical issues such

as production cost and the ability to develop stable

dry formulations. In co-operation with the Swedish

National Yeast Company (Jastbolaget AB) we found

that the production cost for P. anomala biomass was

in fact similar, or even lower than that for bakers

yeast Saccharomyces cerevisiae (Rejholt and Schnu-

rer, unpublished). For practical reasons, the develop-

ment of a dry formulation with long shelf life is

essential for most starter culture applications. For

P. anomala, this appears to be less of an issue as dry

formulations with long-term stability can be obtained

using several techniques, such as fluidised bed drying

or freeze drying (Melin et al. 2010).

A critical constraint for the development of novel

biopreservatives is a very complex regulatory situa-

tion, in particular in the European Union. Sundh and

Melin (2010) discuss the situation, with focus on the

use of P. anomala and other yeasts in feed and food

applications. The Qualified Presumption of Safety

(QPS) approach for safety assessments of microor-

ganisms intentionally added to food or feeds has been

developed by the European Food Safety Authority


123

(EFSA). The intention is to provide efficient assess-

ments of microbial species where a sufficient body of

knowledge or long-term experience testifies to their

safety. P. anomala is one of several yeast species that

have been given QPS status, although QPS was later

restricted to the use of this yeast for production

purposes (Sundh and Melin 2010).

Other barriers can be divided into those related to

patent issues, market awareness, size and ability to

absorb both novel ideas and novel costs, as well as

the structure of the agriculturally oriented starter

culture industry and the structure of agricultural

production. To develop and produce a biopreser-

va tive starter culture, we obtained a patent for a

P. anomala J121 biopreservation applications, but it

soon became too costly to maintain without seeing a

near-future development of a substantial market.

In farming regions with comparatively small units

of animal production, the change from a system with

drying of feed grain to an airtight storage system may

require a complete shift of preservation technology at

the individual farm level. Such a shift is generally

perceived as dangerous by the farmer, especially if it

is a completely new technology. A particular aspect

of developing new biopreservatives for grain storage,

compared to, for example storage of table fruit, is the

comparatively low value of the crop. This gives the

operation a low margin for new developments. In

regions with larger farming units, a shift may be

performed stepwise, with the possibility to keep the

cost reasonably low in spite of handling two feed

storage systems. However, under those conditions the

farmer expects to obtain a secure and well-developed

system that can be operated with few employees only,

commonly having limited formal education. Another

barrier is a traditional, and understandable, hesitation

of successful farmers to replace a safe, although

energy demanding and fossil fuel dependent, storage

system, operating at reasonable profit level, with a

completely new system.

Today, the microbial starter culture industry that

supply inoculants and technical knowledge is reluc-

tant to invest, since few customers are prepared to

shift feed grain handling systems and buy large

quantities of inoculants. While access to stable, dry

starter culture formulations is absolutely necessary

for wide market penetration, this is insufficient.

Purely technical systems for homogeneous grain

inoculation and loading of silos are also needed.

These systems must be very reliable and have high

capacity since the silos need to be filled during a few

days of an intense harvest period. To develop such

complete biopreservation systems, agricultural, tech-

nical and biotechnical industries with a limited

tradition of co-operation need to work closely

together.

We believe that the increasing cost of energy, and

the general desire to reduce dependence on fossil

fuels and minimise carbon dioxide emissions, even-

tually will be a major driver for the changing of grain

treatment and storage systems. This would be of

particular importance in situations of replacement of

out-dated grain storage systems, especially if a turn-

key system would be available. In spite of the

demonstrated biopreservative ability of the yeast

P. anomala, it has to date been very difficult to create

the right constellation of technical, agricultural and

biotechnical industries necessary to commercially

launch P. anomala J121 based biopreservation sys-

tems. The complications caused by the EU regulatory

systems remains another very significant barrier.

Concluding remarks

We have now studied P. anomala J121 biology,

ecology and grain preservation ability for 30 years.

Over this period, more 40 scientific publications and

no less than five PhD theses (Petersson 1998; Boysen

1999; Druvefors 2004; Fredlund 2004; Olstorpe

2008) have been written on different aspects of this

yeast strain, extending from fundamental research on

metabolism, genetics and molecular biology, all the

way to the practical farm scale level. The P. anomala

theses are all available in electronic form from the

SLU library web page (Epsilon-section). An increas-

ing number of P. anomala publications from other

authors, sometimes under different taxonomic desig-

nations (Wickerhamomyces anomalus, Hansenula

anomala), have highlighted other facets and potential

biotechnical applications of this physiologically ver-

satile yeast, many of which are discussed in this

Special Issue. The research on P. anomala in our

laboratory has mainly been in the context of biopre-

servation in energy saving storage systems for feed

grain. The growing awareness of climate change in

relation to fossil fuels use has now perhaps made the

time ripe for introducing energy efficient and carbon


123

dioxide neutral storage solutions in the practical

handling of cereal grains. Along this line, the

‘‘30 year overnight near-success biopreservation

story’’ of P. anomala J121 described here should

then develop into large scale practical use, with

important positive environmental impacts on agricul-

ture. We now look forward to follow both future

fundamental scientific developments and biotechnical

applications and commercialisation of P. anomala, a

truly fascinating yeast species.

Acknowledgments Our research on P. anomala as a

biocontrol organism has received financial support from

MISTRA (The Foundation for Strategic Environmental

Research), FORMAS (The Swedish Research Council for

Environment, Agricultural Sciences and Spatial Planing), SLF

(The Swedish Farmers’ Foundation for Agricultural Research)

and from the European Union (research programme

BIOPOSTHARVEST). The thematic research programme

MicroDrivE (Microbially Derived Energy) at the Faculty of

Natural Resources and Agricultural Sciences has also

contributed financially. Over the years, a large number of

undergraduates, PhD students, scientists, farmers and companies

have been cooperating partners in biopreservation projects. Their

combined contributions to P. anomala research are gratefully

acknowledged.

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Pichia anomala J121: a 30-year overnight near success biopreservation story