INTERNATIONALBIODETERIORATION +BIODEGRADATION

International Biodeterioration + Biodegradation 31 "0887# 14Ð17

S9853Ð7294:87:,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved[PII] S9853Ð7294"87#999319

A microcalorimetric method of studying mould activity as afunction of water activityNatalia Markovaa\ Lars Wadso�b\�

aDivision of Thermochemistry\ Lund University\ Lund\ SwedenbDivison of Building Materials\ Lund University\ Lund\ Sweden

Received 17 August 0886^ revised 7 December 0886^ accepted 15 March 0887

Abstract

This paper presents a new method of studying mould activity as a function of water activity by measuring the heat produced bythe fungal metabolism[ During a measurement a small sample "³0 g# of a moulded substrate is moved between a humidity generator\where it is conditioned to a certain water activity\ and a microcalorimeter\ where the heat production rate is measured[ This isrepeated for di}erent water activities[ A conditioning and the subsequent thermal measurement takes approximately one day foreach water activity[ Results are presented from a measurement with Penicillium mould growing on a bread substrate[ The resultscorrelate well with literature data indicating that the present method is a rapid way of assessing mould activity as a function of wateractivity[ Þ 0887 Elsevier Science Ltd[ All rights reserved

Keywords] Mould activity^ Microcalorimetric^ Water activity

0[ Introduction

Mould growth on foodstu}s\ building materials\ textilesand other materials is a problem in all parts of the world[The most common measure taken to prevent mouldgrowth is to lower the water activity "aw# of the material\as it is well!known that mould cannot grow on drymaterials[ The critical aw for mould growth depends onmould species\ substrate and temperature as has beeninvestigated by Ayerst "0858#^ Block "0842#^ Gervais etal[ "0877#^ Viitanen "0883# and others[

Studies of mould activity as a function of various par!ameters are usually made by exposing a large number ofsamples to di}erent "constant# conditions and afterwardsassessing the fungal activity by measurements of suchparameters as radial growth and toxin production[ Thesetypes of studies are time consuming as a large number ofsamples and climate chambers have to be used[ Dynamice}ects\ such as ~uctuations in aw\ are usually notconsidered[ In the present paper we describe a new mic!rocalorimetric technique to monitor mould activity as afunction of aw by measuring the heat generated by themould metabolism[

Calorimetry is the measurement of heat and heat pro!duction rate "thermal power#[ This is a very general

�Corresponding author[ lars[wadsoÝbyggtek[lth[se

measurement technique as nearly all processes "physical\chemical\ biological# produce heat[ Sensitive isothermalcalorimeters\ microcalorimeters\ have been used in anumber of microbiological applications\ such as in thestudy of yeast "Gustafsson\ 0880#\ needle litter deterio!ration in forest soil "Becker et al[\ 0880# and microbialactivity in food "Nunomura et al[\ 0875#[ These appli!cations rely on the relatively large heat quantities pro!duced by microbial metabolism[ When one gram of acarbohydrate such as a sugar is metabolised aerobically\approx[ 05 kJ of heat is released[ This corresponds toapprox[ 1mg of sugar metabolised per hour to give aheat production rate of 09mW in a sample[ As modernmicrocalorimeters can measure thermal powers wellbelow 0mW it is clear that microcalorimetry is a sensitivetechnique for studying microbial activity[

We use relative humidity "RH# to describe the moisturestate of air\ and water activity "aw# for the moisture stateof materials[ Although their de_nitions di}er\ in thepresent type of study they can be considered to have thesame numerical values at equilibrium conditions[

1[ Method

An ampoule with a mould sample is moved between ahumidity generator for conditioning\ and a calorimeterfor thermal activity measurements[ The humidity gen!

N[ Markova\ L[ Wadso�:International Biodeterioration + Biode`radation 31 "0887# 14Ð1715

erator can then operate at a high ~ow rate without dis!turbing the thermal balance of the calorimeter "Wadso� \0886#[ For the thermal measurements a TAM micro!calorimeter "Thermometric\ Ja�rfa�lla\ Sweden# was placedin a water bath controlled to approx[ 20mK[ The samplewas kept in a steel ampoule "diameter 03mm\ volume2[4ml#[ When the ampoule was in the humidity generator\air was purged through two steel tubes in its lid[ Whenthe ampoule was in the microcalorimeter\ the tubes wereclosed[ The sample was protected from contaminationduring the conditioning by a sterile _lter^ it was onlyunprotected for a few seconds while the ampoule wasbeing transferred from the humidity generator to thecalorimeter[

The humidity generator is a fairly simple and inex!pensive device built from standard components[ It oper!ates by mixing di}erent proportions of air from twosources with di}erent RH|s[ Figure 0 shows a schematicdrawing of the device[ Air ~ows through two 19L plasticboxes "A\ C# and two 199ml glass ~asks "B\ D# containing1L and 099ml\ respectively\ of saturated aqueous saltsolutions[ At 14>C these solutions regulate the RH of theair to 42) in A and B "Mg"NO2#1#\ and 83) in C and D"KNO2# "Greenspan\ 0866#[ A two!way valve "E^ Nep!tune Research\ Caldwell\ NJ\ USA# governs the RH ofthe air stream by allowing di}erent proportions of airwith 42 and 83) RH through the system[ It is operatedwith a total period of 09 s by a timer "Comat\ Worb\Switzerland# and can thus generate RH|s between 42 and83)[ The air stream passes a micro_lter "F^ MilliPore\Bedford\ MA\ USA# before ~owing through a 2[4mlstainless steel ampoule "G# containing the sample "H#[An RH sensor "I^ Vaisala\ Helsinki\ Finland# is pos!itioned after the ampoule G and a peristaltic pump "J\Alitea\ Stockholm\ Sweden# provides a ~ow rate of 4L:h[All parts of the humidity generator except the valve andthe pump are contained in an insulated box with highthermal inertia to decrease temperature ~uctuations[

Fig[ 0[ A schematic drawing of the humidity generator "the di}erentparts are described in the text#[ The heat!generating parts of the instru!mentation are placed outside the insulated box "dotted line#[

As the temperature in the salt solutions "A!D# is thesame as in the ampoule with the sorbing sample "G!H#\the RH of the air entering the ampoule may be calculatedas a weighted mean between the RH|s of the two airstreams "with weights proportional to the times the valvewas open to each ~ow#[ The humidity generator wasoperated long enough to bring the sample close to equi!librium with the humidi_ed air^ the procedure is describedin some detail in Wadso� "0886#[ In no case was the samplein the humidity generator less than three times the timeneeded to bring the sample within 9[994 of the equi!librium aw[ The RH|s of the saturated salt solutions as afunction of temperature were obtained from Greenspan"0866#[

When the ampoule with the sample was in the calor!imeter\ the humidity generator was checked with satu!rated aqueous solutions of the following _ve "calibration#salts "RH|s at 14>C from Greenspan 0866#] Mg"NO2#1"41[8)#^ NaCl "64[2)#^ NH3Cl "67[5)#^ KCl "73[2)#^KNO2 "82[6)#[

2[ Materials

Mould "Penicillium Spp[# that had been found growingon bread was grown on 1) "weight:volume# malt extractagar at 08Ð11>C for 6 days in the dark[ Autoclaved sam!ples of sweet wheat bread were inoculated with the Peni!cillium with the help of a needle[ The samples were thenincubated in 89mm sterile Petri dishes for three days untilgermination was visible[ For the measurements a samplehalf covered with mould was taken from the Petri dishand put into the ampoule[ Measurements were also con!ducted on sterile bread samples[ Each sample containedapprox[ 9[1 g bread "dry mass#[ The measurements weremade at 14>C close to the optimum for growth of anumber of Penicillium according to Ayerst "0858# andPasanen et al[ "0880#[

3[ Results and discussion

Figure 1 presents an example of a check of the humiditygenerator[ The agreement is satisfactory\ taking intoaccount that the hysteresis of the sensor probably hascontributed to the scatter[ Note that the sensor output isnot necessarily a linear function of the RH\ and that theRH!values generated by the humidity generator were notthe same as the RH!values of calibration salts[

From the measurements on sterile bread samples it wasfound that the heat production rate of the humid breaditself was negligible[ The heat measured on the mouldedbread was therefore produced by the mould fungi[ Figure2 gives an overview of the thermal power measured onthe moulded sample[ Maximum thermal powers and totalheats are given in Table 0[ The samples used in this

N[ Markova\ L[ Wadso�:International Biodeterioration + Biode`radation 31 "0887# 14Ð17 16

Fig[ 1[ A check of the performance of the humidity generator[ Onthe x!axis are shown the RH|s generated by calibrations salts "×# orcalculated from the timer settings and the two RH|s of the two saltsolutions used in the humidity generator "�#[ The y!axis gives theoutput from the humidity sensor "in arbitrary units#[ Each point for thehumidity generator is a mean of three measurements^ the calibrationsalt measurements are single measurements[

experiment showed no visual di}erence from duplicatesamples kept in the Petri dishes during the measurements[

From the results presented in Fig[ 2 and Table 0 it isseen that the mould activity increased with aw[ This isin good agreement with Ayerst "0858#^ Troller "0879#^Viitanen "0883# and others[ With the present technique it

Fig[ 2[ Thermal powers measured for a bread sample half covered with Penicillium mould as a function of aw "shown in the top of the _gure#[ At thebreaks in the curve no measurements were made as the sample was then in the humidity generator[

is possible to assess critical moisture states for mouldgrowth[

The thermal powers measured in desorption are higherthan those measured in absorption[ This may have beencaused by changes in the nutritional status of the sampleduring the measurement\ but it may also be explained bythe sorption hysteresis found in most materials\ cf[ Skaar"0877#[ For a particular substrate the mould may be moresensitive to the moisture content than to the aw[

Table 0Summary of result from measurements on a bread sample half coveredwith Penicillium mould "cf[ Fig[ 2#[ In the table\ aw is the water activityof the sample\ Pmax is the maximum thermal power measured\ and Qtot

is the integral of the thermal power for those water activities for whichthe measurement was continued until the thermal power dropped tolow values

aw Pmax:W Qtot:J

9[89 169 03[39[74 44 03[99[79 19 *9[64 03 *9[69 6 *9[54 3 *9[64 7 *9[74 39 03[09[89 029 02[8

N[ Markova\ L[ Wadso�:International Biodeterioration + Biode`radation 31 "0887# 14Ð1717

At the two highest levels of aw the measurements con!tinued until the thermal power dropped to low values[The total heats measured as integrals under the curves inFigure 1 have a mean value of 03[0 J "Table 0#[ As thefree volume in the ampoule was approx[ 2[3ml and theenthalpy of aerobic metabolism of carbohydrates isapprox[ 369 kJ:mol O1 "cf[ Gustafsson\ 0880#\ this cor!responds to a decrease in oxygen concentration from theatmospheric oxygen content of 10) to 0Ð1)\ i[e[ nearlyall the oxygen is consumed[ At an aw of 9[74 it is seen inFig[ 1 that the mould activity is quite constant until itsuddenly drops when the oxygen concentration becomestoo low\ i[e[ at least above a few percent of oxygen theoxygen concentration was not rate!determining[ This isin good agreement with data presented by Northolt andBullerman "0871# and Bailey and Ollis "0875#[

To fully interpret the microcalorimetric signal a knowl!edge of the types of metabolism used by the fungi isneeded[ Di}erent types of metabolism may producedi}erent amounts of heat per mole of carbohydratemetabolised[ We have here assumed that only the rate ofmetabolism\ not the type\ is in~uenced by the aw[ Forthe future development of the method it is\ however\important to investigate this assumption by parallelmeasurements of CO1 production or some other par!ameter related to the fungal metabolism[

We have noted four factors important for the properfunctioning of the humidity generator[

0[ The temperature must be stable in the insulated boxduring the time it takes to change the aw of the sample[In the present case the temperature was constantwithin 29[2>C during the 4Ð13 h the sample was inthe humidity generator[

1[ It is important that all tubes\ valves and other partsof the air ~ow path have a large internal diameter tominimise the pressure drop between the salt solutionsand the ampoule[ The ratio between this pressure!drop and the atmospheric pressure is a direct measureof a relative error in the RH produced[ In the presentcase this ratio was 9[995[

2[ The air ~ow rate in the system should not be dependenton which way the air is ~owing through the valve[Serious errors may be introduced if the valve and thetubing are not symmetric[

3[ The moisture capacity of the ~ow system between thevalve and the sample must be high enough to dampenvariations in RH caused by the switching of the valve[In the present instrumentation the tubing had enoughmoisture capacity\ as no variations were seen in the

output from the humidity sensor when the ampoulewas removed from the ~ow path[

With the present calorimetric measurement technique themould activity is monitored continuously as it takesplace^ it is not measured after a period of exposure[ Thesensitivity of the microcalorimeter makes it possible tostudy even very low thermal powers of long!term pro!cesses like mould fouling of construction materials[ Achange of aw and a measurement of thermal activity takesapproximately one day\ so the fungal activity as a func!tion of aw can be measured on one sample in 0Ð1 weeks[

Acknowledgements

This work was supported by the Swedish Institute "toNM#\ the L!E Lundberg Scholarship Fund "to LW# andThe Swedish Building Research Council "to LW#[

References

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