BIOTECHNOLOGY Vol.IV Bioprocess Engineering - Bioprocess Analysis through Calorimetry and Biothermodynamics - Thomas Maskow, Richard B.

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1 BIOPROCESS ENGINEERING BIOPROCESS ANALYSIS THROUGH CALORIMETRY AND BIOTHERMODYNAMICS Thomas Maskow Dpartmnt of Environmntal Microbiology (UMB), UFZ Hlmholtz Cntr for Environmntal Rsarch, Prmosrstr. 15, D Lipzig, Grmany Richard B. Kmp Institut of Biological Scincs, Edward Llwyd Building, Univrsity of Wals, Abrystwyth SY23 3DA, U.K. Kywords: Bioprocss analysis, Calorimtry, Biothrmodynamics, Mtabolic flux analysis, Procss control, Chip-calorimtry, Mgacalorimtry, isothrmal titration calorimtry (ITC) Contnts 1. Introduction 2. Roots of Modrn Biocalorimtry 3. Information Contnt of Calorimtric Signals 3.1. Stoichiomtry and Kintics of Microbial Growth and Product Formation 3.2. Analysis of th Mtabolic Carbon Fluxs 4. Localization of Calorimtric Snsors 4.1. Calorimtr Embracing Natural or Tchnical Systms 4.2. Snsors Insid Natural or Tchnical Systms 4.3. Snsors in Masuring Loops 5. Rcnt Dvlopmnts of Calorimtry 5.1. Mgacalorimtry 5.2. Miniaturizd Calorimtry 5.3. High-throughput Calorimtry 5.4. Ultra-Snsitiv Calorimtry 5.5. Photocalorimtry 6. Futur Prospcts of Biocalorimtry Glossary Bibliography Biographical Sktchs Summary: Gibbs nrgy dissipation oftn in th form of hat nrgy is an xplicit consqunc of th scond law of thrmodynamics and a gnral fatur of bioprocsss. This is bcaus organisms convrting CO 2 or organic mattr into biomass or any bioproducts cannot us all th Gibbs nrgy providd by th assimilatd substrats or othr nrgy sourcs, i.. ssntial fractions of th Gibbs nrgy oftn ar wastd as hat. This hat production rflcts mtabolic shifts in ral tim and can b masurd non-invasivly by dirct calorimtry. By applying th first law of thrmodynamics, this tchniqu prmits th usr to tst if procss hat is in accordanc with assumd mtabolic pathways and, if not, thrby to dtct unknown ractions. Hat production is incomparably th idal

2 indicator for monitoring and analyzing bioprocsss. Hithrto, tchnical obstacls hav prvntd th broad distribution among th scintific community of this powrful mthodology. Howvr, rcnt dvlopmnts rmovd from calorimtry th old waknsss and qualify th tchniqu for: high-throughput masurmnts, analyzing bioprocsss in bioractors of diffrnt sizs (µl to m 3 ), monitoring of trac pollutant biodgradation and bioprocss control vn undr dirty tchnical conditions. 1. Introduction Th phnomnon of lif itslf is intimatly and inhrntly tid to Gibbs nrgy dissipation oftn in th form of hat nrgy (J). Th amount of it dpnds on th growth and product formation stoichiomtry and its production rat (J s -1, W) additionally on th kintics. Th masurmnts of this rat, i.. calorimtry, combind with furthr bioprocss analysis, provid insights to th on-going bioprocss (Bioprocss Analysis). Calorimtry is suitd for Bioprocss Control vn for such complx systms that hav growing spcis, food chains, diffrnt phas transitions, and/or larg intrfrnc with physical procsss,.g. aration, stirring, and/or mixing, which ar difficult to intrprt by thrmodynamics. Th advantags of thrmal snsors for th control of simpl bioprocsss control ar fast rspons tims, robustnss and incrasing snsitivity with any up scaling du to incrasing th ratio of hat producing volum to hat xchanging surfac. Th hat production rat rquiring sophisticatd masurmnt tchniqus at th bnch scal can dvlop into a ral problm at larg scal bcaus th hat xchang with th nvironmnt is ngligibl (i.. adiabatic mod) at this scal. This mans that all th hat producd by th cultur will contribut to an incras of tmpratur and a dactivation of th microbial catalyst, unlss th bioractor contains appropriat cooling facilitis. Th quantitativ knowldg of th microbial hat production rat is thrfor crucial to succssful Bioractor Dsign [s also Bioractor Typs]. Th dgradation of high valu chmical nrgy from nutrints as substrats (low ntropy) into hat or disordrd structurs (high ntropy) is a prrquisit for lif. Prcption of th rlationship btwn Gibbs nrgy dissipation and hat production hlps in th gnral undrstanding of th Driving Forcs of Bioprocsss. Th mor Gibbs nrgy of th nutrints is dissipatd, th lss nrgy will b rtaind for th formation of biomass or products and thus th lowr will b th rspctiv yilds. By following th principls of irrvrsibl thrmodynamics, it can b sn that th rats of bioprocsss ar influncd by th amount of th driving forc, i.. th Gibbs nrgy dissipation. In volutionary trms, Natur has had to find a compromis btwn high yild and high rats. Th important practical consquncs of a bttr undrstanding of th thrmodynamics of microbial growth and product formation ar prdictions about maximum possibl yilds, maximum practical yilds, maintnanc cofficints, thrshold concntrations and vn maximum growth rats.

3 Th nd to quantify th rlativly small biological hat production in th microwatt scal spurrd th dvlopmnt and prfction of th modrn isothrmal microcalorimtr (ITMC). Du to its xtraordinary snsitivity, it has contributd a grat dal to biochmical and biophysical rsarch. Nvrthlss, microcalorimtric rsarch has hardly touchd progrss in bionginring progrss in rcnt yars. Th rasons ar: difficultis in agitation and aration; complxitis in th intgration of additional microsnsors lik ph, Rdox, UV/VIS or po 2 ; and small sampl volums du to th rlativly small calorimtric vssl. Ths xprimntal limitations in microcalorimtry lad to xprimntal conditions which oftn divrg in ssnc from th ral procss conditions. Howvr, th main waknss of th convntional ITMC, as wll as of th bnch scal calorimtr, is th rstriction in data collction bcaus it is possibl only to follow rlativly fw bioprocsss in paralll whras, for instanc, modrn biotchnological strain optimizations ar nowadays prformd in up to 1536 wll plats. Furthrmor convntional calorimtrs ar prdominantly dsignd and producd for only a fw applications rsulting in rlativly high prics for good calorimtrs. Thrfor, calorimtry will nvr tap its full potntial until at last a fw of ths waknsss ar ovrcom. Howvr, th rcnt trnds in calorimtric dvlopmnts such as mgacalorimtry, miniaturizd calorimtry (chip-, intgratd circuit-, nano-calorimtry), high-throughput calorimtry (nthalpy arrays), ultra-snsitiv calorimtry and photocalorimtry promis to rmdy th dficincis and provid calorimtry with a brilliant futur as a scintific tool as wll as a tchnical snsor. Th roots, th stat of th art and th potntial of calorimtry will b discussd in th following sctions. 2. Roots of Modrn Biocalorimtry For cnturis or vn millnnia, fir has bn sn as a symbol of lif and its xtinction a synonym for dath. This may xplain why living systms hav bn th obcts for calorimtric xprimnts and thrmodynamic intrprtations sinc th arly days of this tchniqu. Evn th mrging trnds of rcnt calorimtry to analyz and control bioprocsss stand in th tradition of a long history of thrmal analyss. Evr sinc th first calorimtric xprimnts with guina pigs mor than 200 yars ago by Lavoisir (1780) and Crawford (1777), it has bn rcognizd that th hat producd from living mattr is a ral tim masur of mtabolic activity. A mor quantitativ viw basd on studis of hors physiology aros whn Robrt Mayr (1842) stablishd th law of consrvation of nrgy (Th First Law of Thrmodynamics). Grmain Hnri Hss discovry of th additivity of indpndnt dtrmind nthalpy is a spcial statmnt of th First Law allowing th quantitativ intrprtation of biocalorimtric

4 xprimnts. Max Rubnr dmonstratd (1890), using a dog s mtabolism, that Hss law is not only applicabl to chmistry, but also to biology. Th conclusion that thr wr no principl thrmodynamic diffrncs btwn inanimat and living systms pavd th way for th furthr dvlopmnt of biocalorimtry,.g. as a tool for onlin stoichiomtry or for mtabolic flux analysis. Thus quantitativ calorimtry is an important rcnt trnd. It combins calorimtry with othr masurmnts ithr on/atlin (Fourir transform infrard spctroscopy, dilctric spctroscopy, optical dnsity, flow inction analysis, gas analysis) or off-lin (HPLC, nzymatic analysis of mtabolits and products, flowcytomtric analysis of cll stats, tc) in ordr to stablish that th growth stoichiomtry is complt and, if not, to calculat th missing part using nthalpy and lmntal balancs. Th first succssful calorimtric xprimnts usd macroscopic liv forms, but it is not th hat production rat pr unit living mass pr s that maks calorimtry with th biotchnologically mor important microscopic lif mor difficult. In practic, xponntially growing yast (250 W kg -1 ) and vn rsting yast culturs (5 W kg -1 ) show much highr spcific hat production rats than Guina pigs (3 W kg -1 ) or humans (1.3 W kg -1 ). In culturs, th difficulty rsults from th high hat storag capacity of th mdia which usually contain low volum fractions of microorganisms. Th hat capacity of air (1.2 kj m -3 K -1 ) is about thr ordrs of magnitud lowr than that of watr (4200 kj m -3 K -1 ) or soils ( kj m -3 K -1 ) (T=298 K, P= Pa). As arly as 1856, Pirr Dubrunfaut found an imprssivly simpl solution to th problm and h is now rputd as th fathr of what ths days is th quit topical Mgacalorimtry, i.. calorimtry in raction tanks of svral hundrd litrs or vn m 3 usd for procss control. Dubrunfaut prformd an alcoholic frmntation of 2559 kg sugar in a hug oak vssl of L volum. In this way h maximizd th ratio of hat-producing volum to hat-xchanging surfac and obsrvd a tmpratur incras from 23.7 o C to 33.7 o C during 4 days of frmntation at ambint tmpraturs btwn 12 o C and 16 o C. Taking into account hat loss by radiation, convction and vaporation and hat storag by th wood, h calculatd a frmntation hat of kj mol -1 glucos quivalnt, a numbr surprisingly clos to th now stablishd valu of kj mol -1. Nw, mor snsitiv tmpratur probs, lss tmpratur dpndnt lctronic dvics and progrss in ral tim data acquisition furthr th caus of Mgacalorimtry as th scond rcnt trnd in calorimtry. Whras up scaling rducs th influnc of nvironmntal fluctuations, rduction in calorimtr siz diminishs th amount of hat nrgy ndd for th calorimtric dvic whil spding up th masurmnt. Dvlopmnts towards short rspons and quilibration tims,.g. Miniaturizd Calorimtry (rsulting in intgratd circuit (IC) calorimtry) ar a third rcnt trnd in calorimtry dvlopmnt. Th principl of ICcalorimtry is illustratd in figur 1. Th first thin-film calorimtr was dvlopd during th arly svntis but rcnt, nw tchniqus of printd circuit dsign hav boostd th dvlopmnt of thm, as tstifid by th publication of mor than 70 paprs in th last dcad. As IC calorimtrs nowadays masur a fw nw thy ar also nicknamd Nanocalorimtrs. Th xpctd progrss in IC calorimtr dvlopmnt will lad to: (i) xtrmly small thrmal dtctors; (ii) minut sampl mass rquirmnts; (iii) incrasd snsitivity; and (iv) much shortr rspons and

5 quilibration tims. In particular, immdiatly rsponding small dtctors ar crucial for th intgration of calorimtric snsors into stablishd bioractors. At prsnt, vn nanocalorimtry is too slow to compt with th robotic monitoring of mtabolic activitis of clls growing in 384- or 1536-wll microtitr plats, usd as a diagnostic tool in th pharmacutical industry or in biotchnology. Calorimtry suffrs from low throughput in comparison to such rivals. Thrfor, a fourth rcnt trnd in biocalorimtry is th dvlopmnt of th High-Throughput Calorimtr, i.. th socalld nthalpy arrays. Figur 1: Masurmnt principl of a chip calorimtr. On anothr front, thr is ral concrn about th potntially dltrious ffct on human halth of micropollutants or of hydrophobic organic compounds (HOC) du to thir ubiquity in th nvironmnt and thir prsistnc. HOC s dissolving only slightly in aquous phass tnd to intract with non-aquous phass and, as a consqunc, ar poorly bioavailabl for microbial dgradation. To masur this, th novl Ultra- Snsitiv Calorimtry has to b dvlopd in ordr to gain ral tim information on th kintics and th physiology of bioconvrsion of HOC tracs in homognous aquous as wll as in htrognous phass. Th ovrwhlming maority of calorimtric masurmnts hav bn of mtabolic hats originating from chmical bonding nrgis and hav disrgardd othr nrgy forms. Howvr th lif on arth in its prsnt form is unthinkabl without th nrgtic utilization of light. Mag and co-workrs rcognisd this absnc as arly as 1939 and dvlopd small-scal Photocalorimtry. On anothr topic, lctrical nrgy drawn from microbial ful clls (MIC) or put to anarobically growing clls to chang th rdox balanc hav bn known and xpctd to influnc nrgy balancs sinc th arly 1900s. Howvr, no calorimtric masurmnts considring this nrgy form ar known to th authors dspit its importanc and long history. Thrfor, calorimtric tchniqus ar urgntly rquird that tak nrgy forms othr than chmical nrgy into account.

6 In th following, th status quo and th potntial of th six rcnt trnds in biocalorimtry will b discussd in mor dtail. 3. Information Contnt of Calorimtric Signals 3.1. Stoichiomtry and Kintics of Microbial Growth and Product Formation Th most important information for an nginr dsigning a biotchnological procss is its stoichiomtry and kintics. Both charactristics ar tightly connctd with th hat production rat. Howvr, to analyz this corrlation, th dynamic nrgy balanc for th opn tchnical or natural systm of intrst has to known. Eq. (1) dscribs a dynamic nrgy balanc for such a systm: V ρ c p d T d t = P + W + ni c p i, i ( T T ) Δ R H ξ, (1) d T Th trm V ρ cp dscribs th hat accumulation in th whol systm as th d t incras of tmpratur T vrsus tim t. cp is th man hat capacity of th systm, V its volum and ρ its dnsity. Hat P, work W and matrial fluxs n ar xchangd with th nvironmnt. Th xchangd matrial of th spcis i transports nrgy proportional to th hat capacity at constant prssur c P i, and to th tmpratur diffrnc (T T) of th rspctiv ntry or xit port. Th raction trm Δ R H ξ corrlats th nrgy rat gnratd or consumd by th raction with th rspctiv raction nthalpy Δ R H and th absolut rat of th raction ξ. Th raction trm includs mtabolic and chmical ractions as wll as phas transitions. It is of practical importanc that all th componnts in Eq. (1) can b masurd and balancd in ordr to find out how complt or corrct ar th assumptions about th tchnical or natural systm. Th first modrn application of this principl in biotchnology, 150 yars aftr Dubrunfaut s pionring work around 1856, bcam known as balancing calorimtry. Rcntly, svral rsarch groups hav improvd bnch scal and industrial scal calorimtry. Thir tchnical dvlopmnts wr aimd at rducing th complxity of Eq. (1) by liminating abiotic sourcs of rror. This was d T achivd by: (i) rducing th hat accumulation trm V ρ c p through opration d t c T T by undr isothrmal conditions; (ii) rducing th convction trm ( ) i n i p i, thrmostating th ntring and laving strams; and/or (iii) kping W (prdominatly stirrr work) constant. Th facilitis of any off th pg bioractor can b mployd for hat flux masurmnts by using th abov discussd achivmnts combind with modrn tmpratur probs to giv lss ovrall xpnditur in trms of both labor and financ. Such an improvisd calorimtr works with a snsitivity of approx. 50 mw L -1.

7 Figur 2 comparing th calculatd with th masurd hat production rat shows th applicability of such a non-convntional calorimtr. Undr idal conditions, th producd hat rflcts nothing but th progrss of all ongoing mtabolic or chmical ractions (Eq. 2): P Δ H ξ R with ξ = ri ν (2) i whr ri and ν i ar th rat and th stoichiomtric cofficint, rspctivly, of th componnt i participating in raction. Figur 2: Tst of th bioractor usd as an off th pg calorimtr. Comparison of th calculatd with th masurd hat production rat for th arobic growth of H. longata on glycrol. Th hat of non-mtabolic ractions,.g. nutralization hats, can b obtaind by th titrimtric consumption of alkali or acid using tabulatd valus for corrction. Th mtabolic hat production can thn b corrlatd with th stoichiomtry of microbial growth and product formation procsss. Th microbial growth raction in its simplst cas can b dscribd stoichiomtrically by Eq. (3): Y S/X C S1 H S2 O S3 + Y N/X NH Y O/X O 2 -> C X1 H X2 O X3 N X4 + Y H2O/X H 2 O + Y CO2/X HCO Y H/X H + (3) whr C S1 H S2 O S3 is th applid carbon sourc, NH 4 + and O 2 stand for th nitrogn sourc and th trminal lctron accptor, rspctivly, and C X1 H X2 O X3 N X4 is th lmntal composition of th biomass. Th lmntal composition of bactria has bn avragd to C 4 H 8 O 2 N 1 or mor rcntly to CH 1.84 O 0.53 N Both valus can b applid for stoichiomtric considrations vry accuratly if no xact lmntal composition is availabl. Th six unknown yild cofficints (Y S/X, Y N/X,, Y O/X, Y H2O/X, Y CO2/X, Y H/X ) in

8 Eq. (3) ar not indpndnt from ach othr bcaus thy hav to satisfy fiv balancs (four lmntal balancs and on charg balanc). This mans that adding th nthalpy balanc and masuring th raction nthalpy of th growth procss Δ R H x allows th calculation of ach of th unknown yild cofficints. Two rfrnc stats for th nthalpis ar commonly usd for this purpos, namly th constitunt lmnts of all th involvd spcis (nthalpis of formation) and th compltly combustd stat (nthalpis of combustion, Δ C H). Using th lattr simplifis th nthalpy balanc (Eq. 4): Δ R H x = -(Δ C H X + Y H/X Δ C H H Y S/X Δ C H S Y N/X Δ C H N ) (4) Th combustion nthalpis of ach spcis ar tabulatd in th litratur (,.g. s Domalski in th bibliography). Thr ar also som combustion nthalpis of biomass of diffrnt organisms growing undr diffrnt conditions availabl from th sam sourc. If thr ar no data availabl, th combustion nthalpy can b stimatd vry accuratly from th lmntal composition applying th Thornton s rul or mor rcnt corrlations. Th hat production rat P of a microbial growth procss dpnds on th raction hat of th growth procss Δ R H x and on th biomass formation rat r X (Eq. 5), P = r X Δ R H x (5) In th cas of chmostatic growth Eq. (5) rads: P = D 0 ( S S) Y S / X Δ R H X Th dilution rat D and th substrat concntration of th ractor input S 0 ar prdfind by th oprator. Th rsidual substrat concntration S is usually low in comparison to S 0 in chmostatic bactrial or yast culturs and can thus b nglctd in nthalpic or stoichiomtric balancs. Thrfor, th masurd P rflcts Δ R H x and, using th balancs of th nthalpy and th lmnts, provids th ral tim stoichiomtry TO ACCESS ALL THE 27 PAGES OF THIS CHAPTER, Visit: (6) Bibliography Domalski, E.S. (1972) Slctd valus of hats of combustion and hats of formation of organic compounds containing th lmnts C, H, N, O, P, and S. Journal of Physical and Chmical Rfrnc Data 1, [This work provids comprhnsiv collction of combustion nthalpis] Janssn M., Patiño R., von Stockar U. (2005) Photobiocalorimtry: a tool to study light nrgy utilization

9 in phototrophic culturs. Thrmochimica Acta 435, [This work dscribs photocalorimtry at L- scal] Kmp R.B. (1998) Nonscanning calorimtry, in Handbook of thrmal analysis and calorimtry, Vol. 1 Principls and practic (Ed: M. Brown) Elsvir, Amstrdam. [Th work provids comprhnsiv ovrviw about th rcnt advancs in biocalorimtry] Kmp R.B. (2000) Gi m a spark o natur s fir an insight into cll physiology from calorimtry, Journal of Thrmal Analysis and Calorimtry 60, [This rviw shows how physiological insights into cll mtabolism can b gaind by calorimtry] Maskow T, Lrchnr J, Pitzsch M, Harms H, Wolf G (2006) A miniaturizd chip-calorimtr for th monitoring of whol cll biotransformation. Journal of Biotchnology 122, [Th papr dscribs th first chip-calorimtr potntially applicabl at tchnical scal] Mukhanov V.S., Kmp R.B. (2006). Simultanous photocalorimtric and oxygn polarographic masurmnts on Dunalilla maritima clls rval a thrmal discrpancy that could b du to nonphotochmical qunching. Thrmochimica Acta 446, [This work dscribs photocalorimtry at ml-scal] Torrs F.E., Kuhn P., d Bruykr D., Bll A.G., Wolkin M.V., Ptrs E., Williamson J.R., Andrson G.B., Schmitz G.P., Rcht M.I., Schwizr S., Scott L.G., Ho H.J., Elrod S.A., Schultz P.G., Lrnr R.A., Bruc R.H. (2004) Enthalpy arrays. Procdings of th National Acadmy of Scincs USA, 101, [Th work rports th fabrication of miniaturizd nthalpy arrays and thir us to dtct molcular intractions, including protin ligand binding, nzymatic turnovr, and mitochondrial rspiration.] Voisard D., Pugaud P., Kumar A.R., Jnny K., Jayaraman K., Marison I.W., von Stockar U. (2002) Dvlopmnt of a larg-scal biocalorimtr to monitor and control bioprocsss. Biotchnology and Bionginring 80, [This study show first tim how th Mgacalorimtry principl works in practic] von Stockar U., Gustafsson L., Larsson C., Marison I.W., Tissot P., Gnaigr E. (1993) Thrmodynamic considrations in constructing nrgy balancs for cllular growth, Biochimica t Biophysica Acta 183, [Th work shows how an nrgy balanc around a bioractor has to b constructd and how th data can b xploitd for analyzing and controlling th procss] von Stockar U., Marison I.W. (1991) Larg-scal calorimtry and biotchnology, Thrmochimica Acta 193, [This work dscribs th basics of Mgacalorimtry] Biographical Sktchs Thomas Maskow, born in 1962, studid Thortical and Physical Chmistry at th Tchnical Univrsity Carl Schorlmmr Luna-Mrsburg ( ). H achivd his Doctoral dgr at th Martin- Luthr Univrsity Hall-Wittnbrg for th dvlopmnt of nw analytical tools and thrmodynamic modls to charactriz th phas bhavior of crud oil or oil fractions undr guidanc of Prof. H. Khln and Prof. M. Rätzsch. Aftrwards h dirctd th rsarch division of a mdium sizd company (R. Myr Laboranalysn und Umwlttchnik GmbH Zitz) daling with analytical srvics, costumrspcific dvlopmnts and functioning as consulting nginr in th nvironmntal fild ( ) h was offrd th chanc to stablish and dvlop biocalorimtry and biothrmodynamics at th UFZ Hlmholtz Cntr for Environmntal Rsarch. His work was strongly supportd by th xcutivs of th Dpartmnt of Environmntal Microbiology (Prof. W. Babl/Prof. H. Harms). In addition to activ rsarch in th filds of bioprocss control, xtrmophils and strss biology h tachs biotchnology and biophysics at th narby univrsitis (Lipzig and Drsdn) and 2004 h was invitd to work as gust scintist at th Institut of Biological Scincs, Univrsity of Wals, Abrystwyth, UK (laboratory of th coauthor) and at th Institut of Chmical Enginring, Swiss Fdral Institut of Tchnology Lausann (EPFL), Switzrland (Laboratory of Prof. U. von Stockar) h qualifid for univrsity lcturr (Habilitation). H taks activ part in diffrnt boards such as Bioprocss Tchnology, Biothrmodynamics and Forum Biotchnology at th DECHEMA (Socity for Chmical Enginring and Biotchnology) and in th Intrnational Socity of Biocalorimtry (ISBC). Sinc 2006 h is a mmbr of th advisory board of th ournal Enginring in Lif Scincs.

10 Richard Kmp, born in 1941, graduatd with a B.Sc. (Hon) in Zoology from London Univrsity (Kings Collg) in 1963 bfor undrtaking postgraduat studis in Cll Biology undr Prof. Bryn Jons at th Univrsity of Wals, Abrystwyth, obtaining a PhD in Aftr priods of postdoctoral rsarch at th Univrsity of Uppsala, Biochmistry Dpartmnt with Profssor J. Hrtén (1966) on th lctrophortic sparation of protins and th Univrsity of Cambridg, Dpartmnt of Radiothraputics with Dr G.V.F. Saman (1967) on cll lctrophorsis, h rturnd to Abrsytwyth as th Scintific Rsarch Council Assistant Dirctor of Rsarch in Cll Biology in 1967, principally working on cll-cll adhsion. H has rmaind thr vr sinc firstly as a Lcturr (1971), thn as a Snior Lcturr (1983) and finally as th Radr in Cll Physiology and Had of th Cll Biology Laboratory (1986). H has had priods of sabbatical lav at th Dpartmnt of Biophysics, Univrsity of Kyoto, Japan with Profssor T.S. Okada on cll adhsion (1972), th John Curtin School of Mdicin, Canbrra, Australia with Dr W. Fild-Smith on th nrgtics of th adhsion procss (1983), th Dpartmnt of Zoology, Univrsity of Innsbruck, Austria with Profssor Erich Gnaigr on th calorimtric masurmnt of mtabolism in mammalian clls (1991) and th Dpartmnt of Biochmistry, Russian National Acadmy of Scincs, Kazan, Russsia with Profssor Lv Gordon on th photocalorimtric masurmnt of photosynthsis in microalga (2000). Ovr th yars, his scintific rsarch has shiftd from cll adhsion to cllular nrgtics. It is for his work on th lattr that h was lctd Fllow of th UK Institut of Biology, 1989, was awardd th Lavoisir Mdal of th Intrnational Socity for Biological Calorimtry, 1992, and gav th NATAS Award lctur at th 11th Intrnational Congrss of th Intrnational Socity for Thrmal Analysis and Calorimtry, Philadlphia, USA, Thrmochimica Acta Volum 394 as a fstschrift was ddicatd to him on th occasion of his 60th birthday, 340pp., and publishd xactly on yar latr (19 Octobr 2002). Currntly, h is working on th calorimtric masurmnt of mtabolism in th bioprocsss of clls ranging from prokaryotic microorganisms to animal clls, but principally using photocalorimtry to study th photosynthtic procss in microalga at both th analytical and biotchnological scals.