In Situ Real-Time Monitoring for Aseptic Drilling: Lessons Learned from the Atacama Rover Astrobiology Drilling Studies Contamination Control Strategy and Implementation and Application to the Icebreaker Mars Life Detection Mission

Open came a o QR eade and scan code o access his a icle and o he esou ces online. In Si u Real-Time Moni o ing o Asep ic D illing: Lessons Lea ned om he A acama Ro e As obiology D illing S udies Con amina ion Con ol S a egy and Implemen a ion and Applica ion o he Iceb eake Ma s Li e De ec ion Mission Rosalba Bonacco si, 1,2 B ian Glass, 2 Me cedes Mo eno-Paz, 3 Mi iam Ga cı´a-Villadangos, 3 Kimbe ley Wa en-Rhodes, 1,2 Vic o Pa o, 3 Juan Manuel Manchado, 3 Ma y Be h Wilhelm, 2 and Ch is ophe P. McKay 2 Abs ac In 2019, he A acama Ro e As obiology D illing S udies (ARADS) p ojec ield- es ed an au onomous o e - moun ed obo ic d ill p o o ype o a 6-Sol li e de ec ion mission o Ma s (Iceb eake ). ARADS d illed Ma s- like ma e ials in he A acama Dese (Chile), one o he mos li e-diminished egions on Ea h, whe e mi iga ing con amina ion ans e in o li e-de ec ion ins umen s becomes c i ical. Ou Con amina ion Con ol S a egy and Implemen a ion (CCSI) o he Sample Handling and T ans e Sys em (SHTS) ha dwa e (d ill, scoop and unnels) included ou -o -simula ion p o ocol es ing (ou -o -sim) o ha dwa e decon amina ion and e i ica ion du ing he 6-Sol simula ion (in-sim). The mos e ec i e i e-s ep decon amina ion combined sa e - o-use s e ilan s (3%_hyd ogen-pe oxide-ac i a ed 5%_sodium-hypochlo i e), and in si u eal- ime e i ica ion by adenosine iphospha e (ATP) and Signs o Li e De ec o (SOLID) Fluo escence Immunoassay cha ac e iza ion o ha dwa e biobu den and ai bo ne con aminan s. The 20- o 40-min p o ocol enabled a 4-log biobu den educ ion down o <0.1 moles ATP de ec ion limi ( unnels and d ill) o 0.2–0.7 moles (scoop) o o al ATP. The (pos -cleaning) ha dwa e backg ound was 0.3 o 1–2 a omoles ATP/cm 2 (cleanliness benchma k backg ound alues) equi alen o ca. 1–10 colony o ming uni (CFU)/cm 2 . Fu he , 60–100% o he in-sim ha dwa e back- g ound was £3–4 bac e ial cells/cm 2 , he h eshold limi o Class <7 asep ic ope a ions. Ac oss he six Sols, he lux o ai bo ne con aminan s o he d ill si es was *5and*22 amoles ATP/(cm 2 $day), accoun ing o an unex- pec edly high Fluo escence In ensi y (FI) signal (FI: *6000) agains aqua ic cyanobac e ia, bu negligible an- h opogenic con ibu ion. The SOLID immunoassay also de ec ed mic oo ganisms om mul iple habi a s ac oss he A acama Dese (anoxic, alkaline/acidic mic oen i onmen s in hali e ields, playas, and allu ial ans) in bo h ai bo ne and pos -cleaning ha dwa e backg ound. Finally, he ha dwa e ATP backg ound was 40–250 imes lowe han he ATP in co es. Simila ly, he FI peaks (FI max ) agains he mic obial axa and molecula bioma ke s de ec ed in he pos -cleaned ha dwa e (FI: *1500–1600) we e 5–10 imes lowe han bioma ke s in d illed sedimen s, excluding signi ican in e e ence wi h pu a i e bioma ke ound in co es. Simila p o ocols enable he acquisi ion o con amina ion- ee ma e ials o ul a-sensi i e ins umen s analysis and he in eg i y o scien i ic esul s. Thei applica ion can augmen ou scien i ic knowledge o he dis ibu ion o c yp ic li e on Ma s-like g ounds and 1 SETI Ins i u e, Moun ain View, Cali o nia, USA. 2 NASA Ames Resea ch Cen e , Mo e Field, Cali o nia, USA. 3 Depa men o Molecula E olu ion, Cen o de As obiologı ´a (INTA-CSIC), Mad id, Spain. Rosalba Bonacco si e al., 2023; Published by Ma y Ann Liebe , Inc. This Open Access a icle is dis ibu ed unde he e ms o he C ea i e Commons License (h p://c ea i ecommons.o g/licenses/by/4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly c edi ed. ASTROBIOLOGY Volume 23, Numbe 12, 2023 Ma y Ann Liebe , Inc. DOI: 10.1089/as .2022.0133 1303 suppo li e-de ec ion obo ic and human-ope a ed missions o Ma s. Key Wo ds: A acama—Mic obial educ ion— Real- ime ATP assay—Robo ic d illing—Li e de ec ion—Ma s’Iceb eake Sample Handling and T ans e Sys em (SHTS)—A acama Ro e As obiology D illing S udies (ARADS). As obiology 23, 1303–1336. 1. In oduc ion Unlike Ea h, he ma ian su ace appea s li eless, wi h he subsu ace p oposed as he bes a ge o de ec ing li e. The A acama Ro e As obiology D illing S udies (ARADS) sys em is a p o o ype o a u u e li e de ec ion d ill mission o Ma s, namely he cu en ly planned Ice- b eake Mission (Da e ´e al., 2013; McKay e al., 2013; Glass e al., 2014; Mann, 2018). ARADS pe o med ield ials in he hype -a id egion o he A acama Dese in he An o a- gas a Region om 2016 o 2019 (Fig. 1), culmina ing in a 6-Sol end- o-end mission simula ion. As obiology obo ic d illing missions on Ma s will ace b oad issues bo h in o wa d plane a y p o ec ion (PP) om e es ial mic obes su i ing s e iliza ion o he spacec a ’s ha dwa e, which is he p ima y conce n (e.g., NAS, 2020; NAS, 2019; NAS, 2018; NRC, 2006) and in he in eg i y o sample analysis. The Commi ee on Space Resea ch (CO- SPAR) PP policy o landed missions in a Ma ian Special Region (Ca ego y IVc PP, p obabili y con amina ion: <10 -4 ) is he mos s ic equi ing ha dwa e biobu den con amina ion con ol o o wa d con amina ion (COSPAR, 2020). Spo es can emain long- e m iable in space (e.g., Ho - neck e al., 2010, 2012; Hallswo h, 2021), be shed on he su ace (Ma shall and Mancinelli, 2011), and en e he unde g ound, becoming me abolically ac i e unde inc ea- sed wa e ac i i y (Rummel e al., 2014; Smi h e al., 2017). A seconda y conce n is mi iga ing he isk o e es ial con aminan s ans e (spo es, amino acids) in o sample analysis gene a ing alse posi i es—li e appea s o be de ec- ed when he e is no —(Kminek e al., 2019), o p o ec ing science da a in eg i y. Human-ope a ed con amina ion emedia ion is a p ima y s ep o obo ic d illing mission planning, beginning wi h clean oom ope a ions du ing Spacec a Assembly, Tes , and Launch Ope a ions (ATLO). The human in ol emen con inues du ing obo ic d illing mission mock-ups, as he hands-o mission echnology eadi- ness is no ye ma u e, equi ing ield enginee s o supe ise FIG. 1. (A) ARADS 2019 S udy Si es. (B) Playa es si e (La . 2406¢08.3†S o Long. 7008¢17.5†W) abou 22 km om he ield suppo headqua e , he Yungay S a ion. (C) Si e loca ion o ‘‘G een Pa o ’’ (GP) La . 245¢37.62†S; Long. 6954¢20.86†W). ARADS, A acama Ro e As obiology D illing S udies; GP, G een Pa o . 1304 BONACCORSI ET AL. decon amina ion (Glass e al., 2023, As obiology his issue). Thus, de eloping and ield- es ing human- ended cleaning e- gimes o payload p o o ypes add essing PP knowledge gaps is manda o y e en o a obo ic mission (e.g., NASA, 2011) and can be ad anced by ield ials such as ARADS. We de eloped ou Con amina ion Con ol S a egy and Im- plemen a ion (CCSI) o e he 4-yea p ojec and in eg a ed lessons lea ned om se e al as obiology and echnology ields d illing simula ions, o example, he 2005 MARTE P ojec a he Rio Tin o Pen ˜a de Hie o, Spain (Glass e al., 2006; Bo- nacco si and S oke , 2008; Mille e al., 2008; Pa o e al., 2008; S oke e al., 2008; Eigenb ode e al., 2009; Amado e al., 2014; Cockell e al., 2018). These es s p o ided oppo uni ies o assess and mi iga e long- and sho - e m biobu den buil -up a *6and 12 mon hs and e alua e cleaning p o ocols o e-con amina ed deep-cleaned ha dwa e ha dwa e a e 1 day o use. This pape desc ibes ield- easible and e ec i e p ac ices o con amina ion isk educ ion du ing he ARADS mission and consis s o se en sec ions. Sec ion 1 in oduces ARADS sample d illing handling and ans e ha dwa e. Sec ion 2 in o ms spacec a ha dwa e con- amina ion con ol. Sec ion 3 ou lines an h opogenic, en i on- men al, and c oss-con amina ion. Sec ion 4 desc ibes (1) he gene al con amina ion emedia ion (du ing o -hou s o e , ins umen main enance, e c.); (2) mul i-s ep cleaning, mic o- bial educ ion, and e i ica ion o he d ill, scoop, and unnels; (3) mul i-s ep p o ocols implemen a ion o in-sim ope a ions; and (4) sou ce- o-sink con amina ion o ai bo ne, sedimen a y, and an h opogenic biomass o he d ill si es. Sec ion 5 epo s he 2019 ARADS esul s. Sec ion 6 discusses alse nega i es in cleaning e i ica ion and en i- onmen al and c oss-con amina ion issues unique o e es- ial ials o a li e-de ec ion d illing mission o Ma s. Sec ion 7 highligh s inno a i e elemen s s emming om ARADS: (1) an e ec i e simpli ied decon amina ion p o- ocol, (2) a sou ce- o-sink con amina ion app oach, (3) a e- assessmen o con amina ion isks in he A acama Dese , (4) he no el use o he immunoassay-based li e de ec- ion ool (Signs o Li e De ec o [SOLID]) o iden i ying o wa d-con aminan s on Ma s, and (5) ha dwa e design o he a m-moun ed scoop analog o he Phoenix soil acquisi ion de ice o mi iga e c oss-con amina ion. 1.1. In oduc ion o ARADS The ARADS p ojec was pa o NASA’s Plane a y Science and Technology Th ough Analog Resea ch (PSTAR) P og am. This p ojec explo es he science, ins umen a ion, and au oma- ion in pe o ming a nea su ace ( o 1 m dep h) d ill ope a ion on Ma s, using he hype -a id A acama (Cab ol e al., 2007; McKay e al., 2003; Na a o-Gonzalez e al., 2003; Cab ol e al., 2007) as an analog d ill si e (e.g., Glass e al., 2019, 2022; Mo eno-Paz e al., 2019; S oke e al., 2023). The inal ARADS ield cam- paign concluded in Sep embe 2019 wi h a 6-day simula ed mission a a no el ‘‘landing si e’’ he ‘‘Playa’’ (Fig. 1), selec ed o mul iple g ound- u h ield s udies (Glass e al., 2023, his issue). The ARADS CCSI cons i u ed an in eg al pa o he o e ield deploymen in bo h in-sim and ou -o -sim ope a ions. The e a e ou majo subsys ems o he ARADS payload: (1) The NASA Ames’ sel -na iga ing midsize o e (KREX-2) as he mechanical in eg a ion pla o m o he 1 m TRIDENT (The Regoli h and Ice D ill o Explo a ion o New Te ains) d ill and he o he sub- sys ems hos ed by he Ro e payload deck (Fig. 2A). (2) The Honeybee Robo ics’ o a y-pe cussi e D ill, whose s ing spi al coils can e ie e ca. 10–60 g o geological cu ings om e e y 25 cm o d illed sub- su ace (Fig. 4C). (3) The Sample Handling and T ans e Sys em (SHTS) in- cludes an a m-moun ed scoop analog o he Phoenix Icy Soil Acquisi ion De ice (ISAD) shown in Fig. 2D (Bo- ni z e al., 2008; Glass e al., 2023) and ou unnels on he op o he o e payload deck, p o ec ing he science payload unde nea h (Fig. 2A): h ee *12 cm diame e ins umen - eeding unnels, and one 13 cm diame e one (aka ‘‘Big_Fun’’) o deli e o e low samples in o a s o age uni o pos - ield analysis (Fig. 2C). (4) The science ins umen s included (i) The Signs o Li e De ec o (SOLID) o p o o ype, buil by he Spanish Cen o de As obiologia (CAB) (Pa o e al., 2008) using luo esce mic oa ay immunoassay de- ec ion o 450 biomolecula compounds (amino acids, nucleic acids, p o eins, and polysaccha ides) a 1–2 pa s pe billion [ppb (ng/mL)] and whole cells, ha is, 10 4 –10 5 cells/mL (Ri as e al., 2008); (ii) he JPL’s Chemical Lap op- o me ly Mic o luidic Li e Analyze (MILA) (Willis e al., 2015; Mo a e al., 2020); (iii) a b assboa d e sion o he Phoenix’s We Chemis y Labo a o y (WCL) o sal chemis y analysis; and (i ) he NASA Godda d Space Fligh Cen e ’s Linea Ion T ap Mass Spec ome e , o LITMS o o ganic compounds de ec ion and iden i- ica ion, which was ield es ed Ma ch 2019 (Cas illo e al., 2023). Nex , we desc ibe he sample handling and ans e o hese ins umen s. 1.2. Li e jou ney o a d illed sample The ARADS’s ully au oma ed o e /d illing/sampling/ analysis sys em aimed a d i ing o loca ions o scien i ic in e es and acqui ing geological ma e ial o geochemical and biological s udy. The sample cap u e occu s in h ee s eps. The s ainless-s eel d ill s ing, nes ed inside he sys- em’s p ima y d ill, accesses he dep h o science in e es and e ie es ca. 10–60 g o powde ed cu ings. Be o e he sed- imen is b ough o he su ace, he a m-moun ed scoop lowe s o he g ound o ecei e he d illed ma e ial (Fig. 2B). Once su icien powde ed sedimen om he a ge dep h en e s he scoop, his ises abo e he o e deck (Fig. 2A) o deli e ma e ial in o he ins umen s’ unnels (Fig. 2C). Each unnel d ops he cu ings in o 1 cm diame e holes aligned wi h he en y po o he analy ical ools benea h he o e payload deck (no isible). Finally, he powde le in he scoop is d opped in o he ou h, la ge unnel (aka ‘‘Big_Fun’’) and ans e ed in o a p e-cleaned bo osilica e glass ja as in p e ious ARADS campaigns (Wilhelm e al., 2018). The unnel-ja uni la ches on o he o e payload deck op (Fig. 2A). The o e ins umen sui e immedia ely analyzes he d opped samples. Al e na i ely, he ins umen is de ached, b ough in o he labo a o y aile , and analyzed immedi- a ely by he science eam. Only one ins umen eam sci- en is can wo k a he dedica ed sani ized lab bench o minimize con amina ion du ing sample analysis. ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1305 Finally, samples in ja s a e s o ed in a clean box o la e sub-sampling and analysis o he a chi ed co ed ma e ials on eques by he science eam. Fo his pu pose, we ope - a ed on he clean bench op using s e ilized s ainless-s eel spa ulas in he dedica ed science aile (Fig. 9A). We excluded a ew low-p io i y d illed in e als om he sim- ula ion o main ain mission ideli y. Howe e , unlike on Ma s, we did no disca d hese low-p io i y samples om he d ill bu manually collec ed hem in labeled s e ile Whi l-Pakbags o la e use by he science eam. 1.3. Rele ance o con amina ion con ol Cleaning, mic obial educ ion, and e i ica ion we e un- damen al o ARADS. Unlike he case o human and obo ic explo a ion o Ma s Special Regions, he o wa d con ami- na ion o he e es ial poly-ex eme A acama Dese poses no eal conce n. Howe e , because he hype -a id su ace o his egion is p edominan ly de oid o mic obes, ins u- men s o de ec molecula indica o s o pu a i e li e mus ope a e unde low de ec ion h esholds (high sensi i i y). E en mino biobu den con amina ion du ing sample acqui- si ion and ans e in o analysis could con amina e scien i ic esul s and con use in e p e a ions. Biobu den is he numbe o mic oo ganisms on he non-s e ile su ace ma e ial. S e - iliza ion e e s o chemical o physical p ocedu es ha kill, deac i a e, o emo e all li e- o ms (i.e., MHRA, 2006). A e minally s e ilized su ace de ice is ‘‘s e ile’’ i he he- o e ical p obabili y o a iable mic oo ganism being p esen is equal o o less han 1 ·10 -6 (Lambe e al., 2021). NASA p o ocols in ol e d y hea o mic obial educ ion (DHMR) and chemical me hods when DHMR is no a ainable. Howe e , long- e m absolu e s e ili y is unachie able o is comp omised a e a ew-minu e exposu e o a con amina ing en i onmen . Fu he , ha dy mic oo ganisms can esis chemical ea - men in clean ooms (Ho neck e al., 2012; Moissl-Eichinge e al., 2015; Sp y e al., 2021) and e en use o ganic com- pounds in cleaning eagen s as an ene gy sou ce (e.g., Mogul e al., 2018). Field ials in ex eme en i onmen s lack clean- oom acili ies, and con inuous human- ended ope a ions en- hance he isk exposu e o biobu den con amina ion o he ha dwa e payload and sample analysis pa hways. FIG. 2. SHTS. (A) Payload deck’s op hos ing he h ee sample deli e y unnels and he la ge unnel (back- g ound) o he ou low sample ans e o he glass ja (no in place); (B) low- e ed scoop ecei ing d illed sample; (C) scoop abo e payload deck deli e - ing cu ings o he unnel eeding he SOLID ins umen below (no isible). The glass ja is isible in he back- g ound. (D) Phoenix ISAD design simila o ARADS scoop (Pho o c edi : NASA). ISAD, Icy Soil Acquisi ion De ice; SHTS, Sample Handling and T ans e Sys em; SOLID, Signs o Li e De ec o . 1306 BONACCORSI ET AL. The isk is enhanced unde a ime-cons ained schedule o decon amina ion. Each Sol, he ARADS simula ion ini- ially alloca ed only a 20-min ope a ional window o he mic obial educ ion and e i ica ion cycle, making c i ical he de ising o a ield- easible and ime-e ec i e decon- amina ion p o ocol (See de ails in Sec ion 4). 2. Con amina ion Con ol P ac ices o Spacec a Ha dwa e Du ing he ARADS campaigns, we ollowed s a e-o - he- a con amina ion con ol p ocedu es ypical o hose o p epa e o Ma s landed missions. The modi ica ions i he equi emen s posed by he e es ial ield ial. Ca osso (2005) o e ed comp ehensi e backg ound in o ma ion on spacec a decon amina ion and e i ica ion echniques sha ed wi h ecen missions a ge ing plane a y samples. Examples include he 2007 Phoenix Mission and o he missions o Ma s such as he 2011 Ma s Science Labo a o y (MSL) (Maha y e al., 2004) and he 2020 missions cen- e ed on he analysis o molecula o ganic compounds, and he 2016 OSIRIS Rex o he ca bonaceous nea -Ea h as e oid Bennu (Lau e a e al., 2017; Dwo kin e al., 2018). Al hough hese missions we e expec ed o ad ance decon amina ion p ocedu es o sampling and sample han- dling mechanisms, hey ha e ye o p og ess beyond he 2007 Phoenix Lande expe ience due o cos caps and slow ad ances in ma e ial echnology (Mille e al., 2008). The Phoenix Mission, ope a ed on Ma s om 2008 o 2009, o e s s a e-o - he-a con amina ion con ol o spacec a ha dwa e analog o ARADS. The Lande ’s obo ic a m-moun ed digging scoop o ISAD collec ed and deli e ed ice-bea ing egoli h samples o he The mal E ol ed Gas Analyze (TEGA) o de e mine indica o s o habi able condi ions in Ma s ice-cemen ed g ound, equi ing s ic con amina ion con ol (10 ppb) on he digging scoop o p o ec he scien i ic esul s om Ma s. Once assembled, he compa men alized scoop su - aces we e inaccessible o cleaning, challenging c oss- con amina ion con ol be ween samples scooped om he ma ian soil. Unlike he 1975–1976 NASA’s Viking lande s, which we e hea ea ed in hei en i e y (e.g., So en and Snyde , 1976), he Phoenix Mission sampling chain (blades and scoop) main ained only low o ganic con amina ion Le el 75 A/10 equi emen , ha is, 100 ng/cm 2 (Bo son, 2005). Con ol and assessmen o con amina ion on he Phoenix TEGA included acuum bake-ou /d y-hea s e iliza ion, con ainmen o anspo and es ing, and con amina ion cha ac e iza ion on an enginee ing mock-up ligh model. Du ing Assembly, Tes , and Launch Ope a ions (ATLO) ligh ha dwa e in an ISO 14644-1 Class 8 con olled en- i onmen , biobu den equi emen is £10 CFU/m 3 (ai samples), 2000 CFU/m 2 (su aces), and £1 CFU/ 5- inge glo e p in (ECSS, 2008a, b). Du ing asep ic op- e a ions (Class <7), ha dwa e assembly occu s on lamina low unde a s ic e NASA S anda d assay (NSA)-based biobu den le el o <1 CFU/m 3 (ai ), £300–400 CFU/m 2 (su aces), and <1 CFU (pe glo ep in ). Assuming ha he NSA can de ec only 1% o he o al iable and non- iable bu den, 400 CFU/m 2 co esponds o he h eshold o asep ic ope a ions o £40,000 mic obial cells/m 2 (o £3–4 cells/cm 2 ) achie able by ARADS. 3. Sou ces o Con amina ion Whe he o no a o eign ma e ial is a ‘‘con aminan ’’ depends on he objec i e o he planned analysis and only i he ma e ial in e e es wi h he ins umen s’ measu emen . Fo eign cellula ma e ials and o ganic bioma ke s de ec ed in ARADS co es and ha dwa e a e con aminan s. ARADS con amina ion con ol (Sec ion 4) a ge ed an h opogenic, en i onmen al, and c oss-con amina ions while add essing hei isk le el be o e and a e mi iga ion. (1) An h opogenic con amina ion by human- ended au oma ed d illing es s is una oidable and a signi i- can conce n o biologically deple ed e es ial and ma ian en i onmen s. In A acama’s biologically lean su ace en i onmen , ai bo ne mic oo ganisms a e lesse conce ning due o ai low dispe sion. Con- e sely, an h opogenic mic obiome con amina ion o soil and ai ia handling, clo hing, and skin pa icles du ing daily ope a ions can comp omise esul s om he li e-de ec ion sui e. (2) En i onmen al con amina ion is a unique p oblem in e es ial ials, wi h h ee po en ial sou ce en ies in o he sample pa h: (i) non-pu a i e biological ( iable spo es, mic obes, cellula emains); (ii) ossil molecula o ganics de ec able by LITMS and SOLID; and (iii) ai bo ne mine al dus and sal s de ec able by he we chemis y ins umen . The e is a simila oppo uni y o ai bo ne ino ganic con- amina ion on he ma ian su ace, which is an un- likely sou ce o biological con aminan s. Ai bo ne dus om he as oligo ophic dese pa emen o d ill si es was ini ially no o g ea conce n wi h bio ba ie s o p o ec sample-handling elemen s when no in use. S ill, ai bo ne biology esul ed in an unexpec ed po en ial con aminan o ARADS (Sec ion 6.2). (3) C oss-con amina ion could occu be ween di e en sedimen a y s a a d illed as powde ed samples and mixed om he a ge dep h; once in he scoop, a new sample could mix wi h sedimen esidue adhe ing o he inside om p e iously d illed in e als. 4. Me hods o Con amina ion Con ol We desc ibe by-design, ope a ional, and con amina ion con ol echniques we es ed in ARADS. 4.1. Con amina ion emedia ion by design and ope a ional me hods To emedia e c oss-con amina ion, we used s ainless-s eel o aluminum sampling ha dwa e, a oided so ma e ials and polyme s poo ly conduci e o ield decon amina- ion, and chose single-use p e-cleaned s ainless s eel un- nels and clean-ce i ied bo osilica e glass ja s. The sample acquisi ion and handling sys em design also enabled con- aminan dilu ion by accep ing a ela i ely la ge sample size, up o 60 g, compa ed wi h he expec ed esidual c oss- con aminan s (Fig. 12). The gene al con amina ion emedia ion ope a ions inclu- ded p o ec i e shields usage and d ill ope a o s aining guidelines ha in ol ed: (1) mind ul walking in ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1307 app oaching he o e downwind o a oid s i ing dus om he playa pa emen (Fig. 3J); (2) only wo ope a o s ending he d ill in-sim (Fig. 3H); (3) use o acemasks and sani ized glo es (e hanol [E -OH]-wiped) when ap- p oaching ha dwa e a close con ac wi h samples o wo king on he ins umen s ou le s; (4) hand-sani iza ion be o e wea ing glo es; (5) ouch no c i ical equipmen su aces and ools o con ac wi h sampling su aces wi h un-sani ized o con amina ed glo es; (6) a oid con ac be ween he clean glo es and body’s su ace, o any comp omised su ace; (7) use o clean/s e ilized ools whene e possible; and (8) wea a p o ec ion sui when app op ia e (Fig. 3F). We used p o ec i e media o p oac i e con amina ion con ol be o e, du ing, and be ween ope a ions. Fo ins- ance, be o e assembly on he o e deck, we anspo ed double-w apped cleaned unnels in sani ized plas ic con- aine s (S e ili e TM) wiped down wi h high-pu i y iso- p opyl alcohol (IPA). While he o e mo ed owa d he d illing si e, we p o ec ed he cleaned d ill, scoop, and unnels wi h hea y-du y Ul a High Vacuum (UHV) Aluminum Foil (AllFoil, Inc.), which was emo ed only be o e use. Be ween each d illing cycle, we kep he bio-ba ie in place un il he d ill was eady o ope a e (Fig. 4C), he scoop was abou o accep d illed samples, and he unnels could ecei e and ans e samples o analysis. We main ained a con amina ion- ee sample pa h o each li e de ec ion ins umen . Fo ins ance, he SOLID’s inle al e opened only be o e he sample’s elease in o he analy ical ool (Pa o e al., 2008). A he end o each Sol, he o e -moun ed d ill s ing and scoop we e insed o wi h dis illed wa e (DW), w apped in Al oil, and le onsi e o e nigh . The unnels we e unclamped om he o e deck, w apped in Al oil, and anspo ed o he Yungay S a ion (La . 244¢53.05’’S, Long. 6955¢9.51’’W) o a new cleaning cycle be o e he successi e Sol’s ope a ions. Each day, he o e -moun ed d ill s ing was ‘‘ ouched up’’ and cleaned (done on p e i- ously deep-cleaned su aces) be o e use. 4.2. Chemical cleaning and disin ec ion We es ed he deep-cleaning e ec i eness on wo Ice- b eake d ill segmen s: A (d ill ip, 0–25 cm) and C (40– 60 cm) in Tes 2 (Ma ch) and segmen B (20–40 cm) in Tes 3 (Sep embe ) oge he wi h he sample handling ha dwa e elemen s (Tes 4). We used chemical p ecision cleaning, a co e componen o bo h Quali y Assu ance o obo ic landed missions o Ma s and PP (e.g., F ick e al., 2014). In 2019, we did no use hea s e iliza ion because su - aces could be easily e-con amina ed in he ield, and decon amina ion cycles would equi e addi ional ime. Fu he , hea s e iliza ion would no emo e o ganic con- aminan s. Tables 1 and 2 summa ize he chemical cleaning FIG. 3. (A) Disassembled d ill s ing, scoop, and unnel p epa ed o ield deep cleaning and es ing; (B) p elimina y s ep (dis illed wa e inse) o emo ing pa icula e ma e ial om he d ill s ing; (C) ATP swab assay o one ins umen ’s unnel ha dwa e; (D) Su ace Swab Assay samples o SOLID co-analysis; (E) ATP molecule (le ) and hand-held lu- minome e wi h liquid (le ) and su ace ( igh ) sampling de ices. ATP, adenosine i- phospha e. 1308 BONACCORSI ET AL. and mic obial educ ion s eps we applied; hei speci ic e ec i eness o ARADS is add essed in Sec ion 5 and Figs. 6A–H and 7. 4.2.1. Cleaning and disin ec ing agen s. The p ecision cleaning in ol ed (1) adenosine iphospha e (ATP)- ee dis illed wa e (DW) o liquid ch oma og aphy/mass spec- ome y (LC/MS) g ade wa e o emo al o isible di (adhe ing clays and dus pa icles); (2) sol en s wi h de- c easing pola i y (pu e g ade ace one, me hanol, and E -OH) o emo e he a oma ic and alipha ic molecula o ganics; and (3) mic obial educing agen s, o example, 60–80% E - OH, high-pu i y IPA, 0.5–5% household bleach (NaClO; esh ba ch, unopened bo le as di ec sunligh and o ganic ma e inac i a e NaClO), and 3% hyd ogen pe oxide (H 2 O 2 ) o dis up ing bac e ial bio ilms and o inac i a e/kill mic oo ganisms. NaClO and H 2 O 2 we e applied indi idually o simul a- neously. We used a ious dispensing me hods and mul iple applica ions o maximize he chemical exposu e o he a ge ha dwa e, o example, bleach-soaked (imp egna ed) poly- p opylene wipes w apped a ound he s ing’s su ace, wi h w aps molded a ound he d ill’s spi als o 1 h. A e wa d, he d ill was wiped o , ai -d ied, and ho oughly insed wi h ca. 0.5–1 L o ATP- ee DW. 4.2.2. Dispensing and wiping. We adap ed echniques based on ha dwa e opog aphy; in some cases, we used p o- essional sp ay bo les o mis , sp ay, and s eam each che- mical. S ill, highly ola ile sol en s e apo a e quickly om he a ge ha dwa e, dec easing hei cleaning e ec i e- ness. In some cases (d ill deep cleaning), we p e e ed s oke-wiping ha dwa e wi h lin - ee anhyd ous sol en - imp egna ed wipes o emo e con aminan s ins ead o di ec sp aying on o he ha dwa e. The s anda d Quali y Assu ance o obo ic landed mis- sions o Ma s equi es pe iodic p ecision cleaning o expo- sed ligh ha dwa e su aces wi h high-pu i y IPA-soaked lin - ee swabs and wipes (e.g., Ca osso, 2005). We im- p egna ed wipes (by sp aying su icien eagen s on o hem) o he mos e icien disin ec ion (Panousi e al., 2009). In o he cases (e.g., unnel’s na ow ou le s), we nebu- lized aqueous IPA se e al imes o maximize exposu e o he disin ec ing agen . Finally, a las i e a ion wi h (IPA)- soaked low-lin Kimwipes (KIMTECH KimWipes Task Wipe s) emo ed cellula emains om he su ace o max- imum disin ec ion. The wiping bes p ac ice we applied in ol ed consis en p essu e on e e y s oke, wo king om op o bo om ( o he d ill s ing) and ‘‘pull and li ’’ om he in e io (cleanes ) o he ex e io (di ies ) a eas ( o scoop and unnels). FIG. 3. (Con inued) (F) Mi iga- ion con amina ion du ing ARADS 2016 g ound u h sampling o soil pi s (Pho o c edi : NASA Ames); (G) con ainmen se up a Yungay Base- camp be o e ield- es ing o he NASA Godda d’s Ion T ap Mass Spec ome e . (H–K) ARADS p ac- ices o con amina ion mi iga ion examples. See desc ip ion in Sec ion 4.1. (H) Reduced ield pe sonnel du ing o e ops; (J) minimized un- necessa y walking on playa owa d o e s; and moni o ed oo s eps on g ound si es o scien i ic in e es , ha is, pos - lood desicca ed bio ilms (K). ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1309 When olded, he wipes p o ide up o eigh clean, us- able sides o p e en e-con amina ion ac oss he cleaned su ace. 4.2.3. Wa e inse. A hi d s ep included ho oughly insing wi h a s eam o ATP- ee gas ch oma og aph–mass spec ome e (GC-MS) g ade wa e o he comple e emo- al o cleaning agen s, any Kimwipes’ ibe esidue, dead/ inac i a ed mic obes, and cellula cons i uen s. 4.3. Ha dwa e con amina ion e i ica ion assays We used wo independen con amina ion e i ica ion me hods: he ATP luminome y assay o iable o al bio- bu den and he Fluo escence Sandwich Mic oa ay FIG. 4. (A) Ou -o -sim biobu den con amina ion es s wi h he TRIDENT d ill in wo king posi ion; (B) Top: Iceb eake d ill segmen including he D ill bi (0–25 cm). Bo om: d ill Segmen 20–40 cm wi h a smoo he su ace inish han he D ill bi ; (C) moun ed d ill a e a 24-h exposu e o he GP en i onmen and Al oil bio-ba ie p o ec ing d ill be o e ield es ing; (D) po en ial sou ces o en i onmen al con amina ion om su ace biology a he GP Si e: Vascula plan biomass; and (E) ield o cyanobac e ia-colonized hali e. TRIDENT, The Regoli h and Ice D ill o Explo a ion o New Te ains. Table 1. Mul i-S ep P o ocol, as Desc ibed in Table 2 These s eps in ol e mechanical cleaning (1–5), sol en -based molecula o ganics emo al (6–8), physical and chemical mic obial educ ion/s e iliza ion (9–15), and p e en i e ac ions (16–17). Household bleach is 5.25% sodium hypochlo i e (NaClO). DW =dis illed/deionized wa e ; E -OH =e hanol; IPA =isop opyl alcohol; LC/MS =liquid ch oma og aphy/mass spec ome y; Me - OH =me hanol. 1310 BONACCORSI ET AL. Immunoassay (FSMI) o b oade cha ac e iza ion o mi- c obial axa and hei molecula and cellula con aminan s. Bo h echniques, desc ibed nex , in ol e a sol en swab assay o he pos -cleaning ex e io d ill s ing segmen s and he in e io su ace o he scoop and unnel. Bes p ac ices include a consis en swabbing echnique (ho izon al, e ical, and diagonal), di ec ional p essu e, and ime ca. 5 min (see e.g., Ba engol z, 2005). Swab samples equi e subsequen we ex ac ion o con aminan s analysis. Depending on he su ace oughness, we used wo swabs o e ie e he ATP om he ha dwa e su ace and combined he esul ing alues o assayed smalle su aces i co e ed by mine al dus . 4.3.1. ATP luminome y assay. We elied on he ATP Swab assay o he in si u eal- ime, p e-sc eening con amina ion isk- educ ion and disin ec ion ce i ica ion because all li ing o ganisms use ATP as he uni e sal en- e gy ca ie (Lundin and Tho e, 1975a, 1975b), and his molecule quickly deg ades a e cellula b eakdown (Holm- Hansen and Boo h, 1966). The assay is used o he biobu den moni o ing o spacec a ha dwa e in clean ooms (NASA P ocedu al Requi emen s 8020.12D; 2011, Venka eswa an e al., 2003). The Hygiena ATP assay sys em (Fig. 3E) includes a sel -calib a ing (25–28C) handheld Luminome e (En- SURE) wi h a dynamic ange o 0.1–2000 em omoles, ope a ing wi h an ul asensi i e ATP su ace swab (Supe - Snap), whose limi o de ec ion (LOD) is 0.1 ·10 -15 moles, o 0.1 moles ATP. The luminome e measu es he ligh emi ed by he i e- ly’s (Pho inus py alis) luci e in-luci e ase (L-L) enzyma ic eac ions (pH 7.6-bu e ed) binding wi h he ATP eleased by li ing cells in he p esence o oxygen and magnesium (Balkwill e al., 1988), p opo ionally o he ATP in he sample (Lundin and Tho e, 1975a; McEl oy and DeLuca, 1983). The pho ode ec o con e s he gene a ed pho- ons in o Rela i e Luminosi y Uni s, o RLUs (15-s ead- ings), ha can ansla e in o ATP concen a ions (1 RLU equi alen o 0.1 moles ATP) wi h dilu ions o ATP s anda d (Na_ATP sal ). ATP þDLuci e in þO2þLuci e ase Mg2þ  >Oxyluci e in þAMP þPy ophospha e þCO2 þh (560 nm) 4.3.2. Fluo escence sandwich mic oa ay immunoassay. The Quali y Assu ance o obo ic landed missions o Ma s (Phoenix, MSL, and 2020) equi es ollow-up s anda d molecula cleaning e i ica ion by Sol en Swab Analysis (SWA) o ligh ha dwa e’s swabbed su aces wi h a F eon- based sol en ia Fou ie ans o m in a ed (FTIR) spec- oscopy o GC-MS (Ande son e al., 2002; Maha y e al., 2004; Blakkolb e al., 2014). A subse o pos -cleaned ha dwa e (Sol 5 and Sol 6) ana- lyzed o ATP (Table 6) was also co-analyzed epu posing he SWA o use by he SOLID’s FSMI a ge ing SOLID- ele an biological con amina ion in place o con amina ing compounds de ec able by FTIR o GC-MS. We used Te lon Table 2. Con amina ion Con ol P ac ices Used o he Ro e As obiology D illing S udies Subsys ems See Table 1 o an explana ion o each p o ocol’s coded i ems. SHTS =Sample Handling and T ans e Sys em. ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1311 analyzed wi h he LDChip200 immunosenso o b oade cha ac e iza ion o he Sol 5 and Sol 6 ha dwa e backg ound esidue (ATP syn hase and cellula p o ein bioma ke s). The ull da ase and immunogens’ in ensi y peaks a e p o ided in Supplemen a y In o ma ion Fig. S1, Table S2a and S2b. We could pe o m only one sample ex ac ion and con- cen a e he olume o imp o e de ec ion. In ou expe ience, no di e ences in luo escence alues esul ed when using he same ex ac wice. E o ba s co espond o he s anda d de ia ions o h ee in e nal eplica es o each an ibody a ay we es ed. 5.2.2.1. ATP syn hase an ibodies. ATP syn hase subuni 1 (ASB and 20_p o ASF1 [ATP syn hase subuni 2]) is a p o ein ha o ms he in acellula ene gy s o age molecule ATP in he mi ochond ia. We de ec ed ATP syn hase in all ha dwa e su aces co-analyzed wi h he ATP Luminome y Assay (Table 6). Al hough he signal in he blank swab (nega i e con ol) was high, a e sub ac ing he nega i e con ol, he immunoassay yielded nea -ze o FI signals o ATP syn hase (20_p o ASF1) in he scoop and d ill (Sep embe 20 assay). The only FI signal o he p o ein’s o he subuni ASB (FI: 2347 –850) ela es o Sample 1 (scoop, Sep embe 19) and subuni ASF1 in ai bo ne samples 9 and 10, in ag ee- men wi h alues o ATP abo e 6.0 RLUs (Table 6). High RLU luminescence ma ches he inc eased luo escence signal o ASB 2, whe eas low RLU co esponds o nega i e de ec ion (FI: 0) o ace signals (FI: 55–235). The wo signals appea posi i ely co ela ed (R=0.836) when inc- luding he wo ai samples, bu he co ela ion d ops when conside ing only ha dwa e samples (N=5), and mo e anal- ysis is necessa y o a obus co ela ion. 5.2.2.2. Cellula and p o ein bioma ke an ibodies. The FI peaks (max alues) agains he mic obial axa and molecula bioma ke s de ec ed by he LDChip200 we e highe han hose agains ATP syn hase in he pos -cleaned ha dwa e (Table 7 and Fig. 8). Fu he , we compa ed each bioma ke de ec ed in he ha dwa e wi h hose iden i ied in he ai dus samples (Supplemen a y Table S1a, b). The compa ison in o ms whe he he backg ound con am- inan was om an ai bo ne sou ce o po en ial c oss- con amina ion om d illed sedimen s esidues ans e ed o he sample handling ha dwa e and analysis. Fi y pe cen o less o axa ( ace esidues) de ec ed in he d ill, scoop, and unnels we e also in he dus . In compa ison, mo e han 50% we e de ec ed only in he backg ound esidue ans e ed om d ill o unnel o scoop (pu a i e subsu ace biome). Ve y low signals (FI <400–600) agains Anabaena sp. a e ubiqui ous in pos -cleaned ha dwa e and dus samples. The highes FI-posi i e alues in he swabbed ha dwa e sam- ples a e agains p o eins G am-nega i e bac e ia ances al be a-lac amase (GNCA), Gammap o eobac e ial ances al be a-lac amase (GPBCA), and Ni S2 (Lep ospi illum e - ooxidans). The pep idoglycan (polyme suga s and amino acids o ming bac e ia cell wall) is ound only in he 19-Sep scoop (FI <800 and 8.4 –1.6 amoles ATP/cm 2 ) bu no in he dus . Ve y weak signals agains Bacillus sub ilis and S ep omyces sp. a e de ec ed only in pos -cleaned ha dwa e om Sol 6 (Sep embe 20), whe eas he peak in ensi y (<700) is agains Planococcus sp., a G am-posi i e psy- ch o ole an , halophilic bac e ium. FIG. 8. Linea plo wi h nega i e co ela ion (R=-0.673) be ween pos -cleaning su ace ATP backg ound (ATP luminescence) and Immunoassay-based mic obial axa backg ound (Fluo escence max in ensi y) o Sol 5 and Sol 6 ha dwa e. No e he weak signals (FI: <700), excep o he d ill bi ’s posi i e signal (FI >1000), as de ailed in Table 7. FI, luo escence in ensi y. Table 7. Pos -Cleaning Mic obial Cells and P o ein Bioma ke s De ec ed by he LDChip_200 in he Ha dwa e Backg ound and Co-Analyzed o Adenosine T iphospha e Bioma ke by Adenosine T iphospha e Luminome y (Thick-Bo de ed Columns) F om le o igh : o al ATP swab Assay o in-sim cleaned HD; ATP pe su ace uni ; maximum alue o FI (peak) agains mic obial axa (whole cell) and molecula bioma ke s (p o eins). ‘‘P esence in ai sample s’’ indica es he ac ion (%) o mic obial axa and p o ein bioma ke s de ec ed in one o bo h ai bo ne sample s and he HD backg ound. We calcula ed he pe cen o molecula bioma ke s in ai sample s om he an ibodies aised agains whole cells, p o eins, bio ilms, and sedimen mix u e cul u es in he HD esidue (Supplemen a y Table S2a and S2b) and hose de ec ed in he wo ai sample s (Table 9). GNCA =g am-nega i e bac e ia ances al be a lac amase. 1318 BONACCORSI ET AL. The su ace swab o he pos -cleaned ha dwa e yielded ela i ely low backg ound con aminan s (Table 7), indica ed by aces (<300) o weak (<1000) FI signals, excep o a ew mode a e signals (1000–3000). The in ensi y signals om p o ein bioma ke s con aminan s (FI peak *1000–1600) a e also highe han hose agains cellula ma e ials (FI peak: *300–1000). A ew high signals (>3000) pe ained only o ma e ials om he ai sample s (Fig. 10 and Table 9). The esul s sugges ha sol en -cleaning and mic obial- educ ion s eps e ec i ely mi iga ed esidual and ac i e biobu den con amina ion in he ARADS ha dwa e, wi h a ew excep ions. The chip de ec ed a mode a e FI signal agains Geobac e sp. (FI: 1008) and he Gammap o eo- bac e ial lac amase p o ein (FI: 1472) in he pos -cleaned d ill bi (Sol 6, Sep embe 20). Howe e , his highes backg ound in ensi y signal co e- sponds o one o he lowes signals de ec ed agains ATP syn hase (FI: <300), which co ela es wi h he ATP bio- ma ke (1.2 –0.6 amoles/cm 2 ). The ATP backg ound con- amina ion was equi alen o aces o iable biobu den o ca. £1 cell/cm 2 , o a o al o *100–200 cells in he d ill bi (0.6 –0.0 amoles/cm 2 ATP) based on *1–2 amoles ATP pe bac e ial cell (Okanojo e al., 2017). Fu he , a nega i e co ela ion exis s be ween he FI peak agains mic obial cells (g ay columns) and he ATP back- g ound signal ( =-0.673) as shown in Fig. 8. The ela- ionship sugges s ha only a negligible ac ion (i any) o he esidual mic obes de ec ed by he LDChip200 may be iable (ATP ac i i y) and ha he FI ela es o esidual aces o dead o pa ially agmen ed cells. The e is no signi ican co ela ion be ween ATP backg ound and FI peak agains p o eins ( =-0.196), whe eas no co ela ion exis s be ween he cells’ and he p o eins’ FI peak ( =-0.017). Co ela ion da a a e no p esen ed. 5.3. Ai bo ne con amina ion Figu e 9A–D and Table 8 illus a e moni o ing se up o ai bo ne biomass lux o collec o s 1 and 2. 5.3.1. An h opogenic and en i onmen al ai bo ne bio- mass. The ATP-based ai bo ne biomass lux a Collec o 1 anged om 23.7 amoles o 47.4 amoles/cm 2 ac oss he 6-Sol simula ion. The deposi ion a es o an h opogenic ATP a basecamp, a e age o 217.8 moles/(m 2 $day), we e 4.2 imes highe han he a es o en i onmen al ATP, which a e aged 51.6 moles/(m 2 $day). The lux o ai bo ne an h opogenic deli e ed o e 5 days a basecamp (Coll. 1) was 110.7 amoles, a e aging 22.1 amoles/ (cm 2 $day), o 221 moles/(m 2 $day), whe eas Playa Collec o 2 ecei ed a o al amoun o 46.4 amoles/cm 2 ATP o e he same pe iod exposu e. A e backg ound sub ac ion (Bkg : 20.6 amoles/cm 2 ), he o al lux o ai bo ne en i onmen al biomass o he playa (Coll. 2) was 25.8 amoles/cm 2 , equi a- len o 5.2 amoles/(cm 2 $day) o 51.6 moles ATP/(m 2 $day). Fu he , by assuming ha basecamp Collec o 1 would ecei e ai bo ne dus in simila amoun s o he Playa Col- lec o 2, he heo e ical ne an h opogenic componen could be calcula ed as he di e ence be ween he wo ai bo ne luxes a he wo collec o s (Coll 1 Flux -Coll 2 lux), which is 166.2 moles ATP/(m 2 $day). Table 8 epo s he calcula ed ai bo ne lux (en i on- men al biomass) based on ATP swab da a and no malized FIG. 9. (A, C) Ai bo ne collec o s’ loca ion and deploymen . (A) Basecamp Collec o 1 ou side he Science aile (da k ed); (B) Collec o 2, wi h basecamp in he backg ound; (C) Top iew o he 23 ·23 cm collec ing su ace and sub-sampling a eas (A–H). Supplemen a y Tables S1a and b in he Supplemen a y In o ma ion epo he su ace a ea and ATP swab da a used o lux calcula ions. (D) Moni o ed 6-Sol ai bo ne con amina ion in collec o s 1 and 2 as ATP a omoles/cm 2 . Es ima ed daily lux da a a e om Table 8. Squa e symbols and e o ba s indica e he a e age and STD (Column F, Table 8) o indi idual measu emen s (no shown he e; see he g ay-shaded Column J in he Supplemen a y Table S2 ins ead). The solid ba s indica e he o al amoun o su ace ATP measu ed o e 5 days (Column D). STD, s anda d de ia ion. ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1319 o he en i e collec o ’s su ace (23 ·23 cm). Fo collec- o s 1 and 2 ai bo ne lux calcula ions, see Supplemen a y In o ma ion o p e-deploymen se ups, pos -sani a ion ATP backg ound de e mina ion (Supplemen a y Table S0a and b), and daily ai bo ne biomass aw da a (Supplemen a y Table S1a, b and Fig. S3). 5.3.2. Mic obial axa in ai bo ne samples. The wo col- lec o s we e loca ed downwind om he Playa and ecei ed a highe lux o con aminan s om he wind-scou ed playa su ace sedimen s (Fig. 13 [3, 4]). LDChip200 de ec ed in si u bac e ia, a chaea, and molecula bioma ke s a he wo si es, wi h mos ly e y weak signals o he ela i e FI (FI <600–1000) ac oss he wo si es, bu a ew excep ions (Table 9 and Fig. 10). The e a e a ew in ensi y signals (weak signals) in bo h Basecamp Collec o 1 (sample #9) and he dis al allu ial an/Playa Collec o 2 (Sample #8), al hough #9 has mo e posi i e ones. The 10 m ack sample is a li le mo e con amina ed gi en he mode a e signals (FI >1000) o an ibodies agains De- chlo omonas,Desul o ib io (Coll 1); he highes luo escence peak (FI >6000) ela ed o he PhaC1 (a p o ein om aqua ic cyanobac e ia, Anabaena sp., Nos oc sp.) and he Fe idoxin enzyme. The aqua ic mic o lo a was p esen in bo h collec o s, bu he cyanobac e ium Xenococcus, cha ac e is ic o semi-a id hypoli hic communi ies ( he A acama B sequence, Wa en- Rhodes e al., 2006), occu ed only in he dis al Collec o 2. O e all, he posi i e immuno- eac ions occu ed wi h an- ibodies aised agains : (1) G am-nega i e acidophilic i on- and sul u -oxidizing bac e ia (Acidi hiobacillus,Desul o ib io); (2) pe chlo a e- educing Be ap o eobac e ia (Dechlo omonas, Ideonella), Gammap o eobac e ia (Shewanella,Pseudomonas, Gammap o eobac e ia ances al be a-lac amase); (3) Cyano- bac e ia: aqua ic (Anabaena,Nos oc) and hypoli hic (Xeno- coccus); (4) G am-posi i e Ac inobac e ia (S ep omyces)and Fi micu es (Bacillus,Desul ospo osinus); (5) s ic ly anae obic sul a e- and me al- educe s bac e ia (Desul ospo osinus,She- wanella); and (6) halophilic he e o ophic a chaea and me- hanobac e ia (Me hanobac e ium). The immunog am da a in Table 9 show ha he mic obes exclusi ely p esen a Collec o 2 a e G am-nega i e bac e ia, wi h G am-posi i e (Fi micu es, ac inobac e ia) and A chaea in he basecamp’s ai sample . The immunoassay da a a e no s a is ically ele an (only a collec o o each a ea), whe eas he immunog ams ma ch well he ATP da a. 5.4. Mic obial biomass o d illed and su ace sedimen Figu e 11 shows he downco e dis ibu ion o ATP bio- ma ke om h ee Playa d illing si es. The a e age ATP bioma ke is 100–250 moles/g and appea s o dec ease wi h dep h in Hole 5 (60 o <0.2 moles/g, below de ec ion limi [DL]) and Hole 2 ( om 350 o 5 moles/g). The dis ibu ion o ATP bioma ke s in hese co es app- ea s o be om complex pu a i e li e (e.g., Wa en-Rhodes e al., 2019) a he han om con aminan s ans e ed du ing d illing ia ha dwa e, pos -d illing sample manipu- la ion (e.g., hand skin eading 200 moles), o ai bo ne biomass- ich sedimen nea by (Fig. 13), wi h hese con- aminan s being p e en ed by ou s ic con amina ion con ol p ac ices du ing ope a ions. Table 8. Flux o Ai bo ne En i onmen al (Collec o 2) and An h opogenic (Collec o 1) Biomass F om le o igh : Column B: Swabbed su ace a ea ou o he o al collec o su ace (529 cm 2 ); Column C: ATP swab alues no malized o 529 cm 2 ; Column D: ai bo ne biomass in a omoles ATP pe su ace uni (amol cm 2 ). Columns E and F: Summa y s a is ics (min, max, A g., and STD) o da a ela i e o indi idual su aces sub-a eas A, B, C, D, e c. (no included, see Table S1 in he Supplemen a y In o ma ion). The las wo ows epo a e aged ATP lux as em omoles pe squa e me e pe day [ moles/(m 2 $day)] and a omole/ (cm 2 $day). 1320 BONACCORSI ET AL. FIG. 10. Immunog am wi h he ela i e FI o he LDChip200 posi i e de ec ion o mic oo ganisms and polyme ic biological ma ke s a he basecamp and dis al collec o s. No e ha some immunogens a e p esen only in ei he one o he wo collec o s, whe eas o he s occu in bo h ones. See Sec ion 5.3.2. o a desc ip ion and Table 9 o he lis o an ibody names. 1321 The unusually highe alues o ATP in he uppe -le el samples (0–10, 40–50, and 10–20 cm) and he dec eases o ATP wi h he bo ehole dep h (50–60, 60–70, and 30–40 cm) migh be due o na u ally occu ing me abolically ac i e biomass p esen in hese le els as he esul o he la es ponding e en s. Fo compa ison, 15.1–32.4 ng/g, o ppb, o o al amino acids (Leu, Val, Se , Ala, and Gly) we e de ec ed by he MILA ins umen in h ee subsu ace samples acqui ed in 2019 ( alues calcula ed om Mo a e al., 2020; Supple- men a y Table S2). Finally, ‘‘G een Pa o ’’ d illed da k b own clays (Ma ch 2019) yielded highe alues (*2·10 3 o *7·10 4 cells/g) agains he coa se-g ained su ace pa emen (*10 2 –10 3 cells/g), wi h no eco e y o mic obial ATP in i e ou o FIG. 11. Dep h dis ibu ion o mic o- bial ATP in h ee holes exp essed as moles o ATP bioma ke pe g am o sedimen . Ho izon al e o is STD (Table S3 in he Supplemen a y In- o ma ion). Table 9. Peak Numbe s, An ibody Names, and Immunogens o In ensi y Peaks o he Immunog am o Ai Samples om Fig. 10 1322 BONACCORSI ET AL. se en ials (Supplemen a y Table S3). Supplemen a y Figu e S2A–E epo s p o ocol es esul s, and Supple- men a y Table S3 p o ides Summa y s a is ics o he ARADS su ace and d ill samples da ase . 5.4.1. Su ace dis ibu ion o biomass con aminan a Playa d ill si e. We measu ed he su ace dis ibu ion o o al, ee, and mic obial ATP in smec i e clay- ich playa sedimen s as po en ial con aminan indica o s o he ARADS si es (Fig. 12 and Supplemen a y Table S3 in he Supple- men a y In o ma ion). Samples included (1) a 3 mm laye o he desicca ed mic obial ma as posi i e con ol, ha is, 4.7 –0.58 ·10 4 moles o al ATP/g (N=9); (2) exca a ed clay om a nea by pi (3–5 cm-dep h), ha is, 7.2 –0.7 ·10 3 moles/g (N=10); and (3) ne e -ponded su ace clay (0– 3 cm-dep h), ha is, 212 –268 moles/g (N=6). These alues a e equi alen o an es ima ed iable mic obial biomass o 10 3 cells/g (0–3 cm-dep h) and 10 5 cells/g (3–5 cm-dep h) o e y high le els (10 6 cells/g) in he 3 mm laye o he d ied mic obial ma (posi i e con ol samples) a 4-log o a 5-log highe mic obial biomass han in d illed sedimen s o he majo i y o he Playa su ace. This unexpec edly highly ac i e su ace biomass ela ed o a hin c us o desicca ed mic obial ma (Fig. 3K) we sampled om small a eas o he pos - looded Playa and was s ill p ese ed a e a sho -li ed pond o med a i s ma - gin du ing an ex eme lood e en in 2017 (Pe sonal com- munica ion; Kim Wa en-Rhodes, 2019), 2 yea s be o e ou campaign. In epheme al ponds, spo e- o ming cyanobac- e ia can p oli e a e and lea e pos - looded desicca ed ye iable biomass on he su ace. The desicca ed, e y b i le bio ilm could be e oded, ca ied, and deli e ed by wind, inc easing he con amina ion isk when en aining exposed co ed ma e ial o he scoop and unnels du ing he sample ans e (Fig. 13). Howe e , mos su ace biomass was 3- o a 4-log lowe han bo eholes sedimen s o he high biomass pa ches. 5.5. Con amina ion ans e o d illed samples An in eg al componen o he con amina ion plan included a sou ce- o-sink assessmen o po en ial con amina ion in he d illed samples (ha dwa e- o-sample ans e , ai bo ne bio- mass con amina ion on sampling ha dwa e, e c.). Resul s om hese asks a e in used in o a concep ual model (Fig. 13) and in eg a ed in o he Discussion (Sec ion 6). Figu e 12 shows he ha dwa e- o-sample con amina ion ans e calcula ed using pos -cleaning ha dwa e back- g ound da a (Table 4), and by assuming an a e age d illed sedimen (60 g) ge ing in o con ac wi h a 50 cm segmen o he d ill s ing (616 cm 2 ), 100% o he scoop (217 cm 2 ), and unnel (196 cm 2 ), and 100% con amina ion ans e (d ill o scoop o unnel). 6. Discussion 6.1. E ec i eness o ARADS me hods We de eloped in eg a ed CCSI p ac ices o ARADS o p e en and mi iga e con amina ion o he SHTS and he acqui ed samples and lea n abou po en ial con amina ion pa hways ac oss he ope a ing en i onmen s. Table 10 sum- ma izes he mi iga ion asks, key indings, and hei appli- cabili y o ield condi ions. The ARADS p ac ices a e simila o hose es ablished o mi iga e con amina ion du ing a landed mission o Ma s (Sp y e al., 2021). A e y close ela ionship exis s be ween mic obial educ- ion and o ganics emo al o p o ec science. Backg ound con aminan s in spacec a ha dwa e pose wo isks o a li e de ec ion mission d illing in o he ma ian ice-cemen g ound Table 10. Desc ip ion and Field Applica ion o Ro e As obiology D illing S udies Mi iga ion P ac ices (I ems #1–6) and C i ical Resul s PPE =pe sonal p o ec i e equipmen . ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1323 like Iceb eake (Heldmann e al., 2013; McKay e al., 2013) and he Ma s Li e Explo e (NAP, 2021). Fi s , o wa d con amina ion om e es ial mic obes su i ing spacec a ’s ha dwa e s e iliza ion is he p ima y conce n (e.g., NRC, 2006; NAS, 2018, 2019, 2020). Spo es can emain long- e m iable in space, e en wi hou me a- bolic ac i i y a he low empe a u es o Ma s’ su ace (e.g., Ho neck e al., 2010, 2012; Hallswo h, 2021), be shed on he su ace du ing landing (Ma shall and Mancinelli, 2011), and en e he unde g ound, becoming me abolically ac i e unde inc eased wa e ac i i y (Rummel e al., 2014; O osei e al., 2018); his will inc ease he o wa d con amina ion isk should mo e habi able condi ions exis unde g ound o nea he su ace Ca ego y IV biobu den le el limi s a e £30 +(2 ·10 5 ) spo es om he landed spacec a ’s su - ace, ma ed, and encapsula ed ha dy biobu den su i ing decon amina ion. Second, whole mic obes o cellula agmen s could elease mission- ele an analy es such as amino acids—o any o he a ge bioma ke along he ‘‘ladde o li e de ec ion’’ (Wes all e al., 2015; Ne eu e al., 2018)—in o he analy ical sui e. Fo ins ance, amino acids a e a p ima y a ge o he Iceb eake li e analyze s and o he li e de ec ion missions. Bacillus spo es con ain ee (l-glu ama e and l-a ginine) and bound amino acids, ha is, mucopep ide and p o ein (Nelson and Ko nbe g, 1970; and e e ences he ein) e- leasable on agmen a ion. I disloca ed and ans e ed o analysis, only one B. sub ilis spo e in he space ha dwa e could elease mo e han 0.24 pg (o 10 -12 g) o al AA. 6.1.1. False nega i es in cleaning e i ica ion. We dem- ons a ed ha alse nega i e de ec ion o con aminan s unde mines mic obial educ ion and e i ica ion, esul ing in mo e conce n o ARADS han alse posi i es, ha is, he le el o eco e y o swabbed ATP decoupled om he ac- ual biobu den backg ound. False nega i es can o igina e om many con ounding ac o s. They a e: (1) The ha dwa e’s su ace cha ac e is ics (discussed in Sec ion 6.1.3). (2) The amoun , dis ibu ion, and complexi y o ha d- wa e biobu den (bio ilms-o ganics laye ing) and mine al coa ings (dus o oxidized). When non isible o ganics and he mine al ilm we e p esen on he d ill, he ATP swab assay yielded alse nega i es o wo plausible easons. (i) Mud pa icles and oily esidues obscu ed he L-L ATP eac ion, and (ii) molecula o - ganic laye s encasing he mic obes p o ec ed hem agains de ec ion (Fig. 6C, D) and mic obial educ ion. The exposed ac i e biobu den could be de ec ed and educed only a e ace one emo es ino ganic and o - ganic polyme s (oils, g ease, lipids) be o e mic obial educ ion. This p oblem did no conce n scoop and unnels, whose biobu den dec eased linea ly a e ea - men s (Fig. 6G, H). Unlike he d ill, no complex o ganic bio( ilms) build-up occu ed du ing he long- e m s o - age o scoop and unnels. The cleaning es esul s we e consis en wi h he common wisdom ha he cleane a su ace can ge , he mo e success ul mic obial educ ion and e i i- ca ion will be (e.g., Mesza os e al., 2005). Gi en wha has been s a ed ea lie , we sugges pe o ming mic obial educ ion and e i ica ion on an ace one-cleaned d ill. Disin ec ion- esis an bio ilm- o ganics laye ing can be a common issue because he d ill sys em in gea s, bea ings, and sliding su aces o he d ill ube connec ions makes ou ine use o s an- da d lub ican s, o example, 3-in-1 SAE 20 mo o oil con aining pe oleum, Lub iMa ic Whi e Li hium G ease, and WD-40 (Mille e al., 2008). (3) Cleaning agen s’ esidues on ha dwa e. We i s mi i- ga ed he alse nega i e issue (a de ec ion one) by conduc ing e i ica ion assays on sol en - insed ha dwa e. Rinsing wi h wa e emo es in e e ing cleaning and disin ec ing agen s and add esses one limi a ion posed o he ATP assay in clean ooms (see e iew by Shama and Malik, 2013). (4) Luminome y quenching e ec s by ch omogenic pig- men s in samples dec ease he ligh emission. Nano- phase clays, FeOx, and sal s can in e e e wi h L-L eac ions and chemiluminescence pho ode ec o s. (5) En i onmen al ield condi ions. High diu nal em- pe a u es (‡25–40C) and di ec sunligh can igge e y high RLU alues gene a ing alse posi i es (Bonacco si and S oke , 2008). The use o chemilu- minescence pho ode ec o s o e pho omul iplie FIG. 12. ATP bioma ke s ( o al, ee, and mic obial) in co ed e sus su ace sedimen s and de i ed alues o mic obial biomass as cells/g sedi- men . No e he negligible con ibu ion o he po en ial con amina ion ans e om ha dwa e (To al ATP, sho es ba s) o d ill samples. 23.7 –2.1 amoles/g o DP and 63.4 –28 amoles/g (6-Sol Bkg ) Bkg : pos -cleaning ha d- wa e backg ound. DP, deep cleaning. 1324 BONACCORSI ET AL. ubes can mi iga e he la e issue. Fu he , wind- gene a ed iboelec ic e ec s could p oduce unusu- ally high RLU alues e en a nigh , as we expe i- enced du ing ield measu emen s. We included posi i e and nega i e con ols o add ess he assay pe o mance. 6.1.2. E ec i eness o mic obial educ ion and e i ica- ion. Coun less cleaning p o ocols exis o labo a o y, clean oom applica ions o asep ic d illing o icy ma e ials (e.g., Ch is ne e al., 2005; Kuhn e al., 2014; Goo dial e al., 2017; Kayani e al., 2018; Coelho e al., 2022). Ye , only a ew ha e been designed o complex as obiology li e-de ec ion ech- nology ials. Mille e al. (2008) applied a mul i-s ep p o ocol o asep ic d illing (Milli-Q wa e , 10% Lysol aqueous solu ion, 70% E -OH, lame s e iliza ion) (Table 3) and immunoassay- based deep biosphe e cha ac e iza ion in he Rio Tin o egion (Pa o e al., 2008), consis en ly wi h o he mul i- eagen cleaning p o ocol ce i ied by immunoassay-based Limulus Amebocy e Lysa e (LAL) and GC-MS swab analyses (Eigen- b ode e al., 2009). ARADS mul i- eagen p o ocols combined immunoassay and ATP luminome y echniques o de ec a b oade ange o iable and non iable biological con aminan s. O e all, he 6-h mic obial educ ion p o ocol enabled a 4-log d op o ATP backg ound om unmi iga ed o asep ic condi ions (1–2 amoles/cm 2 e e ence backg ound wi hin he asep ic h eshold o £3–4 bac e ial cells/cm 2 ); see Sec ion 2. Du ing he six Sols, asep ic d illing was possible by ex ending he p o ocol ime (a e Sol 2) ac oss he ime- cons ained d illing simula ion low (Fig. 7), and consis- en ly wi h o he mul i- eagen cleaning p o ocols (Mille e al., 2008; Eigenb ode e al., 2009). Ac oss he 6-day simula ion, we es ed NaClO-ac i a ed H 2 O 2 o mo e e ec i e mic obial educ ion. A e squi ing 3% H 2 O 2 on a d ill ip sp ayed wi h NaClO, he esul ing single oxygen ( 1 O 2 ) s e ilan agen —decon amina ed he ha dwa e 30 imes as e han bleach alone o le els achie able du ing he ex ensi e 6-h deep cleaning in he lab (Table 4). The ac i a ion o H 2 O 2 by bleach is an easy and ela i ely sa e, ield-e ec i e p ocedu e o educing biobu den a e only a 2-min exposu e, compa ed wi h he long exposu e equi ed by concen a ed bleach alone (10–20¢), 10% bleach (3 h), 70% IPA (1 h), o 3% H 2 O 2 alone (>3 h). The edox eac ion be ween he oxidizing hypochlo i e ion (OCl - ) and he educing H 2 O 2 agen o ms wa e (H 2 O), chlo- ine ion (Cl - ), and 1 O 2 (Held e al., 1978; G ee , 2006) such as: H2O2(aq)þNaCIO (aq)!1O2(g)þNaCI (aq)þH2O(aq) The disin ec ion ac ion was due o he exci ed s a e o 1 O 2 , which con ains mo e ene gy han g ound oxygen (Held e al., 1978; Wi z, 2009). 1 O 2 un olds p o eins (e.g., Del Maes o e al., 1980; Da ies, 2003) and kills bac e ial and euka yo ic cells (Dahl e al., 1987; Djimeli e al., 2014; Taewan e al., 2019) wi h mic oo ganism inac i a ion p o- po ional o 1 O 2 exposu e. Low- empe a u e chemical s e ilan s such as 35% H 2 O 2 Gas Plasma umiga ion, E hylene Oxide (E O), and Ozone (O 3 ) a e all absolu e biocides commonly used in clean ooms (e.g., Go don e al., 2012). Howe e , hese ech- niques a e inapplicable in he ield because hey a e highly oxic and equi e complex equipmen (e.g., con ainmen chambe s, deep acuum d aw zapping, gas moni o ing sys ems) and se e al hou s-exposu e (Mesza os e al., 2005; Go don e al., 2012). 6.1.2.1. Cleanliness e i ica ion and con amina ion knowledge. We used wo me hods o cleanliness e i ica- ion, he ATP Assay, and he SOLID ins umen mic oa ay immunoassay echnology, as a no el app oach o con am- ina ion knowledge o he ha dwa e backg ound. We could no use he cul u e-based NSA, which enume a es (pla e coun ing) d y hea -su i ing ae obes endospo es a e a 72 h g ow h beyond he ARADS eal- ime imeline. In addi ion, he NSA o e looks non-cul i able cells, unde es ima ing he o al mic obial bu den, spo e- o ming s ic anae obes (Clos idium), and ex emophilic a chaea mic oo ganisms o PP conce n (e.g., Moissl-Eichinge e al., 2015). Fo hese easons, Be na dini and Venka eswa an (2016) co-analyzed ATP and NSA da a om he MSL spacec a . They p oposed he ATP assay (pe NASA-HDBK-6022, 2010) as he benchma k h eshold o spacec a su ace cleanliness, ha is, 2.3 ·10 -11 mmol ATP/25 cm 2 (o <0.9 mole/cm 2 ). Se e al me hods exis o de ec and cha ac e ize ha dwa e con aminan s ma ix-assis ed lase deso p ion/io- niza ion- ime o ligh , bu complex se ups limi hei use in he ield. O he eal- ime ools designed o ield de ec ion, such as Minion Ox o d Nanopo e o DNA and RNA se- quencing (10 min) o he LAL assay o G am-nega i e mic oo ganisms, ha e been used (pos -mission) in ield i- als (Eigenb ode e al., 2009; Cockell e al., 2018). Howe e , he LAL assay does no de ec only iable mic obes and equi es mo e ex ended sample p ocessing and analysis ime han he ATP Luminome y assay, ha is, 15–60¢ e sus 15 s -5 min, espec i ely. Thei applica ion is beyond he scope o he quick disin ec ion alida ion equi emen ac oss he six Sols. In si u eal- ime and ul asensi i e ATP assay can de ec 0.1 moles o ATP on ha dwa e su ace as an independen molecula p oxy o ac i e biobu den/mic obial biomass, iable spo es, o in ac ecen ly dead cells. Fu he , he ATP syn hase FI signal was consis en wi h ATP’s pos -cleaning ha dwa e backg ound le els. This independen e i ica ion co obo a es he e ec i eness o in-sim cleaning and mic obial educ ion. Fu he , he luo escence signal agains he p o ein bio- ma ke s was highe han ha agains he whole cells. The wo signals decoupled, likely because mo e agmen ed cellula ma e ials e sus whole cells we e p esen in he wind-ca ied dus (aeolian pa icles subjec o pho o- oxida ion) and subsu ace sedimen . SOLID immunog ams o whole cells, p o eins, and o he cellula compounds p o ided an e ec i e e i ica ion ool o sel -check agains alse posi i es con amina ing science measu emen s. One limi a ion is ha he immunoassay does no di e en ia e be ween li ing o dead cells (a o wa d con- amina ion isk), bu ATP assay co-analysis add esses his gap. Finally, GC-MS and high-pe o mance liquid ch oma o- g aphy iden i y backg ound con aminan s in space ha dwa e (con amina ion knowledge) wi h subs an ial ime elapsed be ween sampling and analysis. Fo he ARADS ha dwa e ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1325 con amina ion knowledge, we used SOLID immunog ams; such an app oach could educe ime and cos s o he si- mul aneous assessmen o o wa ding and science con am- ina ion. 6.1.3. Role o ha dwa e su ace ea u es o geome y. The space ha dwa e design, geome y, su ace opog aphy, po osi y, and oughness can a ec con amina ion con ol e ec i eness. Fo he same eason, hese ea u es can de e mine biobu den e en ion, dislodgemen , ans e , o dispe sal o he en i onmen . Speci ically, he s ainless-s eel a m-moun ed Phoenix-like scoop in e io was di icul o access due o i s 90edges be ween he in e io su aces. The su ace o he ex e nal scoop blade was mo e accessible bu oughe han he scoop in e io . As a esul , he scoop’s in e io and edge exhibi ed a pos -cleaning h eshold back- g ound highe han he unnels. Simila ly, he TRIDENT d ill bi ’s inishing was h ee- dimensional (3D) p in ed and e ched o augmen he eco e y o d illed ines. This d ill bi su ace was oughe han he d ill s ing (Fig. 2), esul ing in high biobu den backg ound (Fig. 6G, H and Tables 4 and 5). Howe e , an augmen ed cleaning p o ocol educed he backg ound. Mi iga ing solu ions could include cu en good manu ac- u ing p ac ices such as ‘‘co ing’’ and polished su ace in- ishing. Co ing minimizes geome y- ela ed con amina ion by eplacing 90and 3D co ne s wi h cu ed su aces and im- p o es s e iliza ion o clean ooms and hospi al loo s. Cu ed co ne s inside he scoop su aces (inne walls) would p e en he accumula ion o biological con aminan s o d illed ma e- ial (conduci e o c oss-con amina ion) and acili a e he pa hway o cleaning and swabbing. An addi ional implemen a ion could use s a e-o - he-a mi o -like polished su aces o he d ill and SHTS. Elec- opolishing ensu es ul a cleaning o embedded con ami- nan s in Ti anium alloys o issue enginee ing and su gical implan s, which mus be s e ile be o e use. In addi ion, elec opolished mic oscopically ea u eless su aces educe mic obial adhesion and su ace con amina ion (Bagno and Bello, 2004; Oshida, 2007; Shimaku a, 2007; Tajima e al., 2008). 6.2. Con amina ion pa hways We p opose a concep ual model (Fig. 13) ha includes he signi ican con amina ion pa hways highligh ed by da a analysis and es s on ARADS ma e ials (ha dwa e, sedi- men s, ai sample s, e c.). In e es ingly, he s udy o ai bo ne ma e ials deli e ed wo su p ising esul s: a highe - han-p edic ed en i onmen al con amina ion (pa hway 8–9 o 3–4); and a lowe - han-expec ed an h opogenic one (pa hway 1–2 o 3). The eme ging pic u e is ascina ingly complex due o he in insic na u e o he A acama Dese . 6.2.1. En i onmen al sou ces o ai bo ne con aminan s. Wind- anspo ed mine al dus can e icien ly ca y and dispe se iable mic obial li e ac oss he A acama (Pa o e al., 2011; and e e ences he ein). Whe he his biomass cons i u es an an h opogenic (human biome) o na u al (en i onmen al) con aminan , a c oss-con aminan , o a ‘‘na i e’’ elemen is no always clea o us o unde s ood (Azua-Bus os e al., 2019, 2022). Ou es s e ealed ha ai bo ne biomass ep esen ed an unusually high sou ce o po en ial con amina ion, unex- pec ed o he hype a id co e o he A acama Dese . The high-in ensi y signal (>6000)— aised agains aqua ic mic o lo a (Anabaena o Nos oc sp. cyanobac e ia) in ai sample s—poin s o a p ima y sou ce o ai bo ne bio- mass consis en wi h ATP da a om he pos - looded Playa (10 6 cells/g sedimen , ATP cell equi alen ) and he ai bo ne FIG. 13. Concep ual model illus a ing sou ce- o-sink con amina ion. Figu e no o scale. Semi-quan i a i e cell es ima es [cells/g, cells/cm 2 ,andcells/(cm$day 1 )] and numbe s nea each pa hway. Base- camp [1] and an h opogenic con amina- ion[2]indus Coll.1[3]andColl.2[4]. The hin dashed a ows indica e Ro e [5] and c oss-con amina ion pa hways: (a) d ill- o-subsu ace [6] (a o wa d con amina ion analog); (a–d) d ill- o-sample ans e and analysis: (a) d ill, (b) scoop, (c) unnel, (d) li e de ec ion ins umen . Blue solid-lined a ows wi h ‘‘X’’ indica e unlikely con amina ion pa hways, o example, desicca ed pos - lood mic obial ma [8] sou cing aqua ic cyanobac e ia [9] o sampling ha dwa e (a–c) o subsu ace sedimen a y laye s [6] and no looded playa su ace [7] delimi- ed by he Playa sho eline (black dashed lines). La ge g een a ows indica e un- expec ed ai bo ne biology, o example, Anabaena sp. [9] in collec o s [3, 4]. Blue and ed pa allel dashed a ows ep esen he e ogeneous (mo e dis al?) sou ces o ai bo ne biomass [10] o he collec o s o sampling ha dwa e. Coll., collec o . 1326 BONACCORSI ET AL. biomass—5–22 amoles/(cm 2 $day) ATP, ac oss he 6-Sol mission (Fig. 12). This sugges s an unexpec edly high iable biomass la e al ans e om an unusual upwind sou ce, ha is, desicca ed ponds wi hin he nea by Playa a ew ens o me e s away om he d ill si e and 300–400 m om Basecamp Collec- o 1. Such an ai bo ne sedimen a y biomass ep esen s a po en ial sou ce o high con aminan (also e y close o one o he d ill si es) ha could ha e en e ed obo ic sampling and analysis i no mi iga ed. Epheme al wa e -induced mic o lo a popula ion g ow h is uncommon and ep esen s a no el phenomenon o he hype -a id co e o he A acama Dese due o he ecen onse (in 2015) o clima ic change b inging yea ly s o ms and loods o he A acama egion (Fe na ´ndez-Ma ı ´nez e al., 2019; K. Wa en-Rhodes 2022, unpublished da a). Howe e , bu s s o mic o lo a g ow h can occu a e ex- eme ain e en s in a id ho dese en i onmen s wo ldwide (e.g., Ci e ´se al., 2017; and e e ences he ein; Bonacco si and McKay, 2022). The weake -in ensi y signals (£1000) wi h LDChip poin o seconda y low-in ensi y sou ces o he han he Playa. Fo ins ance, anspo om he A acama soil mic oen i on- men s con aining s ong acids (Quinn e al., 2005) could explain he de ec ion o bac e ia om acidic habi a s (Pa o e al., 2011; and e e ences he ein). Simila ly, he de ec ed a chaea Me hanobac e ium could ha e been wind-ca ied om he same sou ce o he aqua ic cyanobac e ia, he pos - looded Playa. Me hanogenesis can occu a he wa e -sedimen in e - ace, in anoxic mic opo es o ine-g ained clay- ich sedi- men s du ing loods. Gammap o eobac e ia (Pseudomonas) and hei associa ed bioma ke a e ound in ex eme desic- ca ed en i onmen s (Pa o e al., 2011), including A acama playas and allu ial an samples (Fe na ´ndez-Ma ı ´nez e al., 2019), subsu ace hype saline habi a s (e.g., Sala G ande). The anae obic Shewanella inhabi s hea y me al- con amina ed (i on, lead, and u anium) en i onmen s (mine ac i i y). All hese en i onmen s such as he GP Si e (Fig. 4D, E) exis in closed o dis al p oximi y o he ARADs’ d ill si es and can p o ide biological con ami- nan s o conce n depending on hei ela i e abundance and iabili y. The hype -a id A acama’s su ace soil is mos ly ex e- mely low in iable mic obes and associa ed biomolecula compounds (e.g., Came on, 1969; Na a o-Gonzalez e al., 2003; Skelley e al., 2005; Wa en-Rhodes e al., 2006; Ca ´ce es e al., 2007; Cockell e al., 2008) wi h biomass inc easing wi h some dep h (e.g., Fe na ´ndez-Ma ı ´nez e al., 2019). A ew excep ions can include ex ensi e su ace sou ce a eas such as sala s, mic o lo a-colonized hali e ields (e.g., Da ila e al., 2010; Pa o e al., 2011; Wie zchos e al., 2006, 2011; Da ila and Schulze-Makuch, 2016), g oundwa e - ed oases wi h spa se ascula plan s (Fig. 4D, E), o mic o lo a blooms in esponse o spo adic and ansien ain e en s (e.g., Schulze-Makuch e al., 2018; U i skiy e al., 2018; 2019). Ai bo ne dus om he sou ces ci ed ea lie migh ca y con aminan s o d illed samples o en e he analy ical ins umen , hus con using he scien i ic esul s. A second- a y bu no less impac ul con aminan s a e ‘‘ esh’’ and deg aded ossilized biomolecules, o example, ATP, Phospholipid a y acids (PLFA), DNA, lipopolysaccha ides (LPS), which he hype -a id su ace soil o he A acama Dese can p ese e well (Les e e al., 2007; Bonacco si e al., 2010; Powe s e al., 2018; Wilhelm e al., 2018). 6.2.2. An h opogenic ai bo ne con amina ion. We mon- i o ed he lux o ai bo ne con aminan s du ing he in-sim ope a ions o e alua e he an h opogenic con amina ion isk (expec ed high) o he Playa en i onmen and ARADS science. The biomass lux a he an h opogenic-domina ed basecamp a ea was highe [22.1 amoles/(cm 2 $day) ATP] han ha moni o ed by Collec o 1, 250 m away om basecamp [5.2 amoles/(cm 2 $day) ATP; non-an h opogenic sou ces]. This lux o ATP-based biomass was highe han he a e age ATP backg ound o he (pos -cleaning) SHTS ha dwa e, ha is, 0.9 –1.1 o 3.6 –4.5 amoles/cm 2 (Table 5), implying ha we e ec i ely mi iga ed he deli e y o ai - bo ne con aminan s o he ha dwa e. Howe e , a Basecamp, LDChip200 de ec ed mic oo ganisms na u ally occu ing in he A acama Dese (ai bo ne om p oximal and dis al po en ial sou ce a eas). Only a ew axa de ec ed wi h a weak signal (FI <1000) (B. sub ilis spo es; S ep omyces spp., spo e, and mycelium) migh be ela ed o he human mic obiome. Howe e , B. sub ilis and S ep omyces spp. a e usual bac e ia om soil (also common in he A acama). B.sub ilis spo es can su i e o up o 15 mon hs in he hype -a id co e o he A acama Dese wi h 15% su i al (Dose e al., 2001). S ep omyces is a ilamen ous ac inobac e ium na u ally p esen in he soil. This alkaliphilic and he mophilic genus is ound in a id, hype saline, o hea y me als- ich ex eme habi a s (Kamp e e al., 2014), including he hype -a id A acama (San hanam e al., 2012; Good ellow e al., 2017). S ep omyces can also be ound in a heal hy human mic obiome (a a e occu ence), whe e cul u e-based ech- niques ha e o e looked i s p esence due o low g ow h, bu ecen molecula -based s udies highligh ed i s p esence in he gas o-in es inal (Bolou ian and Moj ahedi, 2018) and espi a o y ac s (Huang e al., 2015; He b ı ´ke al., 2020) and in he heal hy skin (Gallo and Hoope , 2012). In pa - icula , skin lakes a e ich in soma ic cells (5 ·10 8 cells shed e e y day o 2 ·10 7 cells e e y hou (Weschle , 1978)) and associa ed mic obes. Fo ins ance, he hand mic obio a a ies *4·10 4 o 4.6 ·10 6 CFU/cm 2 (Leyden e al., 1987; Message e al., 2004) and 1 ·10 7 cul u able bac e ia pe cm 2 (F ed icks, 2001); ye hese cul u e-based es ima ions a e likely unde - es ima es (Edmonds-Wilson e al., 2015; and e e ences he ein). On a cau iona y no e, Bacillus and S ep omyces axa could come om he en i onmen a he han human biome con aminan s. Howe e , we canno disca d his hypo hesis ei he . An explana ion o he weakness o he ai bo ne an h o- pogenic signal could be ine icien sampling (unde - sampling) o ai bo ne pa icles, ha is, he swab was e ically placed o collec dus pa icles di ec ly om he ai . Ano he possibili y is ha he an h opogenic con ami- nan s (skin, clo hing, and d ople s) shed by he human c ew du ing ope a ions we e non-deposi ed, emo ed a e deposi ion, o dispe sed by ai low and wind. Fu he , he ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1327 signs o li e de ec o -li e de ec o chip du ing a Ma s d illing simula ion campaign in he hype a id co e o he A acama Dese . As obiology 2023;23(12); doi: 10.1089/as .2021 .0174 NAP. Plane a y Science and As obiology Decadal Su ey. Ma s Li e Explo e Mission Concep S udy Repo , 2021. NASA-HDBK-6022. Handbook o he Mic obial Examina ion o Space Ha dwa e. Na ional Ae onau ics and Space Admin- is a ion: Washing on, DC; 2010. Na ional Ae onau ics and Space Adminis a ion (NASA). Pla- ne a y P o ec ion P o isions o Robo ic Ex a e es ial Missions. NPR 8020.12D, Na ional Ae onau ics and Space Adminis a ion: Washing on, DC; 2011. Na ional Academies o Sciences, Enginee ing, and Medicine (NAS); Space S udies Boa d; Commi ee on he Re iew o Plane a y P o ec ion Policy De elopmen P ocesses. Re iew and Assessmen o Plane a y P o ec ion Policy De elopmen P ocesses. The Na ional Academies P ess: Washing on, DC; 2018; doi: 10.17226/25172 Na ional Academies o Sciences, Enginee ing, and Medicine (NAS); Space S udies Boa d; Commi ee o Re iew he Repo o he NASA Plane a y P o ec ion Independen Re iew Boa d. NASA Plane a y P o ec ion Independen Re iew Boa d (PPIRB) Repo o NASA/SMD. 2019. Na ional Academies o Sciences, Enginee ing, and Medicine (NAS); Space S udies Boa d; Commi ee o Re iew he Repo o he NASA Plane a y P o ec ion Independen Re iew Boa d. Assessmen o he Repo o NASA’s Plane a y P o ec ion Independen Re iew Boa d. The Na ional Acade- mies P ess: Washing on, DC; 2020; doi: 10.17226/25773 Na ional Resea ch Council (NRC). P e en ing he Fo wa d Con amina ion o Ma s. The Na ional Academies P ess: Washing on, DC; 2006. doi: 10.17226/11381 Na a o-Gonzalez R, Rainey FA, Molina P, e al. Ma s-like soils in he A acama Dese , Chile, and he d y limi o mic obial li e. Science 2003;302:1018–1021. Nelson DL, Ko nbe g A. Biochemical s udies o bac e ial spo ula ion and ge mina ion. 18. F ee amino acids in spo es. J Biol Chem 1970;245(5):1128–1136. Nesce ecka A, Juhna T, Hammes F. Beha io and s abili y o adenosine iphospha e (ATP) du ing chlo ine disin ec ion. Wa e Res 2016;101:490–497; doi: 10.1016/j.wa es.2016.05 .087 Ne eu M, Hays LE, Voy ek MA, e al. The ladde o li e de ec ion. As obiology 2018;18(11):1375–1402; doi: 10 .1089/as .2017.1773 Okanojo M, Miyashi a N, Tazaki A, e al. A omol-le el ATP bioluminome e o de ec ing single bac e ium. Luminescence 2017;32:751–756; doi: 10.1002/bio.3246 O osei R, Lau o SE, Pe inelli E, e al. Rada e idence o subglacial liquid wa e on Ma s. Science 2018;361(6401): 490–493. Oshida Y. Su ace science and echnology—Ti anium den al im- plan sys ems. J Soc Ti anium Alloys Den is 2007;5:52–53. Pandey S, Cla ke J, Nema P, e al. Ladakh: Di e se, high- al i ude ex eme en i onmen s o o -Ea h analogue and as obiology. In J As obiol 2019;19(1):1–21; doi: 10.1017/ S1473550419000119 Panousi MN, Williams GJ, Gi dles one S, e al. E alua ion o alcohol wipes used du ing asep ic manu ac u ing. Soc Appl Mic obiol Le Appl Mic obiol 2009;48:648–651. Pa nell J, Cullen D, Sims M, e al. Sea ching o li e on Ma s: Selec ion o molecula a ge s o ESA’s Au o a ExoMa s mission. As obiology 2007;7:578–604. Pa o V, Fe na ´ndez-Cal o P, Rod ı ´guez Man edi JA, e al. SOLID2: An an ibody a ay-based li e de ec o ins umen in a Ma s D illing Simula ion Expe imen (MARTE). As o- biology 2008;8(5):987–999. Pa o V, de Diego-Cas illa G, Mo eno-Paz M, e al. A mic obial oasis in he hype saline A acama subsu ace disco e ed by a li e de ec o chip: Implica ions o he sea ch o li e on Ma s. As obiology 2011;11(10):969–996. Pa o V, Puen e-Sa ´nchez F, Cab ol NA, e al. Mic obiology and ni ogen cycle in he ben hic sedimen s o a glacial oligo ophic deep Andean lake as analog o ancien ma ian lake-beds. F on Mic obiol 2019:10:929; doi: 10.3389/ micb.2019.00929 Pa el A, Malino ska L, Saha S, e al. ATP as a biological hyd o ope. Science 2017;356:753–756. Powe s LS, Smi h HD, Kilungo AP, e al. In si u eal- ime quan i ica ion o mic obial communi ies: Applica ions o cold and d y olcanic habi a s. Glob Ecol Conse 2018;16:e00458. Quinn R, Zen AP, G un hane F, e al. De ec ion and cha ac- e iza ion o oxidizing acids in he A acama Dese using he Ma s Oxida ion Ins umen . Plane Space Sci 2005;53(13): 1376–1388; doi: 10.1016/j.pss.2005.07.004 Ri as LA, Ga cı ´a-Villadangos M, Mo eno-Paz M, e al. A 200- an ibody mic oa ay biochip o en i onmen al moni o ing: Sea ching o uni e sal mic obial bioma ke s h ough immu- nop o iling. Anal Chem 2008;80;7970–7979. Rummel JD, Bea y DW, Jones MA, e al. A new analysis o Ma s ‘‘special egions’’: Findings o he second MEPAG special egions science analysis g oup (SR-SAG2). As o- biology 2014;14:887–968. Ru ala WA, Webe DJ; he Heal hca e In ec ion Con ol P ac- ices Ad iso y Commi ee. Guideline o Disin ec ion and S e iliza ion in Heal hca e Facili ies. 2019; p. 163. A ailable om: h ps://www.cdc.go /in ec ioncon ol/pd /guidelines/ disin ec ion-guidelines-H.pd [Las accessed: Decembe 11, 2023]. Sanchez-Ga cı ´a L, Fe na ´ndez-Ma ı ´nez MA, Mo eno-Paz M, e al. Simula ing Ma s d illing mission o sea ching o li e: G ound- u hing lipids and o he complex mic obial bio- ma ke s in he i on-sul u ich Rı ´o Tin o analog. As obiology 2020;20(9):1029–1047; doi: 10.1089/as .2019.2101 San hanam R, Oko o CK, Rong X, e al. S ep omyces a aca- mensis sp. no ., isola ed om an ex eme hype -a id soil o he A acama Dese , Chile. In J Sys E ol Mic obiol 2012;62: 2680. Schulze-Makuch D, Wagne D, Kouna es SP, e al. T ansi o y mic obial habi a in he hype a id A acama Dese . P oc Na l Acad Sci U S A 2018;115(11):2670–2675; doi: 10.1073/ pnas.1714341115 U i skiy G, Ge sin S, Munn, A, e al. Response o ex emophile mic obiome o a a e ain all e eals a wo-s ep adap a ion mechanism. bioRxi 442525, 2018; doi: 10.1101/442525 Shama G, Malik DJ. The uses and abuses o apid bioluminescence-based ATP assays. In J Hyg En i on Heal h 2013;216:115–125. Shimaku a M. E icien polishing me hod o i anium. J Soc Ti anium Alloys Den is 2007;5:32–33. Si agusa SR, Do sa WJ, Cu e CN, e al. Use o a newly de eloped apid mic obial ATP bioluminescence assay o de ec mic obial con amina ion on poul y ca casses. J Bio- luminesc Chemiluminesc 1996;11(6):297–301. Skelley AM, Sche e JR, Aube y AD, e al. De elopmen and e alua ion o a mic ode ice o amino acid bioma ke de ec ion and analysis on Ma s. P oc Na l Acad Sci U S A 2005;102:1041–1046. 1334 BONACCORSI ET AL. Smi h SA, Bena dini JN, Ande l D, e al. Iden i ica ion and cha ac e iza ion o ea ly mission phase mic oo ganisms esiding on he Ma s Science Labo a o y and assessmen o hei po en ial o su i e Ma s-like condi ions. As obiology 2017;17(3):253–265; doi: 10.1089/as .2015.1417 So en GA, Snyde CW. Fi s Viking mission o Ma s. Science 1976;193:759–766. Sp y JA, Siegel B, Kminek G, e al. Plane a y p o ec ion knowledge gaps and enabling science o human Ma s mis- sions. Bull Am As on Soc 2021;53(4):205. S anley PE. Ex ac ion o adenosine iphospha e om mic o- bial and soma ic cells. Me hods Enzymol 1986;133:14–22. S oke CR, Cannon HN, Dunagan SE, e al. The 2005 MARTE Robo ic D illing Expe imen in Rio Tin o Spain: Objec i es, app oach, and esul s o a simula ed mission o sea ch o li e in he Ma ian subsu ace. As obiology 2008;8(5):921–945. S oke CR, Glass BJ, Glass, S ucky TR, e al. A mission si- mula ing he sea ch o li e on Ma s wi h au oma ed d illing, sample handling, and li e de ec ion ins umen s pe o med in he hype a id co e o he A acama Dese , Chile. As obiology 2023;23(12):1284–1302; doi: 10.1089/as .2022-0055 Taewan K, Hyung-eun K, Jiyoon C, e al. Inac i a ion o Esche ichia coli and MS2 coliphage ia single oxygen gene a ed by homogeneous pho osensi iza ion. Ko ean J Chem Eng 2019;36(11):1785–1790; doi: 10.1007/s11814- 019-0353-4 Tajima K, Hi onaka M, Chen K, e al. Elec opolishing o CP i anium and i s alloys in an alcoholic solu ion-based elec- oly e. Den Ma e J 2008;27(2):258–265. U i skiy G, Ge sin S, Munn A, e al. Halophilic mic obial communi y composi ional shi a e a a e ain all in he A acama Dese . ISME J 2019;13(11):2737–2749; doi: 10 .1038/s41396-019-0468-y Venka eswa an K, Ha o i N Duc MTL, e al. ATP as a bio- ma ke o iable mic oo ganisms in clean- oom acili ies. J Mic obiol Me hods 2003;52(3):367–377. Wa en-Rhodes KA, Rhodes KL, Poin ing SB, e al. Hypoli hic cyanobac e ia, d y limi o pho osyn hesis, and mic obial ecology in he hype -a id A acama Dese . Mic ob Ecol 2006; 52:389–398. Wa en-Rhodes KA, Lee KC, A che SDJ, e al. Subsu ace mic obial habi a s in an ex eme dese Ma s-analog en i- onmen . F on Mic obiol 2019;10:69; doi: 10.3389/ micb .2019.00069 Weschle CJ. Cha ac e iza ion echniques applied o indoo dus . En i on Sci Technol 1978;12(8):923; doi: 10.1021/ es60144a011 Wes all F, Fouche F, Bos N, e al. Biosigna u es on Ma s: Wha , whe e, and how? Implica ions o he sea ch o ma - ian li e. As obiology 2015;15:998–1029. Wie zchos J, Ascaso C, McKay CP. Endoli hic cyanobac e ia in hali e ocks om he hype a idco e o he A acama Dese . As obiology 2006;6:415–422. Wie zchos J, Cama a B, de los Rios A, e al. Mic obial colo- niza ion o Ca-sul a e c us s in he hype a id co e o he A acama Dese : Implica ions o he sea ch o li e on Ma s. Geobiology 2011;9(1):44–60; doi: 10.1111/j.1472-4669.2010 .00254.x Wilhelm MB, Da ila AF, Pa en eau MN, e al. Cons ain s on he me abolic ac i i y o mic oo ganisms in A acama su ace soils in e ed om e ac o y bioma ke s: Implica ions o ma ian habi abili y and bioma ke de ec ion. As obiology 2018;10(7):955–966. Willis P, C eame JS, Mo a M. Implemen a ion o mic ochip elec opho esis ins umen a ion o u u e space ligh mis- sions. Anal Bioanal Chem 2015;407:6939–6963. Willson D, Bonacco si R, Gold R, e al. Con amina ion Man- agemen o Sample Collec ion De ices and Ocean Analogs o Li e De ec ion on Icy Wo ld Plume Fly- h ough Missions. Pape ID. B5.3-64-18. 42nd COSPAR Scien i ic Assembly, July 14–22, 2018, Pasadena, CA, 2018. Wi z KPJ. Pho ochemis y o O ganic Compounds: F om Con- cep s o P ac ice. John Wiley & Sons: Chiches e , UK; 2009. Zwa k uis E, Be ay G, Wilke T. Rapid es ing o mic obial su ace con amina ion on eal ca casses using ATP biolu- minescence. Fleischwi scha 1999;79:101–103. Add ess co espondence o: Rosalba Bonacco si NASA Ames Resea ch Cen e MS. 245-3 Mo e Field, CA 94305 USA E-mail: [email p o ec ed] Submi ed 10 Oc obe 2022 Accep ed 2 July 2023 Associa e Edi o : Jack Mus a d Abb e ia ions Used 1 O 2 ¼single oxygen 3D ¼ h ee-dimensional ARADS ¼A acama Ro e As obiology D illing S udies ASB ¼ATP syn hase subuni 1 ASF1 ¼ATP syn hase subuni 2 ATLO ¼Assembly, Tes , and Launch Ope a ions ATP ¼adenosine iphospha e A g. ¼a e age CCSI ¼Con amina ion Con ol S a egy and Implemen a ion CFU ¼colony o ming uni COSPAR ¼Commi ee on Space Resea ch DHMR ¼d y hea o mic obial educ ion DP ¼deep-cleaning DW ¼dis illed/deionized wa e E -OH ¼e hanol FI ¼ luo escence in ensi y FSMI ¼Fluo escence Sandwich Mic oa ay Immunoassay FTIR ¼Fou ie ans o m in a ed GC-MS ¼gas ch oma og aph–mass spec ome e GNCA ¼G am-nega i e bac e ia ances al be a-lac amase GP ¼G een Pa o GPBCA ¼gammap o eobac e ial ances al be a-lac amase H 2 O 2 ¼hyd ogen pe oxide HD ¼ha dwa e IPA ¼isop opyl alcohol ISAD ¼Icy Soil Acquisi ion De ice LAL ¼Limulus Amebocy e Lysa e LC/MS ¼liquid ch oma og aphy/mass spec ome y LITMS ¼Linea Ion T ap Mass Spec ome e L-L ¼luci e in-luci e ase LOD ¼limi o de ec ion LPS ¼lipopolysaccha ides Me -OH ¼me hanol ARADS CONTAMINATION CONTROL STRATEGY IMPLEMENTATION 1335 MILA ¼Mic o luidic Li e Analyze MSL ¼Ma s Science Labo a o y ND ¼no de ec ion NSA ¼NASA S anda d assay PLFA ¼phospholipid a y acids PP ¼Plane a y P o ec ion ppb ¼pa s pe billion PPE ¼pe sonal p o ec i e equipmen PSTAR ¼Plane a y Science and Technology Th ough Analog Resea ch RLU ¼ ela i e luminosi y uni s SHTS ¼Sample Handling and T ans e Sys em SOLID ¼Signs o Li e De ec o STD ¼s anda d de ia ion SWA ¼swab analysis TEGA ¼The mal E ol ed Gas Analyze TRIDENT ¼The Regoli h and Ice D ill o Explo a ion o New Te ains UHV ¼ul a high acuum WCL ¼We Chemis y Labo a o y 1336 BONACCORSI ET AL.