Our recent finding that dilution limits dissolved organic carbon (DOC) utilization in the deep ocean has been criticized based on the common misconception that lability equates to rapid and complete utilization. Even when considering the redefinition of recalcitrant DOC recently proposed by Jiao et al., the dilution hypothesis best explains our experimental observations.This is a contribution to the MALASPINA Expedition 2010 project, funded by the CONSOLIDER-Ingenio 2010 program of the Spanish Ministry of Economy and Competitiveness (ref. CSD2008-00077)Peer Reviewe

Arrieta López de Uralde, Jesús M.

Dittmar, Thorsten

Duarte, Carlos M.

Hansman, Roberta L.

Herndl, Gerhard J.

Mayol, Eva

Digital.CSIC

Response	to	Comment	on	“Dilution	limits	dissolved	organic	carbon	utilization	in	the	deep	ocean”	 	Authors:  Jesús M. Arrieta1,2*, Eva Mayol2, Roberta L. Hansman3, Gerhard J. Herndl3,4, Thorsten Dittmar5 and Carlos M. Duarte1,2 Affiliations: 1 Red Sea Research Center, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia 2 Department of Global Change Research, Institut Mediterrani d'Estudis Avançats (IMEDEA), Consejo Superior de Investigaciones Científicas (CSIC)/Universidad de las Islas Baleares (UIB), 07190 Esporles, Spain 3 Department of Limnology and Bio-Oceanography, Division Bio-Oceanography, University of Vienna, Althanstr. 14, 1090 Vienna, Austria 4 Department of Biological Oceanography, Royal Netherlands Institute for Sea Research (NIOZ), 1790AB Den Burg, The Netherlands 5 Research Group for Marine Geochemistry (ICBM-MPI Bridging Group), Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University Oldenburg, Germany *Correspondence to: J.M. Arrieta e-mail: jesus.arrieta@kaust.edu.sa, Telephone: +966128082566  	Abstract	Our	recent	finding	that	dilution	limits	dissolved	organic	carbon	(DOC)	utilization	in	the	deep	ocean	(1)	has	been	criticized	(2)	based	on	the	common	misconception	that	lability	equates	to	rapid	and	complete	utilization.	Even	when	considering	the	redefinition	of	recalcitrant	DOC	recently	proposed	by	Jiao	et	al.(3)	the	dilution	hypothesis	best	explains	our	experimental	observations.		A	large	reservoir	of	DOC	preserved	within	the	deep	oceanic	water	masses	has	been	termed	“refractory”	or	“recalcitrant”	in	the	bulk	of	the	scientific	literature.	Recalcitrant	DOC	is	defined	as	“resistant	to	rapid	microbial	degradation”(4)	or	“resistant	to	microbial	utilization”(3)	implying	that	the	intrinsic	properties	of	these	substrates	make	them	unavailable	to	oceanic	microbes.	In	a	recent	study(1),	we	tested	the	alternative	hypothesis	that	the	dilution	of	the	different	constituents	of	DOC	rather	than	their	chemical	properties	limits	DOC	utilization	in	the	deep	ocean	(5).	Our	results	show	that	a	substantial	part	of	the	deep-sea	DOC	pool	is	labile	(i.e.,	available	to	the	in	situ	microbial	community)	but	the	low	individual	concentrations	of	its	many	constituents	slow	down	the	overall	rates	of	utilization.	Jiao	et	al.(6)	recently	redefined	recalcitrant	DOC	(RDOC)	as	consisting	of	two	pools:	RDOCc		comprising	labile	compounds	that	are	too	diluted	to	be	efficiently	utilized	by	bacteria	(the	same	mechanism	that	we	demonstrate)	and	RDOCt	consisting	of	labile	compounds	that	cannot	be	used	by	the	actual	microbial	community	in	their	environmental	context.	Our	study	does	not	rule	out	the	existence	of	recalcitrant	molecules,	which	would	be	impossible	to	test	experimentally	for	each	of	the	thousands	of	different	compounds	present.	Yet,	Jiao	et	al.	(6)	implicitly	deny	the	existence	of	structurally	recalcitrant	molecules	in	the	DOC	pool	as	none	of	the	RDOC	fractions	in	their	new	definition	is	inherently	“recalcitrant”.	Thus,	Jiao	et	al.(6)	concur	with	our	assertion	that	much	of	the	deep	oceanic	DOC	is	not	intrinsically	recalcitrant	as	defined	in	the	bulk	of	existing	literature	(3,	4,	7,	8).			In	their	comment,	Jiao	et	al.	(2)	argue	that	our	data	do	not	support	the	dilution	hypothesis	because	increasing	the	DOC	concentrations	in	our	experiments	did	not	result	in	a	much	higher	percentage	of	DOC	utilization.	They	also	model	the	DOC	pool	as	an	even	mixture	of	compounds	present	at	three	concentration	levels.	Based	on	this	crude	model,	they	estimate	that	DOC	availability	should	increase	on	average	from	6	to	90%	when	increasing	the	bulk	DOC	concentration	by	a	factor	of	10	if	all	of	the	DOC	were	labile.	Consequently,	and	based	on	the	fact	that	the	fraction	of	carbon	utilized	at	the	end	of	our	experiments	did	not	exceed	6%	in	most	cases,	Jiao	et	al.	(2)	estimate	that	labile	carbon	represents	at	most	6%	of	the	total	DOC	and	conclude	that	the	remaining	94%	must	be	recalcitrant	or,	according	to	their	new	definition	of	recalcitrant	DOC,	“context-recalcitrant”	DOC.			Unfortunately,	their	model	is	flawed	by	the	underlying	misconception	that	lability	implies	immediate	and	complete	utilization	regardless	of	concentration.	Thus,	they	assume	that	the	amount	of	DOC	consumed	at	the	end	of	the	experiments	represents	the	total	amount	of	labile	DOC	present	over	threshold	concentrations.	This	naïve	expectation	assumes	that	40	days	suffice	to	utilize	all	the	labile	DOC	in	the	samples	and	that	the	utilization	of	a	given	substrate	is	either	inhibited	or	proceeds	at	maximum	speed	in	contrast	with	the	well-established	concentration-dependent	model	of	microbial	substrate	utilization	(9).	A	more	likely	scenario,	consistent	with	microbial	substrate	uptake	kinetics,	is	that	in	the	deep	ocean	DOC	utilization	will	be	relatively	slow,	limited	by	molecular	diffusion,	with	uptake	systems	operating	well	below	their	maximum	velocity	at	the	low	end	of	the	Monod	curve.	Our	experimental	data	indicate	that	labile	DOC	was	not	used	completely	at	the	end	of	the	experiment	and	that	substantial	DOC	consumption	occurred	after	no	apparent	increase	in	cell	abundance	was	detectable	(1).	Although	our	experiment	was	designed	to	test	the	growth	response	of	bacteria	and	not	to	estimate	the	total	amount	of	labile	DOC	a	more	realistic	estimation	of	what	happened	in	our	experiment	can	be	obtained	by	using	rates	of	utilization	rather	than	final	concentrations.		To	better	illustrate	these	processes,	we	performed	a	simulation	in	which	utilization	was	solely	limited	by	concentration	(Figure	1A).	Total	consumption	rates	were	higher	with	increasing	concentrations;	however,	substrate	consumption	did	not	decrease	down	to	the	control	levels	after	one	year.	The	%	of	DOC	utilized	did	increase	with	increasing	concentrations	but	very	slowly.	The	total	DOC	utilized	after	40	d	equaled	2.1,	2.6,	3.7	and	5.3%	of	the	initial	concentration	for	controls,	two-,	five-	and	ten-fold	concentrated	treatments	(Figure	1B),	well	within	the	range	of	our	observations	but	probably	not	enough	to	allow	significant	statistical	comparisons	given	the	scatter	associated	to	a	40-day	incubation	experiment.	Hence,	this	simulation	demonstrates	that	the	small	proportion	of	DOC	used	in	our	experiments	is	consistent	with	the	dilution	hypothesis	although	complete	consumption	of	labile	substrates	would	take	much	longer	as	it	does	in	the	deep	ocean.	Thus,	the	use	of	short-term	incubation	experiments	to	quantify	the	amount	of	labile	DOC	present	in	natural	samples	proposed	by	Jiao	and	colleagues	leads	to	wrong	conclusions	(2).	The	corollary	of	the	dilution	hypothesis	that	“prokaryotic	consumption	is	related	to	DOC	concentration”	still	holds	since	both	microbial	growth	and	total	substrate	consumption	markedly	increased	in	concentrated	samples	as	compared	to	controls	clearly	demonstrating	that	concentration	limited	prokaryotic	growth.	Jiao	et	al.	(2)	argue	that	the	growth	observed	in	our	experiments	could	be	due	to	a	small	amount	of	labile	DOC	already	detectable	in	the	unamended	controls,	implicitly	admitting	that	growth	(and	therefore	DOC	consumption)	was	limited	by	concentration	at	least	for	this	small	fraction	of	DOC	that	they	estimate	to	be	6%	of	the	bulk	concentration.	Yet,	consumption	was	detected	across	thousands	of	different	compounds	in	both	controls	and	concentrated	samples	(1)	which	probably	comprise	a	much	higher	proportion	of	the	total	DOC.	Thus,	a	large	fraction	of	the	DOC	compounds	(and	of	the	bulk	DOC)	in	our	samples	was	neither	intrinsically-	nor	context-recalcitrant	(there	was	no	difference	in	“biogeochemical	context”	between	treatment	and	controls	apart	from	DOC	concentration)	and	dilution	remains	the	mechanism	that	best	explains	our	results.	The	fact	that	we	detected	utilization	of	fewer	compounds	in	the	concentrated	treatment	in	one	of	the	experiments	does	not	make	these	molecules	less	labile.		Jiao	et	al.	also	argue	that	other	sources	of	labile	materials	like	chemolithoautotrophy,	grazing	and	viral	lysis	may	have	played	a	role,	however	it	is	unclear	how	chemolithoautotrophy	would	be	enhanced	by	increasing	DOC	concentrations.	Increases	in	grazing	and	viral	lysis	are	incidentally	derived	from	enhanced	bacterial	growth	caused	by	higher	DOC	concentrations	but	not	the	primary	factor	fueling	bacterial	growth.	Our	results	indicate	that	apparent	recalcitrance	is	largely	related	to	low	substrate	concentrations.	Thus,	deep	oceanic	microbes	may	not	be	functioning	as	a	“carbon	pump”	(2,	3),	transforming	labile	DOC	into	recalcitrant	compounds	prone	to	accumulate	in	the	ocean	but	rather	as	efficient	degraders	of	a	vast	array	of	compounds	even	at	very	low,	growth-limiting	concentrations.	Microbial	production	of	persistent	DOC	by	mechanisms	other	than	substrate	dilution	remains	to	be	demonstrated	although	recent	reports	suggest	that	this	contribution	may	be	minor	(10).		Acknowledgements	This	is	a	contribution	to	the	MALASPINA	Expedition	2010	project,	funded	by	the	CONSOLIDER-Ingenio	2010	program	of	the	Spanish	Ministry	of	Economy	and	Competitiveness	(Ref.	CSD2008-00077).		References		1.		 J.	M.	Arrieta	et	al.,	Dilution	limits	dissolved	organic	carbon	utilization	in	the	deep	ocean.	Science.	348,	331–333	(2015).	2.		 N.	Jiao	et	al.,	Comment	on“Dilution	limits	dissolved	organic	carbon	utilization	in	the	deep	ocean.”	Science	(2015).	3.		 N.	Jiao	et	al.,	Microbial	production	of	recalcitrant	dissolved	organic	matter:	long-term	carbon	storage	in	the	global	ocean.	Nat	Rev	Micro.	8,	593–599	(2010).	4.		 D.	A.	Hansell,	Recalcitrant	Dissolved	Organic	Carbon	Fractions.	Annu.	Rev.	Mar.	Sci.	5,	421–445	(2013).	5.		 H.	W.	Jannasch,	Growth	of	marine	bacteria	at	limiting	concentrations	of	organic	carbon	in	seawater.	Limnol.	Oceanogr.,	264–271	(1967).	6.		 N.	Jiao	et	al.,	Mechanisms	of	microbial	carbon	sequestration	in	the	ocean	–	future	research	directions.	Biogeosciences.	11,	5285–5306	(2014).	7.		 H.	Ogawa,	Y.	Amagai,	I.	Koike,	K.	Kaiser,	R.	Benner,	Production	of	refractory	dissolved	organic	matter	by	bacteria.	Science.	292,	917–920	(2001).	8.		 R.	L.	Sinsabaugh,	S.	Findlay,	in	Aquatic	Ecosystems	(Academic	Press,	Burlington,	2003),	pp.	479–498.	9.		 J.	Monod,	The	Growth	of	Bacterial	Cultures.	Annu.	Rev.	Microbiol.	3,	371–394	(1949).	10.		H.	Osterholz,	J.	Niggemann,	H.-A.	Giebel,	M.	Simon,	T.	Dittmar,	Inefficient	microbial	production	of	refractory	dissolved	organic	matter	in	the	ocean.	Nat.	Commun.	6	(2015),	doi:10.1038/ncomms8422.			 	Figure	legends		Figure	1.	Expected	DOC	utilization	as	a	function	of	DOC	enrichment	in	a	scenario	where	utilization	is	limited	by	dilution.	We	started	our	simulation	with	three	different	DOC	concentration	levels	in	each	sample	(0.8,	1	and	1.2	arbitrary	units	in	the	control	and	the	corresponding	two-,	five-	and	ten-fold	initial	concentrations)	for	consistency	with	the	model	of	Jiao	et	al.(2).	The	rate	of	utilization	for	each	DOC	pool	was	calculated	as	a	function	of	substrate	concentration	and	cell	abundance,	assuming	a	specific	substrate	affinity	of	1x10-11	(l	cell-1	d-1)	and	using	the	maximum	cell	abundances	observed	in	experiment	N	for	each	treatment	(55,	67,	97	and	139	x106	prokaryotes	L-1	for	the	controls,	two-,	five-	and	ten-fold	DOC	treatments,	respectively).	The	average	specific	substrate	affinity	was	estimated	from	the	2%	decrease	in	DOC	concentration	observed	in	control	treatments	of	experiments	K-N	after	40	days.	Our	calculated	utilization	rates	are	proportional	to	substrate	concentration	and	we	have	assumed	one	average	affinity	for	all	the	substrates,	thus	our	model	does	not	change	if	we	use	one	or	many	substrates	and	therefore	only	bulk	DOC	concentrations	are	presented.	This	simplified	model	also	assumes	that	all	of	the	bacteria	present	were	able	to	utilize	all	of	the	substrates	present	with	the	same	average	affinity	and	is	likely	to	result	in	an	overestimation	of	utilization	rates	as	compared	to	the	real	world	scenario	involving	a	diverse	microbial	community	feeding	on	a	very	diverse	molecular	mixture	of	DOC	including	a	large	range	of	concentrations	and	affinities.	Despite	these	limitations	this	is	a	more	realistic	approach	that	more	closely	resembles	the	processes	measured	in	our	experiments	as	well	as	those	occurring	in	the	deep	ocean.	A.	Bulk	consumption	of	DOC	over	time.	Increasing	substrate	concentration	ten-fold	results	in	a	much	higher	bulk	consumption	rate,	however	labile	substrates	are	not	brought	down	to	the	initial	levels	after	one	year	of	incubation.		B.	Percentage	of	substrate	remaining	over	one	year	of	incubation.	At	least	95%	of	the	initial	substrate	remains	unused	after	40	days	(dotted	line)	of	microbial	consumption	even	in	the	most	concentrated	treatments.	Note	that	we	assumed	that	utilization	rates	increase	constantly	with	concentration	and	this	is	only	valid	at	the	low	end	of	the	Monod	model.	If	substrate	concentrations	reached	saturation	levels	in	the	more	concentrated	treatments	as	observed	in	our	experiments,	the	differences	between	the	percentage	of	utilization	in	concentrated	versus	control	treatments	would	be	even	smaller.			10203040remainng DOC concentration(arbitrary units)0 100 200 300 4005060708090100time (d)%intial DOC remainingControl2xDOC5xDOC10xDOCAB

Response to Comment on >dilution limits dissolved organic carbon utilization in the deep ocean>

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