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Journal of Endocytobiosis and Cell Research (2015) 1-7 | International Society of Endocytobiology
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Journal of Endocytobiosis and Cell Research VOL 26 | 2015 1 
Journal of 
Endocytobiosis and
Cell Research
Uptake and biosorption potential of Pistia stratiotes for Cr6+
Aziz-ud-Din1
, Zeshan Ali2
*, Farrakh Meh-
boob2
and Qaiser Mahmood Khan3
1Department	of	Genetics,	Garden	Campus,	Hazara	Universi‐
ty,	Mansehra,	Pakistan;	2,*Ecotoxicology	Research	Institute,	
National	Agricultural	Research	Centre,	Park	Road,	Islama‐
bad,	 PO	 45500,	 Pakistan;	 3Soil	 &	 Environmental	 Biotech‐
nology	 Division,	 National	 Institute	 for	 Biotechnology	 &	
Genetic	Engineering	(NIBGE),	Faisalabad,	Pakistan;		
*correspondence	to:	eco4nd@yahoo.com	
	
Uptake	and	biosorption	potential	of	Pistia	stratiotes	for	
Cr6+	 was	 examined.	 The	 study	 was	 conducted	 in	 two	
phases:	 in	 the	 first	 phase,	 the	 effect	 of	 different	 Cr6+	
concentrations	on	plant	weight,	leaf	number	and	root	
length	was	determined	along	with	plants	uptake	poten‐
tial.	Higher	concentration	of	Cr6+	(>	8	ppm)	had	inhibi‐
tory	effects	on	increase	in	plant	weight	due	to	impair‐
ment	 of	 the	 metabolic	 machinery.	 Leaf	 number	 and	
root	length	showed	no	increase	when	exposed	to	a	Cr6+	
concentration	 of	 ~	 10	 ppm.	 When	 grown	 in	 solutions	
containing	2,	or	4	ppm	Cr6+	concentrations,	P.	stratiotes	
removed	100%	of	Cr6+.	However	at	increasing	concen‐
trations	of	6	and	8	ppm,	only	50%	reduction	in	respec‐
tive	solutions	was	recorded.	In	a	second	phase	biosorp‐
tion	capacity	of	dead	P.	stratiotes	was	determined.	The	
optimal	 pH	 range	 for	 maximum	 Cr6+	 biosorption	 lied	
between	 1‐2.	 Maximum	 biosorption	 capacity	 of	 dead	
biomass	 was	 recorded	 at	 200	 ppm	 of	 the	 initial	 Cr6+	
concentration.	 The	 volume	 and	 pH	 of	 a	 Cr6+	 solution	
passing	through	the	columns	were	critically	important	
affecting	the	biosorption	capacity	of	P.	stratiotes.	These	
findings	 are	 important	 for	 cost‐effective	 and	 environ‐
mental	friendly	removal	of	Cr6+	from	tannery	effluents	
which	 are	 posing	 serious	 risk	 to	 human	 and	 environ‐
mental	health	in	tannery	hubs	of	Pakistan.		
	
Journal	of	Endocytobiosis	and	Cell	Research	(2015)	1‐7	
Category:	Research	paper	
Keywords:	Pistia	stratiotes,	biosorption,	Cr6+,	environmental	
health,	tannery	
	
Accepted:	09	January	2015	
____________________________________________________________________	
	
	
Introduction	
Chromium	(Cr)	is	an	important	environmental	contaminant	
that	generates	from	numerous	anthropogenic	activities,	i.e.	
tanning,	electroplating,	mining,	metal	finishing,	dyes	prepa‐
ration,	 cement	 industries	 and	 preparation	 of	 corrosive	
paints	(Ahn	et	al.	1999).	In	last	decades	its	concentration	
has	consistently	increased	in	different	environmental	com‐
partments	due	to	elevated	municipal	and	industrial	activi‐
ties	reciprocal	to	human	demands	(Ali	et	al.	2013a).	Cr	like	
other	heavy	metals	is	a	serious	health	risk,	i.e.	toxic,	persis‐
tent,	carcinogenic,	mutagenic	and	teratogenic	(Gourdon	et	
al.	1990).		
Environmental	behaviour	of	Cr	is	largely	a	function	of	
its	oxidation	state	(Losi	et	al.	1994).	It	can	exist	in	various	
oxidation	states;	of	which	the	trivalent	form	(Cr3+)	is	most	
stable	 and	 insoluble	 in	 water	 at	 neutral	 pH	 (Losi	 et	 al.	
1994).	 Cr3+	 in	 presence	 of	 mild	 oxidants	 can	 easily	 trans‐
form	to	hexavalent	form	(Cr6+)	which	is	highly	water	solu‐
ble	at	neutral	pH	(Cary	1982).	Cr6+	 is	a	stronger	oxidizing	
agent	 than	 Cr3+	 and	 this	 property	 is	 likely	 related	 to	 its	
higher	 toxicity	 for	 most	 organisms	 (Rapoport	 and	 Mutter	
1995).	Cr6+	 is	described	as	500	times	more	toxic	than	 Cr3+	
(Kowalski	1994).	Transition	between	these	two	forms	of	Cr	
is	important	for	the	accumulation	tendency,	transport	and	
toxicity	in	ecological	compartments	(Smith	et	al.	1989).	
In	Pakistan	the	most	important	anthropogenic	source	of	
Cr	in	the	environmental	compartments	is	tannery	industry	
that	 plays	 a	 significant	 role	 in	 the	 economic	 uplift	 of	 the	
country	(Ali	et	al.	2015).	There	are	around	650	registered	
tannery	units	in	different	cities	of	Pakistan,	i.e.	Gujranwala,	
Multan,	Kasur,	Karachi,	Peshawar,	Sialkot	and	Sahiwal	(Ali	
et	 al.	 2013a).	 Various	 studies	 have	 highlighted	 higher	 Cr	
levels	 in	 the	 soils,	 sediments	 and	 underground/surface	
water	of	these	cities	due	to	unchecked/untreated	tannery	
industrial	effluents	(Tariq	et	al.	2006;	Qadir	et	al.	2008;	Ali	
et	al.	2015).	Tannery	effluents	contain	Cr	in	both	oxidation	
states,	i.e.	Cr6+	and	Cr+3.	Due	to	higher	toxicity,	the	hexava‐
lent	 form	 (Cr6+)	 has	 remained	 a	 pressing	 concern	 in	 tan‐
nery	industrial	effluents	(Gong	et	al.	2010).	Cr6+	has	smaller	
size	which	allows	it	to	breach	through	the	biological	mem‐
branes	adding	to	its	toxicity.	Many	efforts	have	been	made	
to	treat	Cr6+	in	wastewater	to	reduce	environmental	pollu‐
tion	 (Leland	 et	 al.	 1978).	 Conventional	 methods	 used	 for	
the	 treatments	 and	 disposal	 of	 Cr6+	 bearing	 wastewater	
include	 chemical	 precipitation,	 ion	 exchange,	 membrane	
separation,	solvent	extraction	and	adsorption,	etc.	(Lee	et	
al.	1998).	These	methods	either	are	very	expensive	or	re‐
quire	 extensive	 electrical/chemical/mechanical	 inputs	
(Farid	et	al.	2014).	Bioremediation	and	biosorption	on	the	
other	 hand	 offer	 cost‐effective	 and	 environment	 friendly	
disposal	 of	 Cr6+	 bearing	 wastewaters	 without	 any	 further	
environmental	peril.		
The	present	research	is	focused	on	the	bioremediation	
and	biosorption	of	Cr6+	 using	the	indigenous	aquatic	plant	
Pistia	 stratiotes	 (water	 lettuce).	 Water	 lettuce	 exists	 as	
common	weed	in	lentic	habitats	of	Pakistan	and	is	a	recog‐
nized	 metal	 hyperaccumulator	 (Lu	 et	 al.	 2010).	 The	 basic
Potential of Pistia stratiotes for Cr6+
, Aziz-ud-Din et al.
2 Journal of Endocytobiosis and Cell Research VOL 26 | 2015 
aim	of	this	research	work	is	to	develop	an	efficient	biore‐
mediation/biosorption	method	for	Cr6+	removal	that	can	be	
replicated	at	national	scale	in	tannery	industrial	cities	for	
regular	Cr6+	pollution	abatement	strategies.	This	study	will	
also	serve	as	guideline	for	the	legislators	and	environmen‐
tal	 managers	 for	 the	 management	 of	 Cr6+	 contaminated	
environments	around	tanneries.			
	
	
Material	and	methods	
Sample	collection	
Fresh	 and	 healthy	 water	 lettuce	 (P.	 stratiotes)	 plants	 of	
uniform	 size	 (number	 of	 leaves	 per	 plant	 =	 10	 ±	 2;	 root	
length	=	11.5	±	2	cm)	and	weight	(11.2	±	1.5	g	fresh	weight)	
were	collected	from	the	canal	water	near	Ayub	Agricultural	
Research	 Institute,	 Faisalabad.	 Collected	 plants	 were	
washed	 with	 tap	 water	 followed	 by	 distilled	 water	 to	 re‐
move	 mud,	 debris	 and	 other	 foreign	 particles	 attached	 to	
submerged	and	aerial	plant	parts.		
	
Use	of	P.	stratiotes	(living	plants)	for	Cr6+	removal		
The	 experiment	 was	 performed	 in	 a	 greenhouse	 in	 large	
plastic	 tubes	 of	 10	 litre	 capacity.	 Synthetic	 Cr6+	 solutions	
were	 prepared	 in	 distilled	 water	 using	 analytical	 grade	
K2Cr2O7.	Nine	treatments	in	triplicate	were	used	to	investi‐
gate	 the	 phytoremediation	 potential	 of	 P.	 stratiotes.	 The	
experiment	 was	 set	 up	 in	 completely	 randomised	 design	
with	 factorial	 arrangements.	 Essential	 nutrients	 (Cr	 free)	
were	 also	 added	 in	 each	 tube	 to	 support	 normal	 plant	
growth	and	development	(Hewitt	1966).	
	
Treatment	 Concentration
1	 distilled	water
2	 2	ppm	Cr6+	
3	 4	ppm	Cr6+
4	 6	ppm	Cr6+
5	 8	ppm	Cr6+
6	 10	ppm	Cr6+
7	 12	ppm	Cr6+
8	 14	ppm	Cr6+
9	 16	ppm	Cr6+
	
The	experiment	continued	for	a	 period	of	28	days.	Water	
samples	 after	 an	 interval	 of	 7	 days	 were	 carefully	 with‐
drawn	from	the	tubes	to	analyse	Cr6+	 concentration	using	
the	diphenyl	carbazide	(DPC)	method	(Bartlett	and	James	
1988).	At	the	same	time	plant	growth	attributes,	i.e.	plant	
weight,	number	of	leaves	and	roots	length	were	recorded.	
Triplicate	water	Cr6+	 levels	and	plant	growth	results	from	
each	treatment	were	averaged	and	the	results	are	present‐
ed	in	the	Results/Discussion	section.		
	
Use	of	P.	stratiotes	(dry	biomass)	for	Cr6+	removal	
Collected	 plants	 were	 dried	 in	 an	 oven	 at	 60	 °C	 for	 48	
hours.	Plant	material	was	ground	with	the	help	of	grinder	
after	complete	drying.	The	obtained	powdered	dry	biomass	
was	 used	 in	 pH	 and	 biomass‐dependent	 biosorption	 stu‐
dies	 of	 Cr6+	 from	 synthetic	 solutions.	 pH‐dependent	 bio‐
sorption	 experiments	 were	 set	 up	 in	 Erlenmeyer	 flasks	
(250	ml)	containing	Cr6+	synthetic	solution	(100	ppm)	and	
0.15	g	of	dried	plant	biomass.	The	pH	was	adjusted	with	the	
help	of	NaOH	and	H2SO4.	The	experiment	was	conducted	in	
triplicates	at	room	temperature	and	at	different	pH,	i.e.	1,	2,	
3,	4	and	5.	The	flasks	were	placed	in	a	shaker	at	150	rpm.	
Approximately	 1.5	 ml	 of	 water	 samples	 were	 collected	 in	
Eppendorf	tubes	at	different	time	intervals.	Zero	time	sam‐
ples	 were	 taken	 from	 every	 flask	 before	 adding	 the	 plant	
biomass.	 Biomass	 dependent	 biosorption	 experiment	 was	
conducted	as	described	above,	but	the	pH	was	adjusted	at	
1.5.	 Different	 amounts	 of	 plant	 material,	 i.e.	 0.05	 g,	 0.1	 g,	
0.15	g,	0.2	g	and	0.25	g	were	weighed	and	added	in	differ‐
ent	 flasks.	 These	 flasks	 were	 placed	 on	 a	 rotatory	 shaker	
(150	 rpm)	 at	 room	 temperature.	 Zero	 samples	 were	 col‐
lected	 before	 adding	 the	 biomass.	 Samples	 were	 taken	 in	
Eppendorf	tubes	at	different	time	intervals	for	the	analysis	
of	Cr+6.	
	
Cr6+	uptake	capacity	(q)	was	determined	by	the	following	
equation:	
q
V V
g
	
whereas	
Vi	 =	initial	Cr	concentration,	
Vf	 =	final	Cr	concentration,	
g	 =	weight	of	the	biosorbent	in	g.	
	
Column	biosorption	of	Cr6+	by	P.	stratiotes	dry	biomass	was	
studied	using	glass	columns	of	1.5	cm	internal	diameter	at	
room	 temperature.	 Approximately	 2	 g	 of	 uniformly	 pow‐
dered	 Pistia	 biomass	 was	 added	 from	 the	 top	 and	 rinsed	
with	distilled	water.	The	length	of	the	2	g‐packed	column	
was	 6	 cm.	 Synthetic	 Cr6+	 solution	 (100	 ppm)	 was	 passed	
through	the	column	from	the	top	at	a	flow	rate	of	approxi‐
mately	120	ml/h	and	the	eluents	were	collected	in	100	ml	
fractions.	The	working	temperature	was	kept	at	room	tem‐
perature.	 Column	 biosorption	 of	 Cr6+	 by	 P.	 stratiotes	 was	
observed	 at	 different	 pH	 and	 its	 breakthrough	 curve	 was	
studied.	
	
Statistical	analysis	
Statistical	analysis	was	carried	out	using	Randomised	Com‐
plete	 Block	 Design	 (RCBD)	 and	 Duncan’s	 Multiple	 Range	
Test	(DMRT).			
	
	
Results	and	discussion	
Effect	of	Cr6+	on	vegetative	growth	of	P.	stratiotes	
P.	 stratiotes	 weight,	 leaf	 number	 and	 root	 length	 values	
recorded	from	all	treatments	at	increasing	days	were	sub‐
jected	 to	 ANOVA	 (Table	 1a,	 2a,	 3a).	 Duncan’s	 Multiple	
Range	Test	(DMRT)	of	mean	weight,	leaf	number	and	root	
length	 values	 at	 increasing	 days	 were	 estimated	 and	 pre‐
sented	 in	 Tables	 1b,	 2b	 and	 3b.	 The	 results	 showed	 that	
treatments,	the	time	after	treatments	and	their	interaction	
significantly	contributed	in	the	overall	variance	of	weight,	
leaf	 number	 and	 root	 length	 (Table	 1a).	 DMRT	 of	 mean	
values	 of	 weight	 (Table	 1b)	 showed	 that	 the	 first	 three	
doses	 were	 insignificantly	 different	 from	 each	 other	 with	
respect	 to	 weight.	 Weight	 values	 at	 14,	 21	 and	 28	 days	
insignificantly	differed	from	each	other,	but	showed	higher	
values	 of	 weight	 as	 observed	 immediately	 after	 the	 treat‐
ment.	 The	 results	 related	 to	 the	 effect	 of	 Cr6+	 on	 plant	
weight	showed	that	higher	concentration	of	Cr6+	(>	8	ppm)	
had	marked	inhibitory	effects	on	gain	in	plant	weight.	The	
effect	 was	 more	 prominent	 in	 case	 of	 treatment	 with	 10	
ppm	 of	 Cr6+.	 It	 depicts	 that	 higher	 concentration	 of	 Cr6+	
inhibits	the	plant	growth	showing	toxicity	to	the	test	plant.	
In	another	study,	the	Cr6+	concentration	at	around	20	ppm	
killed	 P.	 stratiotes	 in	 three	 days	 of	 exposure	 (Sen	 et	 al.	
1987).
Potential of Pistia stratiotes for Cr6+
, Aziz-ud-Din et al.
Journal of Endocytobiosis and Cell Research VOL 26 | 2015 3 
Table	1a:	ANOVA	of	weight	of	P.	stratiotes	at	various	time	intervals	after	treatment	with	Cr6+	during	its	growth	experiments		
Source	 D.F.	 S.S.	 M.S. F p	
Replication	 1	 0.716	 0.716 1.4045 0.2442	 n.s*	
Treatment	(T)	 6	 112.357 18.726 36.7286 0	 P < 0.001
Day	(D)	 4	 120.343 30.086 59.0086 0	 P < 0.001
T	x	D	 24	 59.176 2.466 4.836 0	 P < 0.001
Error	 34	 17.335 0.51 	
Total	 69	 309.927
n.s*	‐	not	significant	
	
Table	1b:	DMRT	of	mean	weight	of	P.	stratiotes	at	various	time	intervals	after	treatment	with	Cr6+	
Treatments	(mg/l)	↓	
Days→	 Day	0 Day	7 Day	14 Day	21	 Day	28
C B A A	 A	
2.519 3.569 5.455 5.713	 5.709
0	
B	 MN IJKLMN EFG CDEF	 DEFG
4.572	 2.095 3.415 5.625 5.95	 5.775
2	
A	 KLMN HIJKL ABCD AB	 A	
6.082	 2.53 3.94 7.33 8.135	 8.475
4	
A	 KLMN HIJKL BCDE ABC	 AB
5.688	 2.52 3.93 6.74 7.555	 7.695
6	
A	 IJKLMN GHIJK BCDE ABCDE	 AB
5.711	 2.825 4.18 6.71 7.15	 7.69
8	
B	 KLMN IJKLM FGHI DEFG	 EFGH
4.36	 2.51 3.565 4.46 5.765	 5.5
10	
D	 LMN KLMN IJKLMN MN	 N	
2.336	 2.44 2.49 2.95 1.975	 1.825
12	
C	 JKLMN IJKLMN FGHIJ IJKLMN	 IJKLMN
3.401	 2.71 3.46 4.37 3.46	 3.005
All	mean	values	under	a	category	which	share	a	common	letter	are	insignificantly	different,	otherwise	they	differ	at	p	<	0.05	
	
Table	2a:	ANOVA	of	leaf	number	of	P.	stratiotes	at	various	time	intervals	after	treatment	with	Cr6+	during	its	growth	experiments	
n.s*	‐	not	significant	
	
Table	2b:	DMRT	of	mean	leaf	number	of	P.	stratiotes	at	various	time	intervals	after	treatment	with	Cr6+	during	its	growth	experi‐
ments	
Treatments	(mg/l)	↓	
Days→	 Day	0 Day	7 Day	14 Day	21	 Day	28
		 C C B A	 A
		 4.571 6.5 14.93 20	 22.93
0	
AB	 J IJ EFGHIJ CDEF	 ABC
14.1	 4 6.5 13.5 19	 27.5
2	
A	 J IJ CDEFG ABCD	 AB
16.7	 4.5 6.5 18 23.5	 31
4	
A	 J IJ DEFGH BCDE	 A
16.4	 4.5 6.5 16.5 22.5	 32
6	
AB	 J IJ DEFGHI BCDE	 ABC
15.3	 5 6.5 15 22.5	 27.5
8	
AB	 J IJ FGHIJ CDEF	 ABCD
13	 4.5 6.5 12 18.5	 23.5
10	
C	 J IJ FGHIJ FGHIJ	 FGHIJ
8.7	 5 6 10.5 11.5	 10.5
12	
BC	 J HIJ CDEF BCDE	 GHIJ
12.3	 4.5 7 19 22.5	 8.5
All	mean	values	under	a	category	which	share	a	common	letter	are	insignificantly	different,	otherwise	they	differ	at	p	<	0.05	
Source	 D.F.	 S.S.	 M.S. F p	
Replication	 1	 64.129	 64.129 3.9688 0.0544	 n.s*
Treatment	(T)	 6	 464.086	 77.348 4.787 0.0012	 P < 0.01
Day	(D)	 4	 3661	 915.25 56.6438 0	 P	< 0.001
T	x	D	 24	 973.2	 40.55 2.5096 0.0069	 P < 0.01
Error	 34	 549.371	 16.158 	
Total	 69	 5711.786
Potential of Pistia stratiotes for Cr6+
, Aziz-ud-Din et al.
4 Journal of Endocytobiosis and Cell Research VOL 26 | 2015 
Table	3a:	ANOVA	of	root	length	of	P.	stratiotes	at	various	time	intervals	after	treatment	with	Cr6+	during	its	growth	experiments	
	
Table	3b:	DMRT	of	mean	root	length	of	P.	stratiotes	at	various	time	intervals	after	treatment	with	Cr6+	during	its	growth	experiments	
Treatments	(mg/l)	↓	
Days→	 Day	0 Day	7 Day	14 Day	21	 Day	28
		
C B A AB	 A
4.143 6.929 8 7.429	 8.214
0	
C	 IJ DEFGH CDEFG CDEFG	 CDEF
7	 3.5 7 8 8	 8.5
2	
A	 IJ CDEFGH BCD AB	 A
9.2	 3 7.5 10 11.5	 14
4	
AB	 IJ CDEFGH BCD BC	 BC
8.4	 3.5 7.5 10 10.5	 10.5
6	
BC	 HIJ CDEFG CDEF CDEFGH	 CDEFG
7.3	 4.5 8 8.5 7.5	 8
8	
BC	 EFGHI CDEFGH BCDE CDEFG	 CDEFG
7.7	 6 7.5 9 8	 8
10	
D	 IJ FGHIJ FGHIJ IJ	 IJ
4.5	 4 5.5 5.5 4	 3.5
12	
D	 HIJ FGHIJ GHIJ J	 GHIJ
4.5	 4.5 5.5 5 2.5	 5
All	mean	values	under	a	category	which	share	a	common	letter	are	insignificantly	different,	otherwise	they	differ	at	p	<	0.05	
	
	
Cr6+	 toxicity	is	an	established	phenomenon	in	aquatic	and	
terrestrial	plants.	In	Myriophyllum	spicatum,	an	increase	in	
shoot	length	was	recorded	at	0.05	ppm	Cr6+	level,	however	
when	 its	 concentration	 was	 increased	 to	 1	 ppm,	 linear	
reduction	in	shoot	length/weight	was	recorded.	In	a	study	
on	 three	 herbaceous	 plants,	 i.e.	
Trifolium	 repens,	 Festuca	 arundina‐
cea	and	Medicago	sativa,	Wang	et	al.	
(2012)	 demonstrated	 decrease	 in	
plant	 height,	 dry	 weight	 of	
roots/shoots	 when	 exposed	 to	 Cr6+	
levels	 exceeding	 200	 mg/kg	 in	 pot	
soils.	 The	 results	 regarding	 the	
effects	 of	 Cr6+	 on	 leaf	 number	 and	
increase	in	leaf	number	was	record‐
ed	 up	 to	 concentration	 of	 8	 ppm.	
There	was	relatively	small	increase	
in	leaf	number	at	10	ppm	concentra‐
tion.	 At	 higher	 concentration,	 the	
plant	showed	increase	in	leaf	 num‐
ber	 up	 to	 21	 days,	 but	 after	 that	 it	
did	not	survive.	Working	on	wheat,	
Sharma	 and	 Sharma	 (1993)	 also	
showed	 negative	 correlation	 be‐
tween	 Cr	 concentration	 and	 leaf	
number.	DMRT	mean	values	of	root	
length	 showed	 that	 the	 first	 four	
doses	increased	the	root	length	while	the	latter	two	doses	
reduced	it.	As	days	after	treatment	with	Cr6+	are	concerned,	
the	 values	 of	 root	 length	 increased	 with	 increasing	 days	
after	the	treatments.	There	was	no	or	small	increase	in	the	
root	length	of	P.	stratiotes	at	10	ppm	while	at	higher	con‐
centration	 the	 plant	 showed	 reduction	 in	 the	 root	 length	
after	the	first	week.	Negative	correlation	between	Cr	con‐
centration	and	root	length	of	different	crops	has	also	been	
observed	by	others	(Sharma	and	Sharma	1993;	Stiborova	et	
al.	1986).		
	
	
	
	
	
	
	
Source	 D.F.	 S.S.	 M.S. F p	
Replication	 1	 0.057	 0.057 0.0367 	 P	<	0.001
Treatment	(T)	 6	 198.571	 33.095 21.2538 0	 P	<	0.001
Day	(D)	 4	 151.343	 37.836 24.2982 0	 P	<	0.001
T	x	D	 24	 142.857	 5.952 3.8226 0.0002	 P	<	0.001
Error	 34	 52.943	 1.557 	
Total	 69	 545.771
Figure	1:	Reduction	in	Cr6+	concentration	during	growth	studies	
of	P.	stratiotes.	Treatments	were	(T1	=	2	ppm,	T2	=	4	ppm,	T3	=	6	
ppm,	T4	=	8	ppm,	T5	=	10	ppm,	T6	=	12	ppm).	Data	was	collected	
for	four	weeks	at	7	days	interval	by	growing	P.	stratiotes	plants	
on	the	given	concentrations	of	Cr6+. 
Potential of Pistia stratiotes for Cr6+
, Aziz-ud-Din et al.
Journal of Endocytobiosis and Cell Research VOL 26 | 2015 5 
Uptake	of	Cr6+	during	growth	of	P.	stratiotes	
P.	stratiotes	eliminated	Cr6+	 from	the	solution	at	a	concen‐
trations	of	2	and	4	ppm,	while	at	6	and	8	ppm	the	Cr	con‐
centration	is	reduced	to	one	half.	At	10	ppm	the	reduction	
in	 Cr	 concentration	 in	 the	 solution	 is	 negligible,	 whereas	
there	is	no	reduction	beyond	10	ppm	(Figure	1).	P.	strati‐
otes	uptake	of	Cr	is	affected	by	its	oxidation	state.	Cr6+	can	
either	 directly	 pass	 through	 the	 plasma	 membrane	 or	 via	
carriers,	 i.e.  phosphate‐sulfate	 carriers.	 Cr3+	 on	 the	 other	
hand	 cannot	 use	 any	 specific	 membrane	 transporters	 for	
inward	 movements.	 In	 trivalent	 form	 it	 simply	 diffuses	
through	 the	 plasma	 membrane	 by	 forming	 lipophilic	 lig‐
ands.	 It	 is	 evident	 from	 these	 findings	 that	 P.	 stratiotes	
efficiently	 uptakes/concentrates	 Cr6+	 in	 leaves	 below	 10	
ppm	 concentration	 and	 therefore	 can	 play	 a	 vital	 role	 in	
recycling	Cr6+	from	the	contaminated	industrial	effluents.	
	
Cr6+	biosorption	studies	using	dried	biomass	of	P.	
stratiotesEffect	 of	 different	 dosage	 of	 P.	 stratiotes	
biomass	on	the	biosorption	of	Cr6+			
Five	different	concentration	of	biomass	of	P.	stratiotes,	i.e.	
0.05,	 0.1,	 0.15,	 0.2	 and	 0.25	 g/50	 ml,	 were	 used	 for	 Cr6+	
removal	from	100	ppm	Cr6+	solution.	It	was	found	that	with	
increase	 in	 initial	 biomass,	 the	 removal	 of	 Cr6+	 from	 the	
solution	 also	 increased.	 It	 has	 been	 reported	 extensively	
that	the	initial	concentration	of	biomass	is	important	and	
with	 increase	 in	 biomass,	 the	 rate	 of	 biosorption	 is	 in‐
creased	accordingly	(Kuyucak	1990).	
In	 this	 study,	 0.05	 g	 biomass/50	 ml	 solution	removed	
25%	 Cr6+	 and	 0.1	 g	 biomass/50	 ml	 of	 solution	 removed	
63%	Cr6+,	while	the	higher	amounts,	i.e.	0.15	g,	0.2	g	and	
0.25	 g/50	 ml	 removed	 100%	 Cr6+	 from	 the	 solution	 con‐
taining	initially	100	ppm	Cr6+	(Table	4)	after	6	hour	of	shak‐
ing	 (150	 rpm)	 at	 room	 temperature.	 According	 to	 Gadd	
(1990)	the	most	probable	mechanism	is	that	first	the	cell	
wall	comes	in	contact	with	metal	ions	in	solution,	where	the	
metal	 can	 be	 deposited	 on	 the	 surface	 or	 within	 the	 cell	
wall	 structure	 before	 interacting	 with	 cytoplasm	 material	
or	 other	 cellular	 parts	 (Gadd	 1990;	 Pereira	 et	 al.	 2014).	
This	cell	wall	uptake	may	be	directed	by	functional	groups	
like	 phosphates,	 carboxyl	 groups,	 amines	 or	 phospho‐
diester	 species.	 The	 results	 of	 Crist	 et	 al.	 (1988)	 showed	
that	the	biosorption	of	heavy	metals	has	two	phases:	first	
phase	is	attributed	to	surface	adsorption,	mainly	based	on	
anion	exchange	with	the	participation	of	carboxyl	groups	of	
uronic	acids.	The	second	phase	represents	the	diffusion	of	
ions	into	the	cell.	
	
	
Table	4:	Effect	of	different	dosages	of	P.	stratiotes	dried	biomass	on	Cr6+	biosorption	
Biosorbent	amount	(g)	 0.05 0.1 0.15 0.2	 0.25
Initial	concentration	(ppm)	 100 100 100 100	 100
Concentration	after	6	hours	(ppm)	 75 37 0 0	 0
%	decrease	in	Cr6+	concentration	 25 63 100 100	 100
Uptake	capacity	(q)	(mg/g)	 25 31.5 33.33 25	 20
Volume	of	test	sample	=	50	ml;	pH	=	1.5	
	
	
Effect	of	pH	on	Cr6+	biosorption	
The	results	showing	the	effect	of	different	pH	levels	on	the	
biosorption	of	Cr6+	by	P.	stratiotes	are	summarized	in	Table	
5.	It	was	observed	that	maximum	biosorption	takes	place	at	
lower	initial	pH.	Metal	uptake	from	100	ppm/50	ml	solu‐
tion	using	0.15	g	of	dried	biomass	of	P.	stratiotes	is	100%,	
46%,	15%,	2%	and	0%	at	pH	1,	2,	3,	4	and	5,	respectively.	
Earlier	studies	on	heavy	metal	biosorption	showed	that	pH	
was	 the	 single	 most	 important	 parameter	 affecting	 the	
biosorption	 process.	 Our	 finding	 are	 in	 agreement	 with	
Cetinkaya	et	al.	(1999)	who	observed	that	Cr6+	 	was	more	
effectively	adsorbed	at	low	pH	in	different	fungal	species.		
	
	
Table	5:	Effect	of	different	pH	levels	on	Cr6+	biosorption	using	
dried	biomass	of	P.	stratiotes		
	 pH	1	 pH	2	 pH	3	 pH	4	 pH	5	
Initial	concentra‐
tion	(ppm)	
100	 100	 100	 100	 100	
Concentration	
after	6	hours	
(ppm)	
0	 54	 85	 98	 100	
%	decrease	in	
Cr6+	concentra‐
tion	
100	 46	 15	 2	 0	
Uptake	capacity	
(q)	(mg/g)	
33.33	 15.33	 5	 0.67	 0	
Volume	of	test	sample	=	50	ml;	Biosorbent	amount	=	0.15	g	
	
	
Effect	of	initial	Cr6+	concentrations	on	biosorption	
Results	showing	the	effect	of	initial	Cr6+	concentrations	on	
the	biosorption	of	Cr6+	by	dead	P.	stratiotes	are	summarized	
in	Figure	2.	The	initial	metal	ion	concentrations	remarkably	
influenced	the	equilibrium	of	metal	uptake	and	adsorption.	
Maximum	equilibrium	uptakes	of	Cr6+	 ions	were	observed	
at	 49	 mg/g	 for	 P.	 stratiotes	 at	 200	 ppm	 (mg/l)	 of	 initial	
Cr6+concentration.	 The	 increase	 in	 loading	 capacities	 of	
biosorbent	with	the	increase	of	metal	concentration	may	be	
due	to	higher	probability	of	collusion	between	metal	ions	
and	biosorbent.	Our	results	showed	a	positive	correlation	
between	uptake	capacity	and	initial	Cr6+	concentration,	and	
are	in	agreement	with	Cetinkaya	et	al.	(1999).	
Column	 biosorption	 of	 Cr6+	 using	 dried	 biomass	 of	 P.	
stratiotes	
Performance	 of	 P.	 stratiotes	 material	 for	 sorption	 of	 Cr6+	
was	estimated	by	running	a	number	of	columns	at	different	
influent	 pH’s	 and	 varying	 Cr6+	 inflow	 concentration		
(50	ppm	and	100	ppm).	
Column	biosorption	at	different	pH	
The	effects	of	varying	influent	pH	levels	on	sorption	of	Cr6+	
on	P.	stratiotes	biomass	are	represented	in	Table	6.	Three	
pH	were	1.98,	3.02	and	4.0.	The	inflow	Cr6+	 concentration	
was	 100	 ppm	 and	 2	 g	 P.	 stratiotes	 biomass	 was	 used	 in	
these	 columns.	 The	 results	 showed	 that	 at	 low	 pH	 maxi‐
mum	biosorption	took	place,	while	with	increasing	pH;	Cr6+	
biosorption	decreases.
Potential of Pistia stratiotes for Cr6+
, Aziz-ud-Din et al.
6 Journal of Endocytobiosis and Cell Research VOL 26 | 2015 
Table	6:	Column	biosorption	of	Cr6+	at	different	pH	levels	by	P.	
stratiotes	dried	biomass	
pH	(inflow)	 1.98	 3.02	 4.07
%	Cr6+	removal		 95		 6		 0.5	
After	passing	0.5	(L)	Cr	ppm	
in	eluents	
5	 94	 99.5
Uptake	capacity	(q)	(mg/g)	 21.25	 1.5	 0.12
Cr6+	 concentrations	=	100	ppm;	pH	levels	=	1.98,	3.02	and	
4.07;	Flow	rate	=	120	ml/h;	Biosorbent	amount	=	2	g;	Vo‐	
lume	treated	=	0.5	L	
	
At	 pH	 1.98,	 3.02	 and	 4.07,	 95%,	 6%	 and	 0.5%	 Cr6+	 was	
removed	 from	 the	 solution	 in	 the	 columns,	 respectively.	
The	metal	uptake	calculated	after	passing	0.5	L	of	100	ppm	
Cr6+	solution	though	2	g	of	P.	stratiotes	biomass	was	21.25	
mg/g,	1.5	mg/g	and	0.12	mg/g	at	pH	1.98,	3.02	and	4.07,	
respectively.	 The	 results	 from	 column	 sorption	 are	 in	
agreement	with	the	previous	studies	on	an	Azolla	Cr6+	sys‐
tem	which	showed	that	the	optimum	pH	for	Cr6+	binding	is	
2	‐	2.5	(Zhao	and	Duncan	1997).	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
Table	7:	Column	biosorption	of	Cr6+	(50	ppm	and	100	ppm)	by	P.	stratiotes	dried	biomass	
Cr6+	concentrations	=	50	ppm	&	100	ppm;	pH	1.95;	Flow	rate	=	120	ml/h;	Biosorbent	amount	=	2	g;	Volume	treated	=	0.8	L	
	
	
Gradual	breakthrough	of	Cr6+	removal	in	column	oper‐
ations	
A	 gradual	 breakthrough	 curve	 of	 Cr6+	 removal	 was	 ob‐
served	 in	 the	 column	 elution	 profiles	 at	 50	 and	 100	 ppm	
inflow	 Cr6+	 concentration	 (Table	 7).	 Cr6+	 solution	 was	 ap‐
plied	to	the	columns	in	100	ml	fractions	up	to	0.8	L.	It	was	
observed	 that	 the	 P.	 stratiotes	 biomass	 in	 the	 column	 is	
gradually	saturated	until	it	reached	50%	removal	in	case	of	
100	ppm	inflow	Cr6+	solution	after	passing	0.8	liter	through	
the	 column	 (as	 shown	 in	 Figure	 3).	 The	 phenomenon	 of	
gradual	break‐	through	is	of	importance	because	a	column	
run	is	always	terminated	at	some	chosen	value	of	Cr6+	con‐
centration	 in	 the	 effluent.	 Therefore,	 the	 capacity	 of	 the	
sorbents	for	Cr6+	removal	will	not	be	fully	utilized	in	a	sin‐
gle	bed	run.	Other	researchers	have	also	observed	similar	
gradual	 breakthroughs	 of	 Cr6+	 in	 their	 studies	 on	 column	
sorption	of	Cr6+	 with	activated	carbon,	leaf	 mould,	Sphag‐
num	moss	peat,	coconut	husk	and	palm	pressed	fibers	with	
these	 materials.	 Physical	 and	 chemical	 sorption	 was	
deemed	to	be	the	predominant	mechanisms	for	the	remo‐
val	of	Cr6+	(Tan	et	al.	1993;	Sharma	and	Foster	1996).		
	
	
Conclusion	
Plants	as	living	or	dead	biomass	can	be	very	useful	for	the	
removal	of	different	kinds	of	pollutants	from	various	types	
of	 environments.	 The	 present	 study	 is	 an	 attempt	 to	 find	
out	the	solution	to	environmental	pollution	caused	by	Cr6+	
using	P.	stratiotes.	In	the	present	investigation	P.	stratiotes	
has	successfully	demonstrated	the	removal	of	Cr6+	from	the	
water	 with	 both	 living	 and	 dead	 plant	 material.	 It	 can	 be	
effectively	 used	 in	 decontamination	 of	 tannery	 effluents	
containing	 Cr6+	 in	 excess	 quantities.	 Phytoremediation	 of	
Cr6+	in	tannery	effluents	via	P.	stratiotes	is	cost	effective,	an	
economical	 and	 environment	 friendly	 method	 that	 can	 be	
adopted	 on	 large	 scale	 to	 prevent	 environmental	 damage	
caused	by	this	metal.		
Volume	treated	(L)	 0.1 0.2 0.3 0.4 0.5 0.6	 0.7	 0.8
Cr6+	conc.	(50	ppm)	 0 0.07 1.2 3.1 4 12	 17	 21
Cr6+	conc.	(100	ppm)	 2.5 3.7 5 7.4 11.2 16	 25	 48
%	removal	of	Cr6+		from	100	ppm	solution	 97.5 96.5 95 92.6 88.8 84	 75	 52
Figure	 2:	 Biosorption	 at	 initial	 Cr6+	 con‐
centrations	 by	 dried	 biomass	 of	 P.	 strati‐
otes.	50	ml	of	solution	in	a	flask	containing	
different	 concentrations	 of	 Cr6+	 was	 sub‐
jected	to	2	g	of	dried	biomass	of	P.	strati‐
otes	at	pH	1.5 
Potential of Pistia stratiotes for Cr6+
, Aziz-ud-Din et al.
Journal of Endocytobiosis and Cell Research VOL 26 | 2015 7 
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
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Figure	3:	Breakthrough	curve	for	a	2	g	biomass	column	
for	the	removal	of	Cr6+.	A	column	was	used	by	adding	2	
g	of	dried	biomass	of	P.	stratiotes	which	was	packed	in	
a	length	of	6	cm.	a	total	of	0.8	liters	of	100	ppm	at	pH	
1.95.	Cr6+	solution	was	passed	through	the	column	at	a	
rate	 of	 120	 ml/h.	 Eluents	 were	 collected	 in	 100	 ml	
fractions.

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  • 1.   Journal of Endocytobiosis and Cell Research (2015) 1-7 | International Society of Endocytobiology zs.thulb.uni-jena.de/content/main/journals/ecb/info.xml   Journal of Endocytobiosis and Cell Research VOL 26 | 2015 1  Journal of  Endocytobiosis and Cell Research Uptake and biosorption potential of Pistia stratiotes for Cr6+ Aziz-ud-Din1 , Zeshan Ali2 *, Farrakh Meh- boob2 and Qaiser Mahmood Khan3 1Department of Genetics, Garden Campus, Hazara Universi‐ ty, Mansehra, Pakistan; 2,*Ecotoxicology Research Institute, National Agricultural Research Centre, Park Road, Islama‐ bad, PO 45500, Pakistan; 3Soil & Environmental Biotech‐ nology Division, National Institute for Biotechnology & Genetic Engineering (NIBGE), Faisalabad, Pakistan; *correspondence to: eco4nd@yahoo.com Uptake and biosorption potential of Pistia stratiotes for Cr6+ was examined. The study was conducted in two phases: in the first phase, the effect of different Cr6+ concentrations on plant weight, leaf number and root length was determined along with plants uptake poten‐ tial. Higher concentration of Cr6+ (> 8 ppm) had inhibi‐ tory effects on increase in plant weight due to impair‐ ment of the metabolic machinery. Leaf number and root length showed no increase when exposed to a Cr6+ concentration of ~ 10 ppm. When grown in solutions containing 2, or 4 ppm Cr6+ concentrations, P. stratiotes removed 100% of Cr6+. However at increasing concen‐ trations of 6 and 8 ppm, only 50% reduction in respec‐ tive solutions was recorded. In a second phase biosorp‐ tion capacity of dead P. stratiotes was determined. The optimal pH range for maximum Cr6+ biosorption lied between 1‐2. Maximum biosorption capacity of dead biomass was recorded at 200 ppm of the initial Cr6+ concentration. The volume and pH of a Cr6+ solution passing through the columns were critically important affecting the biosorption capacity of P. stratiotes. These findings are important for cost‐effective and environ‐ mental friendly removal of Cr6+ from tannery effluents which are posing serious risk to human and environ‐ mental health in tannery hubs of Pakistan. Journal of Endocytobiosis and Cell Research (2015) 1‐7 Category: Research paper Keywords: Pistia stratiotes, biosorption, Cr6+, environmental health, tannery Accepted: 09 January 2015 ____________________________________________________________________ Introduction Chromium (Cr) is an important environmental contaminant that generates from numerous anthropogenic activities, i.e. tanning, electroplating, mining, metal finishing, dyes prepa‐ ration, cement industries and preparation of corrosive paints (Ahn et al. 1999). In last decades its concentration has consistently increased in different environmental com‐ partments due to elevated municipal and industrial activi‐ ties reciprocal to human demands (Ali et al. 2013a). Cr like other heavy metals is a serious health risk, i.e. toxic, persis‐ tent, carcinogenic, mutagenic and teratogenic (Gourdon et al. 1990). Environmental behaviour of Cr is largely a function of its oxidation state (Losi et al. 1994). It can exist in various oxidation states; of which the trivalent form (Cr3+) is most stable and insoluble in water at neutral pH (Losi et al. 1994). Cr3+ in presence of mild oxidants can easily trans‐ form to hexavalent form (Cr6+) which is highly water solu‐ ble at neutral pH (Cary 1982). Cr6+ is a stronger oxidizing agent than Cr3+ and this property is likely related to its higher toxicity for most organisms (Rapoport and Mutter 1995). Cr6+ is described as 500 times more toxic than Cr3+ (Kowalski 1994). Transition between these two forms of Cr is important for the accumulation tendency, transport and toxicity in ecological compartments (Smith et al. 1989). In Pakistan the most important anthropogenic source of Cr in the environmental compartments is tannery industry that plays a significant role in the economic uplift of the country (Ali et al. 2015). There are around 650 registered tannery units in different cities of Pakistan, i.e. Gujranwala, Multan, Kasur, Karachi, Peshawar, Sialkot and Sahiwal (Ali et al. 2013a). Various studies have highlighted higher Cr levels in the soils, sediments and underground/surface water of these cities due to unchecked/untreated tannery industrial effluents (Tariq et al. 2006; Qadir et al. 2008; Ali et al. 2015). Tannery effluents contain Cr in both oxidation states, i.e. Cr6+ and Cr+3. Due to higher toxicity, the hexava‐ lent form (Cr6+) has remained a pressing concern in tan‐ nery industrial effluents (Gong et al. 2010). Cr6+ has smaller size which allows it to breach through the biological mem‐ branes adding to its toxicity. Many efforts have been made to treat Cr6+ in wastewater to reduce environmental pollu‐ tion (Leland et al. 1978). Conventional methods used for the treatments and disposal of Cr6+ bearing wastewater include chemical precipitation, ion exchange, membrane separation, solvent extraction and adsorption, etc. (Lee et al. 1998). These methods either are very expensive or re‐ quire extensive electrical/chemical/mechanical inputs (Farid et al. 2014). Bioremediation and biosorption on the other hand offer cost‐effective and environment friendly disposal of Cr6+ bearing wastewaters without any further environmental peril. The present research is focused on the bioremediation and biosorption of Cr6+ using the indigenous aquatic plant Pistia stratiotes (water lettuce). Water lettuce exists as common weed in lentic habitats of Pakistan and is a recog‐ nized metal hyperaccumulator (Lu et al. 2010). The basic
  • 2. Potential of Pistia stratiotes for Cr6+ , Aziz-ud-Din et al. 2 Journal of Endocytobiosis and Cell Research VOL 26 | 2015  aim of this research work is to develop an efficient biore‐ mediation/biosorption method for Cr6+ removal that can be replicated at national scale in tannery industrial cities for regular Cr6+ pollution abatement strategies. This study will also serve as guideline for the legislators and environmen‐ tal managers for the management of Cr6+ contaminated environments around tanneries. Material and methods Sample collection Fresh and healthy water lettuce (P. stratiotes) plants of uniform size (number of leaves per plant = 10 ± 2; root length = 11.5 ± 2 cm) and weight (11.2 ± 1.5 g fresh weight) were collected from the canal water near Ayub Agricultural Research Institute, Faisalabad. Collected plants were washed with tap water followed by distilled water to re‐ move mud, debris and other foreign particles attached to submerged and aerial plant parts. Use of P. stratiotes (living plants) for Cr6+ removal The experiment was performed in a greenhouse in large plastic tubes of 10 litre capacity. Synthetic Cr6+ solutions were prepared in distilled water using analytical grade K2Cr2O7. Nine treatments in triplicate were used to investi‐ gate the phytoremediation potential of P. stratiotes. The experiment was set up in completely randomised design with factorial arrangements. Essential nutrients (Cr free) were also added in each tube to support normal plant growth and development (Hewitt 1966). Treatment Concentration 1 distilled water 2 2 ppm Cr6+ 3 4 ppm Cr6+ 4 6 ppm Cr6+ 5 8 ppm Cr6+ 6 10 ppm Cr6+ 7 12 ppm Cr6+ 8 14 ppm Cr6+ 9 16 ppm Cr6+ The experiment continued for a period of 28 days. Water samples after an interval of 7 days were carefully with‐ drawn from the tubes to analyse Cr6+ concentration using the diphenyl carbazide (DPC) method (Bartlett and James 1988). At the same time plant growth attributes, i.e. plant weight, number of leaves and roots length were recorded. Triplicate water Cr6+ levels and plant growth results from each treatment were averaged and the results are present‐ ed in the Results/Discussion section. Use of P. stratiotes (dry biomass) for Cr6+ removal Collected plants were dried in an oven at 60 °C for 48 hours. Plant material was ground with the help of grinder after complete drying. The obtained powdered dry biomass was used in pH and biomass‐dependent biosorption stu‐ dies of Cr6+ from synthetic solutions. pH‐dependent bio‐ sorption experiments were set up in Erlenmeyer flasks (250 ml) containing Cr6+ synthetic solution (100 ppm) and 0.15 g of dried plant biomass. The pH was adjusted with the help of NaOH and H2SO4. The experiment was conducted in triplicates at room temperature and at different pH, i.e. 1, 2, 3, 4 and 5. The flasks were placed in a shaker at 150 rpm. Approximately 1.5 ml of water samples were collected in Eppendorf tubes at different time intervals. Zero time sam‐ ples were taken from every flask before adding the plant biomass. Biomass dependent biosorption experiment was conducted as described above, but the pH was adjusted at 1.5. Different amounts of plant material, i.e. 0.05 g, 0.1 g, 0.15 g, 0.2 g and 0.25 g were weighed and added in differ‐ ent flasks. These flasks were placed on a rotatory shaker (150 rpm) at room temperature. Zero samples were col‐ lected before adding the biomass. Samples were taken in Eppendorf tubes at different time intervals for the analysis of Cr+6. Cr6+ uptake capacity (q) was determined by the following equation: q V V g whereas Vi = initial Cr concentration, Vf = final Cr concentration, g = weight of the biosorbent in g. Column biosorption of Cr6+ by P. stratiotes dry biomass was studied using glass columns of 1.5 cm internal diameter at room temperature. Approximately 2 g of uniformly pow‐ dered Pistia biomass was added from the top and rinsed with distilled water. The length of the 2 g‐packed column was 6 cm. Synthetic Cr6+ solution (100 ppm) was passed through the column from the top at a flow rate of approxi‐ mately 120 ml/h and the eluents were collected in 100 ml fractions. The working temperature was kept at room tem‐ perature. Column biosorption of Cr6+ by P. stratiotes was observed at different pH and its breakthrough curve was studied. Statistical analysis Statistical analysis was carried out using Randomised Com‐ plete Block Design (RCBD) and Duncan’s Multiple Range Test (DMRT). Results and discussion Effect of Cr6+ on vegetative growth of P. stratiotes P. stratiotes weight, leaf number and root length values recorded from all treatments at increasing days were sub‐ jected to ANOVA (Table 1a, 2a, 3a). Duncan’s Multiple Range Test (DMRT) of mean weight, leaf number and root length values at increasing days were estimated and pre‐ sented in Tables 1b, 2b and 3b. The results showed that treatments, the time after treatments and their interaction significantly contributed in the overall variance of weight, leaf number and root length (Table 1a). DMRT of mean values of weight (Table 1b) showed that the first three doses were insignificantly different from each other with respect to weight. Weight values at 14, 21 and 28 days insignificantly differed from each other, but showed higher values of weight as observed immediately after the treat‐ ment. The results related to the effect of Cr6+ on plant weight showed that higher concentration of Cr6+ (> 8 ppm) had marked inhibitory effects on gain in plant weight. The effect was more prominent in case of treatment with 10 ppm of Cr6+. It depicts that higher concentration of Cr6+ inhibits the plant growth showing toxicity to the test plant. In another study, the Cr6+ concentration at around 20 ppm killed P. stratiotes in three days of exposure (Sen et al. 1987).
  • 3. Potential of Pistia stratiotes for Cr6+ , Aziz-ud-Din et al. Journal of Endocytobiosis and Cell Research VOL 26 | 2015 3  Table 1a: ANOVA of weight of P. stratiotes at various time intervals after treatment with Cr6+ during its growth experiments Source D.F. S.S. M.S. F p Replication 1 0.716 0.716 1.4045 0.2442 n.s* Treatment (T) 6 112.357 18.726 36.7286 0 P < 0.001 Day (D) 4 120.343 30.086 59.0086 0 P < 0.001 T x D 24 59.176 2.466 4.836 0 P < 0.001 Error 34 17.335 0.51 Total 69 309.927 n.s* ‐ not significant Table 1b: DMRT of mean weight of P. stratiotes at various time intervals after treatment with Cr6+ Treatments (mg/l) ↓ Days→ Day 0 Day 7 Day 14 Day 21 Day 28 C B A A A 2.519 3.569 5.455 5.713 5.709 0 B MN IJKLMN EFG CDEF DEFG 4.572 2.095 3.415 5.625 5.95 5.775 2 A KLMN HIJKL ABCD AB A 6.082 2.53 3.94 7.33 8.135 8.475 4 A KLMN HIJKL BCDE ABC AB 5.688 2.52 3.93 6.74 7.555 7.695 6 A IJKLMN GHIJK BCDE ABCDE AB 5.711 2.825 4.18 6.71 7.15 7.69 8 B KLMN IJKLM FGHI DEFG EFGH 4.36 2.51 3.565 4.46 5.765 5.5 10 D LMN KLMN IJKLMN MN N 2.336 2.44 2.49 2.95 1.975 1.825 12 C JKLMN IJKLMN FGHIJ IJKLMN IJKLMN 3.401 2.71 3.46 4.37 3.46 3.005 All mean values under a category which share a common letter are insignificantly different, otherwise they differ at p < 0.05 Table 2a: ANOVA of leaf number of P. stratiotes at various time intervals after treatment with Cr6+ during its growth experiments n.s* ‐ not significant Table 2b: DMRT of mean leaf number of P. stratiotes at various time intervals after treatment with Cr6+ during its growth experi‐ ments Treatments (mg/l) ↓ Days→ Day 0 Day 7 Day 14 Day 21 Day 28 C C B A A 4.571 6.5 14.93 20 22.93 0 AB J IJ EFGHIJ CDEF ABC 14.1 4 6.5 13.5 19 27.5 2 A J IJ CDEFG ABCD AB 16.7 4.5 6.5 18 23.5 31 4 A J IJ DEFGH BCDE A 16.4 4.5 6.5 16.5 22.5 32 6 AB J IJ DEFGHI BCDE ABC 15.3 5 6.5 15 22.5 27.5 8 AB J IJ FGHIJ CDEF ABCD 13 4.5 6.5 12 18.5 23.5 10 C J IJ FGHIJ FGHIJ FGHIJ 8.7 5 6 10.5 11.5 10.5 12 BC J HIJ CDEF BCDE GHIJ 12.3 4.5 7 19 22.5 8.5 All mean values under a category which share a common letter are insignificantly different, otherwise they differ at p < 0.05 Source D.F. S.S. M.S. F p Replication 1 64.129 64.129 3.9688 0.0544 n.s* Treatment (T) 6 464.086 77.348 4.787 0.0012 P < 0.01 Day (D) 4 3661 915.25 56.6438 0 P < 0.001 T x D 24 973.2 40.55 2.5096 0.0069 P < 0.01 Error 34 549.371 16.158 Total 69 5711.786
  • 4. Potential of Pistia stratiotes for Cr6+ , Aziz-ud-Din et al. 4 Journal of Endocytobiosis and Cell Research VOL 26 | 2015  Table 3a: ANOVA of root length of P. stratiotes at various time intervals after treatment with Cr6+ during its growth experiments Table 3b: DMRT of mean root length of P. stratiotes at various time intervals after treatment with Cr6+ during its growth experiments Treatments (mg/l) ↓ Days→ Day 0 Day 7 Day 14 Day 21 Day 28 C B A AB A 4.143 6.929 8 7.429 8.214 0 C IJ DEFGH CDEFG CDEFG CDEF 7 3.5 7 8 8 8.5 2 A IJ CDEFGH BCD AB A 9.2 3 7.5 10 11.5 14 4 AB IJ CDEFGH BCD BC BC 8.4 3.5 7.5 10 10.5 10.5 6 BC HIJ CDEFG CDEF CDEFGH CDEFG 7.3 4.5 8 8.5 7.5 8 8 BC EFGHI CDEFGH BCDE CDEFG CDEFG 7.7 6 7.5 9 8 8 10 D IJ FGHIJ FGHIJ IJ IJ 4.5 4 5.5 5.5 4 3.5 12 D HIJ FGHIJ GHIJ J GHIJ 4.5 4.5 5.5 5 2.5 5 All mean values under a category which share a common letter are insignificantly different, otherwise they differ at p < 0.05 Cr6+ toxicity is an established phenomenon in aquatic and terrestrial plants. In Myriophyllum spicatum, an increase in shoot length was recorded at 0.05 ppm Cr6+ level, however when its concentration was increased to 1 ppm, linear reduction in shoot length/weight was recorded. In a study on three herbaceous plants, i.e. Trifolium repens, Festuca arundina‐ cea and Medicago sativa, Wang et al. (2012) demonstrated decrease in plant height, dry weight of roots/shoots when exposed to Cr6+ levels exceeding 200 mg/kg in pot soils. The results regarding the effects of Cr6+ on leaf number and increase in leaf number was record‐ ed up to concentration of 8 ppm. There was relatively small increase in leaf number at 10 ppm concentra‐ tion. At higher concentration, the plant showed increase in leaf num‐ ber up to 21 days, but after that it did not survive. Working on wheat, Sharma and Sharma (1993) also showed negative correlation be‐ tween Cr concentration and leaf number. DMRT mean values of root length showed that the first four doses increased the root length while the latter two doses reduced it. As days after treatment with Cr6+ are concerned, the values of root length increased with increasing days after the treatments. There was no or small increase in the root length of P. stratiotes at 10 ppm while at higher con‐ centration the plant showed reduction in the root length after the first week. Negative correlation between Cr con‐ centration and root length of different crops has also been observed by others (Sharma and Sharma 1993; Stiborova et al. 1986). Source D.F. S.S. M.S. F p Replication 1 0.057 0.057 0.0367 P < 0.001 Treatment (T) 6 198.571 33.095 21.2538 0 P < 0.001 Day (D) 4 151.343 37.836 24.2982 0 P < 0.001 T x D 24 142.857 5.952 3.8226 0.0002 P < 0.001 Error 34 52.943 1.557 Total 69 545.771 Figure 1: Reduction in Cr6+ concentration during growth studies of P. stratiotes. Treatments were (T1 = 2 ppm, T2 = 4 ppm, T3 = 6 ppm, T4 = 8 ppm, T5 = 10 ppm, T6 = 12 ppm). Data was collected for four weeks at 7 days interval by growing P. stratiotes plants on the given concentrations of Cr6+. 
  • 5. Potential of Pistia stratiotes for Cr6+ , Aziz-ud-Din et al. Journal of Endocytobiosis and Cell Research VOL 26 | 2015 5  Uptake of Cr6+ during growth of P. stratiotes P. stratiotes eliminated Cr6+ from the solution at a concen‐ trations of 2 and 4 ppm, while at 6 and 8 ppm the Cr con‐ centration is reduced to one half. At 10 ppm the reduction in Cr concentration in the solution is negligible, whereas there is no reduction beyond 10 ppm (Figure 1). P. strati‐ otes uptake of Cr is affected by its oxidation state. Cr6+ can either directly pass through the plasma membrane or via carriers, i.e.  phosphate‐sulfate carriers. Cr3+ on the other hand cannot use any specific membrane transporters for inward movements. In trivalent form it simply diffuses through the plasma membrane by forming lipophilic lig‐ ands. It is evident from these findings that P. stratiotes efficiently uptakes/concentrates Cr6+ in leaves below 10 ppm concentration and therefore can play a vital role in recycling Cr6+ from the contaminated industrial effluents. Cr6+ biosorption studies using dried biomass of P. stratiotesEffect of different dosage of P. stratiotes biomass on the biosorption of Cr6+ Five different concentration of biomass of P. stratiotes, i.e. 0.05, 0.1, 0.15, 0.2 and 0.25 g/50 ml, were used for Cr6+ removal from 100 ppm Cr6+ solution. It was found that with increase in initial biomass, the removal of Cr6+ from the solution also increased. It has been reported extensively that the initial concentration of biomass is important and with increase in biomass, the rate of biosorption is in‐ creased accordingly (Kuyucak 1990). In this study, 0.05 g biomass/50 ml solution removed 25% Cr6+ and 0.1 g biomass/50 ml of solution removed 63% Cr6+, while the higher amounts, i.e. 0.15 g, 0.2 g and 0.25 g/50 ml removed 100% Cr6+ from the solution con‐ taining initially 100 ppm Cr6+ (Table 4) after 6 hour of shak‐ ing (150 rpm) at room temperature. According to Gadd (1990) the most probable mechanism is that first the cell wall comes in contact with metal ions in solution, where the metal can be deposited on the surface or within the cell wall structure before interacting with cytoplasm material or other cellular parts (Gadd 1990; Pereira et al. 2014). This cell wall uptake may be directed by functional groups like phosphates, carboxyl groups, amines or phospho‐ diester species. The results of Crist et al. (1988) showed that the biosorption of heavy metals has two phases: first phase is attributed to surface adsorption, mainly based on anion exchange with the participation of carboxyl groups of uronic acids. The second phase represents the diffusion of ions into the cell. Table 4: Effect of different dosages of P. stratiotes dried biomass on Cr6+ biosorption Biosorbent amount (g) 0.05 0.1 0.15 0.2 0.25 Initial concentration (ppm) 100 100 100 100 100 Concentration after 6 hours (ppm) 75 37 0 0 0 % decrease in Cr6+ concentration 25 63 100 100 100 Uptake capacity (q) (mg/g) 25 31.5 33.33 25 20 Volume of test sample = 50 ml; pH = 1.5 Effect of pH on Cr6+ biosorption The results showing the effect of different pH levels on the biosorption of Cr6+ by P. stratiotes are summarized in Table 5. It was observed that maximum biosorption takes place at lower initial pH. Metal uptake from 100 ppm/50 ml solu‐ tion using 0.15 g of dried biomass of P. stratiotes is 100%, 46%, 15%, 2% and 0% at pH 1, 2, 3, 4 and 5, respectively. Earlier studies on heavy metal biosorption showed that pH was the single most important parameter affecting the biosorption process. Our finding are in agreement with Cetinkaya et al. (1999) who observed that Cr6+ was more effectively adsorbed at low pH in different fungal species. Table 5: Effect of different pH levels on Cr6+ biosorption using dried biomass of P. stratiotes pH 1 pH 2 pH 3 pH 4 pH 5 Initial concentra‐ tion (ppm) 100 100 100 100 100 Concentration after 6 hours (ppm) 0 54 85 98 100 % decrease in Cr6+ concentra‐ tion 100 46 15 2 0 Uptake capacity (q) (mg/g) 33.33 15.33 5 0.67 0 Volume of test sample = 50 ml; Biosorbent amount = 0.15 g Effect of initial Cr6+ concentrations on biosorption Results showing the effect of initial Cr6+ concentrations on the biosorption of Cr6+ by dead P. stratiotes are summarized in Figure 2. The initial metal ion concentrations remarkably influenced the equilibrium of metal uptake and adsorption. Maximum equilibrium uptakes of Cr6+ ions were observed at 49 mg/g for P. stratiotes at 200 ppm (mg/l) of initial Cr6+concentration. The increase in loading capacities of biosorbent with the increase of metal concentration may be due to higher probability of collusion between metal ions and biosorbent. Our results showed a positive correlation between uptake capacity and initial Cr6+ concentration, and are in agreement with Cetinkaya et al. (1999). Column biosorption of Cr6+ using dried biomass of P. stratiotes Performance of P. stratiotes material for sorption of Cr6+ was estimated by running a number of columns at different influent pH’s and varying Cr6+ inflow concentration (50 ppm and 100 ppm). Column biosorption at different pH The effects of varying influent pH levels on sorption of Cr6+ on P. stratiotes biomass are represented in Table 6. Three pH were 1.98, 3.02 and 4.0. The inflow Cr6+ concentration was 100 ppm and 2 g P. stratiotes biomass was used in these columns. The results showed that at low pH maxi‐ mum biosorption took place, while with increasing pH; Cr6+ biosorption decreases.
  • 6. Potential of Pistia stratiotes for Cr6+ , Aziz-ud-Din et al. 6 Journal of Endocytobiosis and Cell Research VOL 26 | 2015  Table 6: Column biosorption of Cr6+ at different pH levels by P. stratiotes dried biomass pH (inflow) 1.98 3.02 4.07 % Cr6+ removal 95 6 0.5 After passing 0.5 (L) Cr ppm in eluents 5 94 99.5 Uptake capacity (q) (mg/g) 21.25 1.5 0.12 Cr6+ concentrations = 100 ppm; pH levels = 1.98, 3.02 and 4.07; Flow rate = 120 ml/h; Biosorbent amount = 2 g; Vo‐ lume treated = 0.5 L At pH 1.98, 3.02 and 4.07, 95%, 6% and 0.5% Cr6+ was removed from the solution in the columns, respectively. The metal uptake calculated after passing 0.5 L of 100 ppm Cr6+ solution though 2 g of P. stratiotes biomass was 21.25 mg/g, 1.5 mg/g and 0.12 mg/g at pH 1.98, 3.02 and 4.07, respectively. The results from column sorption are in agreement with the previous studies on an Azolla Cr6+ sys‐ tem which showed that the optimum pH for Cr6+ binding is 2 ‐ 2.5 (Zhao and Duncan 1997). Table 7: Column biosorption of Cr6+ (50 ppm and 100 ppm) by P. stratiotes dried biomass Cr6+ concentrations = 50 ppm & 100 ppm; pH 1.95; Flow rate = 120 ml/h; Biosorbent amount = 2 g; Volume treated = 0.8 L Gradual breakthrough of Cr6+ removal in column oper‐ ations A gradual breakthrough curve of Cr6+ removal was ob‐ served in the column elution profiles at 50 and 100 ppm inflow Cr6+ concentration (Table 7). Cr6+ solution was ap‐ plied to the columns in 100 ml fractions up to 0.8 L. It was observed that the P. stratiotes biomass in the column is gradually saturated until it reached 50% removal in case of 100 ppm inflow Cr6+ solution after passing 0.8 liter through the column (as shown in Figure 3). The phenomenon of gradual break‐ through is of importance because a column run is always terminated at some chosen value of Cr6+ con‐ centration in the effluent. Therefore, the capacity of the sorbents for Cr6+ removal will not be fully utilized in a sin‐ gle bed run. Other researchers have also observed similar gradual breakthroughs of Cr6+ in their studies on column sorption of Cr6+ with activated carbon, leaf mould, Sphag‐ num moss peat, coconut husk and palm pressed fibers with these materials. Physical and chemical sorption was deemed to be the predominant mechanisms for the remo‐ val of Cr6+ (Tan et al. 1993; Sharma and Foster 1996). Conclusion Plants as living or dead biomass can be very useful for the removal of different kinds of pollutants from various types of environments. The present study is an attempt to find out the solution to environmental pollution caused by Cr6+ using P. stratiotes. In the present investigation P. stratiotes has successfully demonstrated the removal of Cr6+ from the water with both living and dead plant material. It can be effectively used in decontamination of tannery effluents containing Cr6+ in excess quantities. Phytoremediation of Cr6+ in tannery effluents via P. stratiotes is cost effective, an economical and environment friendly method that can be adopted on large scale to prevent environmental damage caused by this metal. Volume treated (L) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Cr6+ conc. (50 ppm) 0 0.07 1.2 3.1 4 12 17 21 Cr6+ conc. (100 ppm) 2.5 3.7 5 7.4 11.2 16 25 48 % removal of Cr6+ from 100 ppm solution 97.5 96.5 95 92.6 88.8 84 75 52 Figure 2: Biosorption at initial Cr6+ con‐ centrations by dried biomass of P. strati‐ otes. 50 ml of solution in a flask containing different concentrations of Cr6+ was sub‐ jected to 2 g of dried biomass of P. strati‐ otes at pH 1.5 
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(1997) Column sorption and desorption of hexavalent Chromium from aqueous solution and electroplating effluent using Azolla filiculoides. Res Environ Biotechnol. 2:51‐64. Figure 3: Breakthrough curve for a 2 g biomass column for the removal of Cr6+. A column was used by adding 2 g of dried biomass of P. stratiotes which was packed in a length of 6 cm. a total of 0.8 liters of 100 ppm at pH 1.95. Cr6+ solution was passed through the column at a rate of 120 ml/h. Eluents were collected in 100 ml fractions.