c







c


 
 


  
a
Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India
Received 14 September 2008;
revised 5 December 2008;
accepted 6 December 2008.
Available online 6 January 2009.
?
c  ?
Biological H2 production has an edge over its chemical counterpart mainly because it is environmentally benign. Despite
having simpler technology, higher evolution rate of H2 and the wide spectrum of substrate utilization, the major deterrent
of anaerobic dark fermentation process stems from its lower achievable yields. Theoretically, the maximum H2 yield is
4 mol H2/mol glucose when glucose is completely metabolized to acetate or acetone in the anaerobic process. But it is
somewhat difficult to achieve the complete degradation of glucose to carbon dioxide and H2 through anaerobic dark
fermentation. Moreover, this yield appears too low to be economically viable as an alternative to the existing chemical or
electrochemical processes of hydrogen generation. Intensive research studies have already been carried out on the
advancement of these processes, such as the development of genetically modifiedmicroorganism, improvement of the
reactor designs, use of different solid matrices for the immobilization of whole cells, development of two -stage
processes, and higher H2 production rates. Maximum H2 yield is found to be 5.1 mol H2/mol glucose. However, major
bottlenecks for the commercialization of these processes are lower H 2 yield and rate of H2 production. Competent
microbial cultures are required to handle waste materials efficiently, which are usually complex in nature. This will serve
dual purposes: clean energy generation and bioremediation. Scale-up studies on fermentative H2 production processes
have been done successfully. Pilot plant trials of the photo-fermentation processes require more attention. Use of
cheaper raw materials and efficient biological H2 production processes will surely make them more competitive with the
conventional H2 generation processes in near future.
j
Biohydrogen; ‰

 ; Dark fermentation; Photo-fermentation; Fe-hydrogenase
c 
1.
Introduction

2.
Hydrogen research at IIT Kharagpur
2.1. Selection of microorganisms
2.2. Metabolic engineering: redirection of metabolic pathways
2.3. Improvement of bioH2 production under decreased partial pressure of H2
2.4. Key role of some biocatalysts on H2 production
2.4.1. Identification and characterization of Fe-hydrogenase coded gene
2.4.2. Isolation and purification of Fe-hydrogenase
2.5. Feasibility studies of fermentative hydrogen production using recombinant strain
2.6. Suitability of different carbon sources and the process development
2.6.1. H2 production from different carbon sources
2.6.2. Hydrogen production from cheaper carbon sources
2.6.3. Suitability of lignocelluloses as solid matrix and performance of continuous process
2.7. Mathematical modeling and simulation of hydrogen production using E. cloacae IIT-BT 08
2.8. Studies on scaling up processes
2.9. Production of H2 using hybrid bioreactor

3.
Summary and conclusion

4.
Future scope
Acknowledgements
References
 


Man has sought to fuel his energy needs since the beginning of human history. Mankind's fuels
have continually evolved as better, more efficient, safer, and cleaner fuels. From wood, to animal
fat, to coal, to petroleum, to natural gas, to H 2, a clear trend to lighter and cleaner fuels is apparent.
Fossil fuels have unanimously ruled the world for more than one century. The indiscriminate use of
fossil fuels has gone to such an extent that it has not only polluted the environment but also
exhausted the limited fuel reserves, necessitating the quest for a clean alternative
energy. Hydrogen (H2) is the most promising in the succession of fuel evolution, with several
technical, socio-economic and environmental benefits to its credit. It has the highest energy content
per unit weight of any known fuel (142 kJ/g or 61,000 Btu/lb) and can be transported for
domestic/industrial consumption through conventional means. H2 gas is safer to handle than
domestic natural gas. H2 is now universally accepted as an environmentally safe, renewable energy
resource and an ideal alternative to fossil fuels that does not contribute to the greenhouse.
Presently, 40% H2 is produced from natural gas, 30% fr om heavy oils and naphtha, 18% from coal,
and 4% from electrolysis and about 1% is produced from biomass. However, today, biological
H2 production processes are becoming important mainly due to two reasons: utilization of renewable
energy resources, and usually operated at ambient temperature and atmospheric pressure.
The microbial production of hydrogen by fermentation can be broadly classified into two main
categories ± one is light independent and the other is light-dependent. The light independent
fermentation processes, commonly known as dark fermentation, employ both obligate as well as
facultative anaerobic bacteria for the production of H2 from a variety of potentially utilizable
substrates, including refuse and waste products. It generally gives a high rate of H2 evolution and
does not rely on the availability of light sources. In contrast, in photo-fermentation, small-chain
organic acids are used by photosynthetic bacteria as electron donors for the production of H2 at
the expense of light energy [1], [2] and [3]. However, the reported H 2 production rates, stabilities
and efficiency of these processes are far below for commercialization. The major bottleneck of the
biohydrogen production processes for the commercialization is use of efficient microbial strains
which can use different carbonaceous organic materials present in the wastes and also the rate of
H2 production. Keeping these limitations in view Biohydrogen Group of IIT Kharagpur has been
striving towards a stable hydrogen economy for over a decade now.

 
  j  
This group has been engaged in carrying out research on different bioenergy production
processes like bioethanol, biomethanation and biohydrogen for several years. Recently, major
emphasize has been on the fermentative hydrogen production using not only a high yielding strain
of ‰

  IIT-BT 08 but also mixed microbial consortium and two-stage process for
the use of cheaper raw materials as well as for the impro vement of H2 production. One patent has
already been awarded on the biohydrogenation process [4]. The salient features of the research
work are discussed in the present paper.

  




The success or failure of a process often depends on the initial choice of the host organism. In
view of this a high H2 yielding strain ‰
  IIT-BT 08 was isolated from leaf extracts [5]. The
organism was characterized by a set of standard tests carried out according to Bergey's manual.
The most suitable physico-chemical parameters like inoculum age, initial pH, optimal temperature,
and substrate concentration were 12 h, 6.5, 36 °C and 1% w/v glucose, respectively, for the
maximization of H2 production. The pH profiles of the organism were found to be almost similar
both in aerobic and anaerobic conditions up to about 10th hour of fermentation. In case of
anaerobic conditions, pH remained almost constant even after 10 h. On the other hand, the pH
profile changed significantly up to 15 h of incubation in aerobic condition. It was found that the
H2 yield of the organism was 2.2 mol H2/mol glucose considering 1% glucose as substrate in a
batch system at 36 °C. The organism was also found to adapt to a wide range of pH from 4 to
11 [6], however, the range of hydrogen production was within the pH range of 4±7. Recent studies
conducted on the effect of regulated pH on biohydrogen production by the organism showed that
at a pH of 6.5 maximum H 2 yields of 2.9 mol H2/mol glucose, higher H2 production rates of
55.2 ± 0.4 mmol/L h and a glucose degradation efficiency of around 80% w/w could be
 achieved [6]. Comparative analysis with other facultative and obligate anaerobes showed
superiority of the isolated strain in terms of H2 production as shown in Table
1 [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16],[17], [18], [19], [20], [21], [22] and [23].
Table 1. Comparative study of the various dark and photo-fermentation processes.

 
   
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Glucose Batch studies 20.0 2.2 [5]
  IIT-BT 08
‰


Glucose Regulated pH 55.2 2.9 [6]
  IIT-BT 08
Mutated strain;
mutations
‰

 introduced in acid
Glucose ± 3.4 [7]
  DM11 and alcohol
pathways of the
organism
Immobilization of
‰

 cells on
Glucose 75.6 ± [8]
  IIT-BT 08 lignocellulosic
material
Automated-logic-
18.0 mol
‰

 Cane control-system in a
375.5 H2/ kg CODr [9]
  DM11 molasses bench scale
emoved
bioreactor (20 L)
‰  
Glucose Recombinant strain 66.0 3.2 [10]
 BL-21
   
Glucose Batch studies 11.8 2.3 [11]

‰


Pretreated
  IIT-BT
raw 45.4 mL H2/
08, 


sludge + 1 Mixed consortia ± g CODremove [9]
  and  
% d
   IIT-BT
molasses
08
Studies on photo-fermentation processes at IIT Kharagpur
Malic acid Employment of

 and jacketed glass 80 mL
± [3]
 
 sodium column H2/L h
glutamate photobioreactor

 Spent Two-stage photo- ± 3.3 mol H2/ [12]
 
   
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  O.U. media fermentation mol glucose an
001 from dark + 1.7 mol H d [1
fermentati 2/mol acetic 3]
on acid
Comparative studies with other reported literature

 Continuous nitroge
Glucose ± 0.065 [14]

 n sparging
 
   Non-vacuum
Glucose ± 1.4±2.0 15

 ‰ culture
Vacuum culture 1.3±2.2
Sucrose
added to

 Continuous hydrog 0.001
sugar
 
 en production for L H2/L ± [16]
refinery
O.U. 001 100 days culture
wastewate
r
Continuous hydrog
‰

 en gas evolution by
Glucose 58 1.1 [17]
  AY2 self-flocculated
cells in PBR
 Intermittent
Glucose ± 2.8 [18]
 
  P4 sparging with argon
Enzymatic
Hydrogenase
conversion of 4.5 ȝm
from  Glucose 8.7 ȝmol [19]
sucrose ol/h
 
to hydrogen
Synthetic
enzymatic pathway
Synthetic enzyme consisting of 13 8.4 H2/g G-
Starch ± [20]
pathway enzymes was used 6-P
to
produce hydrogen
Noodle
manufactu
Anaerobic 1.5 mol H2/
ring Effect of pH ± [21]
microflora mol hexose
wastewate
r
‰
  E. Glucose Effects of formate 15 mm 0.8 [22]
 
   
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H2 production y
wt cell
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3.1 mL
H2/mi
 
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n g bio
mass
Full-size table

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‰
  IIT-BT 08 produces H2 at a higher rate and yield than that of other reported strains
using different carbon sources as substrate, however, it was still low for commercial exploitation.
Therefore an attempt was made to redirect the biochemical pathways for further improvement of
the process. It was done by blocking alcohol and some of the organic acids formation in ‰

  IIT-BT 08 during their metabolism. The principle being that NADH is usually generated by
catabolism of glucose to pyruvatethrough glycolysis. The conversion of pyruvate to ethanol,
butanediol, lactic acid and butyric acid involves oxidation of NADH. The concentration
of NADH would be increased if the formation of these metabolites could be blocked. This will no
doubt enhance H2 production through the oxidation of NADH. On this basis the alcohol
dehydrogenase and the lactate dehydrogenase were mutated. It was seen that the wild
type
 was more susceptible to allyl alcohol and combined effect of NaBr and NaBrO 3. On the
other hand, the double mutants of ‰
  IIT-BT 08 with defects in both alcohol and organic
acid formation pathways were able to tolerate the lethal concentration of these reagents and
had better H2 yield than wild type strain (3.8 mol H2/mol glucose) [7].





 


        
 
Modification of operating conditions needs to be implemented in order to overcome the
thermodynamic limitations of 4 mol H2 /mol glucose. Decrease of partial pressure was considered
as one of the approaches towards improvement of hydrogen productivity [24]. It was observed that
when the partial pressure of H2 was decreased by lowering the total pressure in the headspace of
the reactor in a batch fermentation pr ocess from 760 mm Hg to 380 mm Hg containing ‰
 ,
the molar yield of H2 increased by 34% using glucose as a substrate. The lag period as well as
total batch time of H2 production decreased significantly using decreased partial pressure. This
approach is considered as a significant feature towards commercialization of the process since
yields close to the theoretical have been achieved. One ³automated-logic-control-system´ was
developed and found suitable to regulate the pressure.

  j


 




    
   

()

 
Fe-hydrogenase gene is the principle gene involved in H2 production. An attempt was made to
characterize the gene at the molecular level. Degenerate primers were designed from the
conserved zone of  structural gene encoding for catalytic subunit of Fe-hydrogenase of
different H2 producing bacteria. A 750 bp of PCR product was amplified by using the above-
mentioned degenerate primers and genomic DNA of ‰
  IIT-BT 08 as template. The
amplified PCR product was cloned and sequenced. The sequence showed the presence of an
ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-
cluster) of Fe-hydrogenase.  ORF was then amplified and cloned in frame with GST in
pGEX4T-1 and overexpressed in a non-hydrogen producing ‰   BL-21 to produce
a GST-fusion protein of calculated molecular mass of about 42.1 kDa. Recombinant protein was
purified and specifically recognized by anti-GST monoclonal antibody through Western blot.
Southern hybridization confirmed the presence of this gene in ‰
  IIT-BT 08 genome. In
vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is
catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence
of H2 gas in the gas mixture obtained from the batch culture of recombinant ‰
 BL-21. A
tentative molecular mechanism has been proposed about the transfer of electron from electron
donor to H-cluster without the mediation of the F -cluster [25] and [26].

   

  

()

Purification and characterization studies of Fe-hydrogenase from ‰
  IIT-BT 08 were
carried out. Fe-hydrogenase was purified to near homogeneity by four chromatographic steps
under strictly anaerobic conditions. After the final chromatographic separation the enzyme was
í1
purified approximately 1300-fold with specific activity of 334.8 U mg . The purified enzyme was in
its active form as evidenced from single band on native PAGE after activity staining. The
molecular weight of the enzyme was approximated as 51 kDa. The Fe content of the enzyme was
determined both by colorimetric assay and by atomic absorption spectroscopy. Ferridoxin (Fd)
were partially purified from ‰
  IIT-BT 08 using successive steps of Q-Sepharose and gel
filtration chromatography. The molecular weight of Fd was determined as 8 kDa by SDS-PAGE
analysis [27] and [28]. Fd was found to be the most probable physiological electron donor.

  (  

 



 
 
 
Fermentative studies were carried out using recombinant ‰
 BL-21. Effect of various process
parameters like inoculums' age, pH, and initial substrate concentration was analyzed. A
comparison of H2 production characteristics between the wild type strain ‰
  IIT-BT 08 and
that of the recombinant ‰
 BL-21 indicated that optimum initial pH, initial glucose concentration
and reaction temperature were almost same for both the strains. However, the yield of H 2 with the
recombinant strain was 3.1 mol H2/mol glucose which was much higher than that reported for the
wild strain [10] and also with that of other reported recombinants/mutated strains ( Table
2) [29], [30], [31], [32], [33] and [34].
Table 2. Comparison of different recombinant/mutant strains reported for biohydrogen production.

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Overexpression of hydrogenase
‰   Recombi
encoded gene from ‰
  IIT- 66 [10]
 BL-21 nant
BT 08 in pGEX T1 vector
‰
 SR13 Recombi Formate hydrogen lyase (FHL) 1339 [29]
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nant overexpressing strain
‰


Chemica Double mutation to block alcohols
  A 58 [30]
l mutants and organic acids
Y-2


Chemica Glutamine auxotrophic mutant by
  0.63 [31]
l mutants EMS treatment
AR-3
Anaerobic induction of the
‰   Recombi formate hydrogen lyase (FHL) of 144 mmol
[32]
 SR 14 nant aerobically grown ‰
 was /g h
established
Recombi Glucose metabolism was driven
‰
 SR13 793 [33]
nant towards formate hydrogen lyase


Recombi
  Metabolic engineering 2.8 [34]
nant
SMV089
Full-size table

   
  

  
 



    


  

 
The industrial feasibility of any organism increases with the ability of the organism to utilize
differentcarbon sources for H2 production. In accordance cellobiose, L-arabinose,
fructose, maltose, potato starch, CMC, D-xylose, dextrose and sucrose were explored as
substrates for H2 production using ‰
  IIT-BT 08. The H2 yield from different C-sources and
maximum H2 production rate achieved are shown inFig. 1. Among different C-sources, sucrose
showed maximum rate of hydrogen production which was 660 mL H2/L h with the corresponding
H2 yield of 6.0 mol H2/mol sucrose. Cellobiose also showed a relatively good H2 yield of
5.4 mol H2/mol substrate and the maximum rate of hydrogen production was 650 mL/L h [5].
These observations clearly indicate that the organism produced several hydrolytic enzymes like
amylases, cellulases, lipase, etc. and is capable of utilizing various types of carbon present in the
wastes for H2 production [9] and [35].
 Full-size image (35K)

Fig. 1. ?
Suitability of different carbon substrates on hydrogen production in a batch system using ‰

 IIT-BT 08?

   



 

 
The attraction of bioH2 as a fuel emanates from the fact that it can be produced from different
waste resources. In view of this, recent studies were accomplished on the use of sewage sludge
as substrate for hydrogen production using a mixed microbial consortium consisting of
three bacteria, ‰
  IIT-BT 08, 

  IIT-BT L139 (isolated from high oil
containing soil) and     IIT-BT S1 (which was isolated from sewage sludge itself).
Among these organisms 
   was found to be the dominating species [9] and [11]. The
maximum H2 yields from MYG medium and sludge, using mixed microbial consortium were found
to be 16.1 mmol H2/g COD reduced and 1.8 mmol H2/g COD reduced, respectively. This yield
of hydrogen was nearly 25% higher than the maximum hydrogen yield from sewage sludge
obtained using pure cultures. The H2 yields achieved from sewage sludge using the consortium in
the present study were higher than that reported earlier [9].
The potentiality of the bioH2 production process by ‰
  IIT-BT 08 in a continuous
immobilized whole cell system using different types of industrial wastes like distillery effluent,
starch wastewater and whey has been studied in detail [8], [36] and [37]. The main objective of
this research work was not only to produce H 2 but also to stabilize the environmental pollutants.
Recently, work is under progress with cane molasses, by-product of the sugar industry, as
substrate for biohydrogen production [9].

 
 



  *  




 
In order to produce H2 continuously from biological resources for industrial use, the H2 evolution
rate is as important as its overall yield. Therefore, for the practical operation of a bioreactor, a high
cell density is required. To achieve this end, a variety of reactor systems with immobilized
cells using several microbial support carriers have been reported. These immobilized cultures
have advantages over cell suspensions as they provide a higher cell concentration, eliminate cell
wash out and promote cell recovery and recycle. In view of this immobilized studies were carried
out using environmental friendly natural polymers such as lignocellulosic material [8]. The effect of
immobilization on various polymers such as rice straw, bagasse and coir was tried. The work was
intended to increase both the rate of H2 production and substrate utilization
efficiency. Coconut coir as carrier showed the highest H2 production rate (72 mmol/1 h) among all
the carrier materials used.
Gas hold-up appears to be a major problem in tubular bioreactor with immobilized cells resulting in
reduction of working volume and hence the efficiency of the process. The tapered
and rhomboidbioreactors were found to give better r esults in terms of rate of hydrogen production
and gas hold-up as compared with tubular reactor. The gas hold-up was reduced by 67% by
changing bioreactor configuration[36]. These studies thus emphasized the importance of
immobilization on natural polymers against a back drop of usage of synthetic polymers which
 created disposal problems as represented in Table
3 [8], [38],[39], [40], [41], [42], [43], [44], [45] and [46].
Table 3. Comparison of different immobilization processes and organisms used for biohydrogen
production.





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Packed Conti
‰

 Gluc Lignocellulosic
bed nuou 75.6 [8]
  DM 11 ose material
reactor s
‰

 Column Conti
Mola Immobilized on
  E.8200 bioreac nuou 13 [38]
sses polyurethane
5 tor s
Immobilized on
Column Conti
‰
 !" Gluc agar gel and
bioreac nuou 37.9 [39]
#$ ose porous glass
tor s
beads
Column Conti
 
   Gluc Porous glass
bioreac nuou 51.4 [40]

  ose beads
tor s
Packed Conti
Microflora Sucr polyethylene±
bed nuou 59 [41]
(sewage sludge) ose octane elastomer
reactor s
 
   Trickle Conti 1270 mL/g
Gluc Porous glass


  AT bed nuou glucose L [42]
ose beads
CC 824 filter s of reactor
Packed Conti
Acclimated Sucr
Activated carbon bed nuou 330.4 [43]
sewage sludge ose
reactor s
 
  
  Conti
Starc Porous glass Stirred
 and ‰
 nuou 58.0 [44]
h beads reactor

  s
Packed Conti
    Gluc
Brick dust bed nuou 1.1a [45]
  ose
reactor s
Packed
‰


 Gluc Porous glass Batc
bed 49.1 [46]
%&$ ose beads h
reactor
Full-size table
a
Yield 1.07 mol/mol glucose.
  !
 




+ 

),-.
Substrate inhibition model using glucose as a substrate was found suitable for H2 generating
system as compared to that using Monod's model. A modified Andrew's model for the substrate
inhibition has been proposed and was found to be suitable for the existing system [47].
In another type of the study kinetics of the two-stage process for the production of H2 were
studied. Monod model, with incorporation of substrate inhibition term, was used to determine the
growth kinetic parameters for the first stage. The values of maximum specific growth rate ( max)
and s (saturation constant) were 0.4/h and 5.51 g/L, respectively, using glucose as substrate.
Modified Gompertz equation was applied to estimate the H2 production potential, rate and lag
phase time in a batch process for various initial concentrations of glucose, based on the
cumulative H2 production curves. Both the curve fitting and statistical analysis showed that the
equation was suitable to describe the progress of cumulative H2 production [48].
Modeling and simulation of continuous H2 production by ‰
  IIT-BT 08 using different
bioreactor configurations have been carried out and a suitable kinetic model for the substrate
degradation has been proposed, assuming ideal plug flow, no axial dispersion, steady state and
first-order substrate degradation kinetics. The degradation kinetics was investigated as a function
of flow rates considering the gas hold-up in all the bioreactors. The external film diffusion model of
the type

is proposed for a tubular bioreactor and 'D is found to be the constant in the
present system. The same model was applied to both tapered and rhomboidal bioreactors and
was found to give reasonably good fit with experimental results with respect to substrate
conversion. Comparison of the external mass transfer coefficient with the observed first-order
reaction rate constant predicted by the model shows that the effect of external film diffusion on the
observed substrate degradation rate decreases with increasing flow rates[49].
In studies of sewage sludge as substrate and mixed consortia, using the extended Logistic
equation, the regression analysis for
(as response) and in (
, ( and (2 (as predictors) for the four
2
types of cultures gave the  values as 
  IIT-BT L139 ± 93.8, 
   IIT-BT S1 ±
96.8, ‰
  IIT-BT 08 ± 98.7 and constructed consortium ± 99.7. The data were modeled for
Haldane model, Monod model and the Contois model as well [9].

 .  


The suitability of any process lies in its capacity to be scaled up to the industrial level. The scale-
up studies were conducted to explore the suitability of cane molasses for continuous
bioH2 production using a 20 L immobilized anaerobic bioreactor. The system was equipped with
an indigenously developed ³automated-logic-control-system´ to operate at reduced partial
pressure of H2 ranging from 380 to 760 mm Hg. Organic loading rates (OLR) were varied from
0.005 to 0.03 kg COD/L h and corresponding rate of hydrogen production was recorded between
0.12 and 8.41 L H2 /L h. The H2 content of the gas fluctuated from 58 to 66% v/v. Maximum
specific hydrogen production rate of 0.093 L H2/g VSS h was achieved at a dilution rate of 0.068/h
and recirculation ratio of 6.4. This rate, however, was observed to be inversely proportional to
specific OLR when dilution rate was more than 0.07 hí1. The maximum hydrogenyield was
17.94 mol H2/kg COD removed at an OLR of 0.02 kg COD/L h, which was comparatively higher than
earlier reported values (Table 4) [9], [50], [51] and [52]. Automation in maintaining reduced partial
pressure, which in turn was found to enhance the H 2 yield, purity of the gas, H 2 production rate
and cell loading [9].

/
012
Max. yield of H2 in the process = 45.4 mL/g COD reduced
Average COD value of the sludge = 88 g/L
COD reduction = 46%
Heating value of H2 = 142,000 kJ/kg of H2
Heating value of sewage sludge = 18,640 kJ/kg
H2 yield = 45.4 mL/g COD reduced = 45.4 × 460 mL/kg sludge = 0.0019 kg H2/kg sludge
Energy recovery from the substrate = {(heating value of H2 × yield of H2
?