As the remedy to overcome the crisis following depleting fossil fuels and global climate change, a variety of alternative fuels emerged. Among all the alternative fuels or energy, hydrogen attracted more and more attention due to its being clean, efficient and renewable nature. This study evaluates the potential of employing food and temple waste for fermentative hydrogen production.

1. FERMENTATIVE HYDROGEN PRODUCTION FROM
TEMPLE AND FOOD WASTE BY BATCH MODE USING
Enterobacter aerogenes
Submitted by-
Ingole Charan Uttamrao
Supervisor
Prof. Subir Kundu
School of Biochemical engineering
Indian Institute of Technology (Banaras Hindu University)
Varanasi -221005 1

2. Introduction
2
• As the remedy to overcome the crisis following depleting fossil fuels
and global climate change, a variety of alternative fuels emerged.
Among all the alternative fuels or energy, hydrogen attracted more and
more attention due to its being clean, efficient and renewable nature.
• Hydrogen is harmless to humans and the environment and is the only
carbon-free fuel, on the oxidation produces water alone.
H2+1/2 O2 H2O (1)
• It gives secure elasticity for a sustainable energy system, in nature only,
bacteria and algae have the gift to produce hydrogen, considering the
present energy crisis and environmental tribulations.

3. Introduction cont.
• Biological processes, unlike their chemical or electrochemical
counterparts, are catalyzed by microorganisms in an aqueous
environment at ambient temperature and atmospheric pressure.
• These techniques are well suited for decentralized energy production in
small-scale installations in locations where biomass or wastes are
available, thus avoiding energy expenditure and costs for transport
• Intensive research on Biohydrogen is underway, and in the last few years
several novel approaches have been proposed and studied to surpass
these drawbacks. Among the available biological methods, microbial
fermentation of organic carbon sources has been comprehensively
studied (Ilgi Karapinar and Kargi 2006).
• Glucose is a monomer of cellulose, the most abundant biomass (Lattin
and Utgikar 2007).
3

4. Introduction cont.
• This study evaluates the potential of employing food and temple waste
for fermentative hydrogen production. For the optimum hydrolysis of
these waste materials to release reducing sugars, different concentrations
(1-5%, v/v) of HCl were used.
• Released reducing sugar is further used as substrate for biohydrogen
production by using Enterobacter aerogenes.
• The fermentative H2 production mainly depends on temperature, pH and
substrate concentration.
• In the world a good source for H2 production is considered as
microorganisms.
4

5. Introduction cont.
Enzymes involved in hydrogen production
• There are four fundamentally different hydrogen producing and
metabolizing enzymes found in Microorganism:
(1)The reversible or classical hydrogenase
(2) The membrane-bound uptake hydrogenase
(3) The nitrogenase enzymes and
(4) Hydrogenase
5

6. Introduction cont.
• General reaction implicated in the microbial conversion of
Biomass to Biohydrogen
6
Process General reaction Microorganism
Direct Biophotolysis 2H2O+Light2H2+O2 Algae
Photo fermentation CH3COOH+2H2O+Light4H2+2CO2 Purple bacteria, Microalgae
Indirect Biophotolysis a. 6H2O+6CO2+LightC6H12O6+6O2
b. C6H12O6+2H2O4H2+2CH3COOH+2CO2
c. 2CH3COOH+4H2O+Light8H2+6O2
Overall reaction:12H2O+Light12H2+6O2
Microalgae,Cynobacteria
Water gas shift reaction CO+H2OCO2+H2 Fermentative bacteria
+Methanogenic bacteria
Two phase H2+CH4
Fermentation
a. C6H12O6+2H2O4H2+2CH3COOH+2CO2
b. 2CH3COOH=2CH4+2CO2
Fermentative bacteria
High yield dark fermentation C6H12O6+6H2O12H2+6CO2 Fermentative bacteria

7. Introduction cont.
Anaerobic decomposition of organic matter (Zehnder et al.
1982)
7

8. Literature and review
• Hydrogen gas is a one of the hopeful and alternate source for reduction
of greenhouse effect (Christopher and dimitrios 2012).
• Approximately 95% of hydrogen produced is consumed at the site of
production, with 1.5 million tons being sold for industrial and chemical
uses (Lattin and Utgikar 2007).
• Carbohydrate rich, nitrogen deficient solid wastes such as cellulose and
starch containing agricultural and food industry wastes and some food
industry wastewaters such as cheese whey, olive mill and baker’s yeast
industry wastewaters can be used for hydrogen production by using
suitable bio-process technologies.
• Hydrogen was produced at an average rate of 6 ml/h per g (dry weight)
of cells with whey as a hydrogen donor. In continuous cultures with
glutamate as a growth-limiting nitrogen source and lactate as a hydrogen
donor, hydrogen was evolved at a rate of 20 ml/h per g (dry weight)
(Hans and Reinhard 1979).
8

9. Literature and review cont.
• Despite being a clean and high energy fuel, currently only 50 million
tons of Hydrogen is traded every year with a growth rate of about 10%
(Winter 2005).
• The majority of this hydrogen is used to produce ammonia fertilizer, as
feedstock for chemical and petroleum refining areas, plastics, solvents
and other commodities (Dunn 2002).The technology currently used to
make hydrogen has been well established, but the majority of hydrogen
produced uses fossil fuels in the production process.
• Project to turn flower waste into organic fertilizers – showing the
technology for the waste would be amassed and dumped into this dip
places to produce cheap vermicompost fertilisers.
• During the time of special occasions or pujas the amount of wastes
increases leading to major difficulties for us in terms of clearing them.
But in this project all the wastes would be cleared and stored (Kenya
flower council 2013).
9

10. Literature and review cont.
• The food processing industry in the United States is composed of more
than 20,000 companies (Elitzak 2000).
• It is estimated that the average large food processing industry annually
produces about 1.4 billion liters of wastewater (Van Ginkel et al. 2005).
• Wastes from these industries are usually high in organic matter and
normally contain sufficient nitrogen, phosphorus, and trace elements for
biological growth (Gray 2004).
• The biodegradability of food waste is mainly related to carbohydrate
materials, which are the main source of hydrogen production (Noike and
Mizuno 2000).
• India wastes Rs 44,000 cr worth food by food processing industry and
others every year. (Deccan herland Aug. 2013).
• These waste streams usually require treatment practices before being
discharged into local sewer districts.
10

11. Literature and review cont.
• Approximately 50% of hydrogen production globally comes from
natural gas, 30% oil, and 20% coal; see Figure.03 (Romm 2005).
• Current research has studied many different types of substrates for the
use of hydrogen production. The major criteria for substrate selection are
the availability, cost, carbohydrate content, and biodegradability
(Kapdan and Kargi 2006; Rai et al 2012). 11

12. Literature and review cont.
• Hydrogen production capability of anaerobic facultative bacterial
culture Enterobacter aerogenes has been widely studied (Yoko and
Saitsu 2001)
• In dark fermentation of hydrogen the enzyme hydrogenase present
in anaerobic organisms oxidizes reduced ferrodoxin to produce
molecular hydrogen, external iron addition is required for
hydrogen production.
• Hydrogen producing microorganisms
• Diverse microbes capable of H2 production by dark fermentation
are distributed across a wide variety of bacterial groups (Lee et al.,
2011).
• The organisms used in dark fermentation studies include
anaerobes, facultative anaerobes and aerobes in a wide
Temperature range (mesophiles, thermophiles and
hyperthermophiles). 12

13. Literature and review cont.
• Mesophiles are mainly affiliated with two genera: facultative
Enterobacteriaceae (Kumar and Das, 2000) and strictly anaerobic
Clostridiaceae (Collet et al., 2004; Evvyernie et al., 2001; Wang
et al., 2003),
• Most thermophiles belong to genus Thermoanaerobacterium (Ahn
et al., 2005; Ueno et al., 2001; Zhang et al., 2003). Also few
aerobes, such as Bacillus (Kalia et al., 1994; Kumar et al.,
1995; Shin et al., 2004),
• Aeromonons spp., Pseudomonos spp. and Vibrio spp. (Oh et
al., 2003b) have been characterized for H2 production under
anaerobic conditions but they show H2 yields less than 1.2
mol H2/mol-glucose.
13

14. Literature and review cont.
• Various wastewaters viz., paper mill wastewater (Idania, et al.,
2005), starch effluent (Zhang, et al., 2003), food processing
wastewater (Shin et al., 2004, van Ginkel, et al., 2005),
• Domestic wastewater (Shin, et al., 2004, 2008), rice winery
wastewater (Yu et al., 2002), distillery and molasses based
wastewater (Ren, et al., 2007, Venkata Mohan, et al., 2008),
• wheat straw wastes (Fan, et al., 2006) and palm oil mill
wastewater (Vijayaraghavan and Ahmed, 2006) have been
studied as fermentable substrates for H2 production along with
wastewater treatment.
14

15. Literature and review cont.
• Hydrogen production capability of anaerobic facultative bacterial
culture Enterobacter aerogenes has been widely studied (Yoko and
Saitsu 2001),in dark
• Fermentation of hydrogen the enzyme hydrogenase present in
anaerobic organisms oxidizes reduced ferrodoxin to produce
molecular hydrogen, external iron addition is required for hydrogen
production.
• Ten milligram per liter iron concentration was determined to be the
optimum in batch hydrogen production by C. Pasteurianum from
starch (Liu Gand Shen J. 2004)
15

16. Biochemical Processes
• There are two main stages in the biochemical process of AD for
hydrogen production: hydrolysis, acidogenesis, which are shown in
Fig. Schematic diagramme for biochemical process modified
(Metcalf and Eddy, 1985).
• Hydrolysis , Acetogenesis
16

17. Literature and review cont.
• Schematic diagramme (mechanism) for Enteric-type mixed-
acid fermentation (Modified from Turcot et al. 2008)
17

18. Objectives
• To characterize the physical properties of the food and temple
waste that is collected from Hostel mess (BHU) Varanasi and
different temples from Varanasi (UP).
• To determine the potential of this food waste and temple waste as
a feedstock for fermentative H2 production.
• The overall objective of this work will be to develop Processing
strategy for the production of hydrogen from flower waste (TW),
and food waste generated during food processing and production.
• Comparative studies of Hydrogen production from different
wastes.
18

19. Plan of work
• Procurement of suitable microorganism for the production of
Hydrogen.
• Collection of different waste materials.
• Pretreatment of the waste materials for the bioconversion.
• Formulation of suitable medium using waste for the production
of hydrogen.
• Determine the experimental setup for anaerobic fermentation
system.
• Testing for the existence of Hydrogen gas
19

20. Materials and methods
 Hydrogen-producing microbial strain and medium.
 Enterobacter aerogenes (MTCC 2822)
• Microorganism and culture condition
• The strains were procured from Microbial type culture collection
and Gene Bank Chandigarh (IMTECH) Enterobacter
aerogenes( MTCC 2822).
• These strains belong to the family of Enterobacteriaceae which are
known for their H2 production ability.
• They are obligate anaerobes capable of producing endospores
Individual cells are rod-shaped, these characteristics traditionally
defined the genus.
20

21. Materials and methods
• The Enterobacter sp. was reactivated in pre-culture basal medium
with the following composition (g/l):
• The basic culture medium for E.aerogens
• Glucose 10.0 g
• Tryptone 5.0 g
• (NH4)2SO4 2.0 g and MgSO4 0.2 g.100mM of monophosphate
(NaH2PO4, Pi) as phosphate sources was added into the basic
culture medium.
• All chemicals used were of analytical grade, and sterilized
individually by autoclave. The initial pH value of medium was
controlled at 6.0 by addition of NaOH or HCl.
21

22. Materials and methods
• Substrate and raw materials
• Temple Waste includes-
• 1. New Vishwanath Temple waste include marigold (Genda)
flower with rose.
• 2. Durga Temple, Durgakund Varanasi the waste of flower include
Hibiscus Rosa sinesis (Gudhal)
• 3. Shiv-Temple (Mrityujaya Mahadev), Visheshwarganj, Varanasi
the waste flower include Calotropis gigantea (Madar)
• 4. Waste food material Food waste from Different Hostel Mess of
Banaras Hindu University (BHU), Varanasi
22

23. Experimental setup
• For biohydrogen production from food waste and temple waste
the modified laboratory setup made as shown in Fig. the batch
reactor with working volume is 120 ml was operated at 32 to 35
°C for hydrogen production the reactor operated till the
production end. The generated biohydrogen measured by the
water disceplacement method.
23

24. Estimation
• Carbohydrate (X-PertTM
Carbohydrates Estimation Teaching Kit)
• Reducing sugar by DNS Method
Standard curve for reducing sugar estimation by DNS method 24

25. Estimation
• Carbohydrate (X-PertTM
Carbohydrates Estimation Teaching Kit)
• Phenol Sulphuric Acid Method:
Standard curve for total sugar by PSA method
25

26. Estimation
• C: N Ratio Analysis (Allison, LE in Black, CA et al. 1965)
• Carbon analysis: Walkley-Black chromic acid wet oxidation
method
• Nitrogen analysis
26

27. Characteristic
• Physico-chemical Analysis of Food and the Temple waste
• Waste food from Hostel mess of BHU and the different temple
waste i.e. Flower waste sample was collected from in zip lock
bags and all were dried under sun natural drying and made
pieces and with make fine powder after all kept all sample by
labeling accordingly in research laboratory biochemical
engineering oven for 1 hour under 90 -105°C.
27

28. .
28

29. Study for MC, TS, VS, and FS [APHA, 1989]
• The food and temple waste sample was finely ground to maintain
homogeneity. Specific amounts of food sample were weighed,
using a laboratory weight box in three separately prepared
crucible dishes and same for all separated sample of temple waste.
• The crucible dishes were also weighed individually using the
same balance and were then placed in an oven at 105ºC overnight
and weighed again the next morning. The crucible dishes were
then transferred to a cool muffle furnace heated to 550ºC ± 50ºC
and ignited for an hour to remove volatile organics.
• Bulk Density Determination (FMP Group (Australia) Ltd.
Information QC 125 Issue 3)
29

30. Theoretical consideration
• The biological hydrogen production by anaerobic condition by
specific microorganism, usually use the organic substrate as
their carbon and energy and hydrogen ion (H+
) as an electron
acceptor, the specified mechanism for biological production of
hydrogen by fermentation is generally associated with the
presence of an iron-sulphur, protein called “ferredoxin, an
electron carrier and low redox potential.
• Different type of microorganism including bacteria and archaea
domain, have the ability to show the potential of hydrogen
production by naturally metabolic process, with that the
metabolically observed product formation in microorganism
with H2 gas with other co-product get
30

31. Theoretical consideration
31
Energetic view of anaerobic bacterial hydrogen production. (Biohydrogen
by Oskar R.Zaborsky)

32. Result and Discussion
• Hydrogen is an alternative energy source is produced
biologically via various methods using different types of
materials. As the case of substrate for hydrogen production the
important consideration is of the economics of biohydrogen
production, there is need to go for cheaper and abundant
feedstock for making the process cost effective. (Rai et.al
2014, Bioresource technology 152:140-146).
32

33. Effects of dilute acids treatment on hydrolysis of flower waste
33
Schematic diagramme for hydrogen production from acid hydrolysate (Rong Chen, et al 2013)

34. References
• Bartlett, GN, Craze, B, Stone, MJ & Crouch, R (Ed) 1994,
Guidelines for Analytical Laboratory Safety. Department of
Conservation & Land Management, Sydney.
• Benemann J (1996) Hydrogen biotechnology: progress and
prospects. Nature Biotechnology 14:1101-1103.
• Benmeman, J.R 2000. Hydrogen Production by Algae. Journal
of Applied Phycology. 12291-300.
• Chen CC, Lin CY, Lin MC. Acid-base enrichment enhances
anaerobic hydrogen process. Appl Micro Biotechnol 2002.
58:224–228.
• Christopher and dimitrios Energy & Environmental Science,
Royal Society of Chemistry Publishing, Energy Environ. Sci.,
2012, 5, 6640.
34

35. References
• Chun-Mei Pan, Hong-Cui Ma,Yao-Ting Fan,Hong-Wei Hou
International journal of hydrogen energy 36 (2011) 4852-4862
• Collet C, Adler N, Schwitzguebel JP, Peringer P. Hydrogen
production by Clostridium thermolacticum during continuous
fermentation of lactose. Int J Hydrogen Energy 2004. 29:
1479–1485.
• Das, D. and T.N. Veziroglu. 2001. Hydrogen production by
biological processes: a survey of literature. International
Journal of Hydrogen Energy.26 (1):13-28.
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