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Production of bioethanol from lignocellulosic biomass

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Production of bioethanol from lignocellulosic biomass Powered By Docstoc
					  Bioethanol production from
    lignocellulosic biomass


Sachin Kumar: SNIRE, Kapurthala
S. P. Singh: DPT, IIT Roorkee, Saharanpur Campus
I. M. Mishra: DChE, IIT Roorkee
A. D. Adhikari: IIP, Dehradun
Energy from biomass is sustainable
• Fuels from biomass
   – Clean, carbon-neutral, and sustainable energy
   – Ethanol has potential of an alternative automotive
     fuel, preferably in a blend with gasoline
   – Use of ethanol in transport sector important for
     countries like India that depend heavily on import of
     crude oil
   – Use of ethanol as fuel reduces CO2 emission
• Fossil fuels
   – Reserves depleting fast
   – Rising price and uncertainty in availability
   – Environmentally unfriendly - blamed for the global
     climate change to a large extent
            The present study
• Screening of a thermophilic strain that can
  ferment glucose and xylose to ethanol. Strain has
  been characterized as yeast Kluyveromyces sp.
  IIPE453.
• Sugars for fermentation obtained from acid
  hydrolysis of sugarcane bagasse.
• Two-stage acid hydrolysis – dilute acid treatment
  followed by strong acid treatment – enabled
  about 92% recovery of the sugars present in
  biomass
• Optimum growth and fermentation were
  observed at 50 C and pH of 5
      Feed-stocks for bioethanol
• Lignocellulosic biomass: available more
  abundantly - forest residues, agricultural
  residues, industrial residues, energy crops
  such as switch grass
• Traditional: such as corn, food grains,
  sugarcane juice, and cane molasses face social
  and economic barriers as these materials are
  used substantially for human and animal
  consumption.
  Composition of lignocellulosic biomass
• Cellulose (20-50 %): Linear polymer of glucose
  units linked by β-(1–4)-glycosidic bonds.
• Hemicellulose (20-40 %): Highly branched and
  complex heteropolymer that contains
  hexoses, pentoses, and uronic acids.
  Hemicellulose is more easily hydrolyzed to its
  constituent monosaccharides than cellulose.
• Lignin (15-25 %): Aromatic polymer containing
  phenolic residues.
• Other components: Small quantity
    Composition of sugars in various
       lignocellulosic biomass
                                   Sugar content, %
 Raw materials
                 Glucose   Mannose    Galactose   Xylose     Arabinose

Wheat straw       36.6       0.8         2.4          19.2     2.4
Rice straw         41        1.8         0.4          14.8     4.5
Sugarcane         38.1        --         1.1          23.3     2.5
Bagasse
Rice hulls       38.1       3.0          0.1      14.0          2.6
Switch grass     31.0       0.2          0.9       0.4          2.8
Rye grass        23.9        --           --      14.7          2.8
Cotton gin       37.1       1.1          2.4       9.4          2.3
Beet pulp        24.1       4.6          0.9      18.2          1.5
Hybrid poplar    44.7       2.2         0.97      14.56        0.82
Eucalyptus       49.5      1.27         0.76      10.73        0.31
Pine             44.55     11.43        2.56       6.3          1.6
 Conversion of biomass to biofuels
• There are several biochemical and chemical
  routes to produce biofuels from biomass
• The main processes are fermentation of
  sugars to alcohol, gasification and chemical
  synthesis, and direct liquefaction.
• Many different fuels such methanol, ethanol,
  hydrogen, synthetic diesel, biodiesel, and bio-
  oil can be produced from biomass.
           Conversion of biomass to biofuels
                       Hydrolysi       Fermentable        Fermentation    Ethanol,
                       s               sugar                              Butanol,
Biochemical process                                                       Hydrogen
                                       Residue            Bio-oil
                       Anaerobi
                                                                          SNG
                       c                    Biogas        Purification    CH4
                       digestion

                                                                         FT Diesel
    Biomas
    s                                                                    DME
                      Gasificatio      Syn gas       Catalytic
                      n                              synthesis           Methanol

                      Pyrolysis        Bio-oil       Hydro               Hydrocarbo
Chemical                                             treating and        n
process                                              refining

                      Hydrotherma
                      l liquefaction
                                                                         Power
                                          Residu                         generation
                      Extraction          e
   Oil plant
                      of oil                         Trans-              Biodiesel
                                          Oil        esterificatio
                                                     n
     Biochemical processes for
   converting biomass to ethanol

• Hydrolysis of hemicellulose/ cellulose to
  monomer sugars
• Separation of sugars
• Fermentation of sugars
• Product recovery and concentration by
  distillation
Major differences in biochemical processes
• Hydrolysis of biomass
  By acid or by enzymes
• Selection of microbes
  For fermentation of sugars to ethanol
  (ethanologens)
  – For economical production of ethanol from
    lignocellulosic biomass, the microorganisms must be
    capable of fermenting both glucose and xylose.
  – Both anaerobic and aerobic ethanologens available
  – Facultative aerobes preferred for industrial
    applications due to difficulties in maintaining strict
    anaerobic conditions in large scale fermentations
    using thermophilic anaerobes
By-products in bioethanol production

• In spite of several breakthroughs, the cost of
  bioethanol produced from lignocellulosic
  feed-stocks remains high.
• Processes having potential of high ethanol
  recovery with some value-added by-products
  may improve the economy.
• Some examples of value-added by-products
  from bioethanol production follow:
 By-products during ethanol production
            from biomass
• Xylitol: conversion of xylose to xylitol during
  hydrolysis of biomass. Useful in prevention of tooth
  decay and ear infection in children, sugar substitute for
  diabetic patients
• Furfural: Is a valuable chemical formed by conversion
  of xylose to furfural
• Single cell protein: Useful for animal feed and can be
  produced by utilization of xylose solution for growing
  Candida utilis.
• Lignin: it is the residue after extraction of sugars.
  Useful as additive, adhesives, adsorptive materials
        EXPERIMENTAL WORK
• Isolation of thermophilic and thermotolerant
  strains
• Screening of isolates for ethanol production
• Growth of screened microbe
• Hydrolysis of sugarcane bagasse
• Fermentation conditions
• Analytical methods
     Isolation of Thermophilic and
        Thermotolerant Strains
• Thermophiles were isolated from soil samples
  in nutrient broth (NB) and yeast extract
  peptone and dextrose (YPD) media
• Soil samples were collected from dumping
  sites of crushed bagasse in a sugar mill.
• Pure colonies were isolated by using 2% agar
  and 1% gelrite as solidifying agent at 45 oC and
  60 oC, respectively.
 Composition of NB & YPD media
                                 Medium
                               NB          YPD
Yeast Extract, (g/l)           10          10

Peptone, (g/l)                 10          20
Sodium Chloride, (g/l)          5          --
Glucose, (g/l)                 10          20
pH                             6.5         5.0
 NB: Nutrient Broth
 YPD: Yeast extract Peptone and Dextrose
   Screening of Isolates for Ethanol
             Production
• Isolated ethanologens screened using different
  sugars: glucose, mannose, galactose, xylose,
  arabinose, sucrose, cellobiose and lactose.
• New isolate inoculated into phenol red broth
  medium and incubated overnight at 45-60 oC
• Change in pH due to acid production by
  ethanologens indicated by color change from red
  to yellow
• One potential yeast Kluyveromyces sp. IIPE453
  (KS) was selected based on faster growth and
  color change in the phenol red medium
    Growth of screened microbe
• The screened strain KS was grown in salt medium
  (SM)
• The cells were grown in 250 ml flasks in shaker at
  50 oC and 150 rpm on glucose, mannose,
  galactose, xylose, arabinose, sucrose, cellobiose
  and lactose, 10 g/l each separately.
• The cells were also produced in large quantity by
  growing in a Bioflow-110 bioreactor (ca. 5 liters)
  on glucose and xylose. The temperature, pH and
  dissolved oxygen were controlled at 50 oC, 5.0
  and 40 % saturation, respectively, during the
  growth phase.
    Composition of salt medium
                                        g/l
di-sodium hydrogen ortho phosphate      0.15
potassium di-hydrogen ortho phosphate   0.15
ammonium sulphate                       2.0
yeast extract                           1.0
carbon source e.g. glucose, xylose      20

  The pH was adjusted 5.0 by 1N hydrochloric
  acid
           Hydrolysis of bagasse
• First stage hydrolysis (acid conc 2-10 %)
  2 kg crushed bagasse with acid solution charged to a
  30-L digester fitted with an agitator (200 rpm), solid to
  liquid ratio 1:8 to 1:4, maintained at100 oC for 90 min.
  Samples withdrawn at 15 min interval. After 90 min the
  digested biomass washed to recover the sugars.
• Second stage hydrolysis (acid conc 18-40 %)
  Residual bagasse of first stage treated with stronger
  acid in the same digester for 90 min at 80 oC and 200
  rpm. Samples withdrawn at 15 min interval. The
  digested biomass wasahed to recover the sugars
       Fermentation Conditions
• Batch fermentation of the hydrolysate in a
  Bioflow-110 bioreactor (ca. 2 liters) by free
  cells of Kluyveromyces sp. IIPE453.
• Temperature: 50 oC, pH: 5.0
• Fermentation medium (g/l): di-sodium
  hydrogen ortho phosphate, 0.15; potassium
  di-hydrogen ortho phosphate, 0.15;
  ammonium sulphate, 1.0; yeast extract, 1.0.
           Analytical Methods
• HPLC used for analysis of sugars (glucose,
  fructose, sucrose and xylose) and xylitol: High
  Performance Carbohydrate Column (Waters) at
  30 oC; Acetonitrile and water mixture (75:25) at a
  flow rate of 1.4 ml/min as mobile carrier;
  Refractive index detector (Waters 2414).
• Gas chromatography used for analysis of
  ethanol: Ashco Neon II Gas Analyzer; 2 m long
  and 1/8" dia Porapak-QS column with 80/100
  mesh packing; Sample injection at 220 oC; Oven
  temperature 150 oC; Flame ionization detector at
  250 oC; Nitrogen gas as carrier.
     RESULTS AND DISCUSSION
• Isolation and characterization of microbe
• Products of 1st stage hydrolysis
• Products of 2nd stage hydrolysis
• Sugar recovery from the hydrolyzate
• Fermentation of 1st stage hydrolysate to
  ethanol
• Fermentation of 2nd stage hydrolysate to
  ethanol
      Isolation and characterization of
                  microbe
• From several thermophilic strains isolated from soil
  samples collected from dumping site of sugarcane
  bagasse where the temperature was usually high, only
  one microorganism showed growth and fermentation on
  glucose, mannose, galactose, xylose, sucrose, cellobiose
  and lactose.
• This strain was selected for further fermentation studies
  and characterized as yeast Kluyveromyces sp. IIPE453
  (deposited in ‘Microbial Type Culture Collection, Institute
  of Microbial Technology, Chandigarh (India)’ with
  deposition no. MTCC 5314).
• The optimum temperature and pH for growth and
  fermentation were found to be 50 C and 5.0,
  respectively.
1st stage hydrolysis: Xylose concentration
        Effect of acid concentration
                     30                                          Temp: 100 °C
                     25                                               Acid Conc
                                                                          %
Xylose conc. (g/l)




                     20
                                                                  ●       2
                     15
                                                                  ■       4
                     10
                                                                 ▲        6
                      5                                           ○       8
                      0                                           □      10
                          0   20   40            60   80   100
                                        Time (min)
                      1st stage hydrolysis: Glucose concentration
                              Effect of acid concentration
                      2.5
                                                                 Temp: 100 °C
                       2                                              Acid Conc
Glucose conc. (g/l)




                                                                          %
                      1.5
                                                                  ●       2
                       1                                          ■       4
                                                                  ▲       6
                      0.5
                                                                  ○       8
                       0
                                                                  □      10
                            0   20   40          60   80   100
                                       Time (min)
                       1st stage hydrolysis: Furfural concentration
                               Effect of acid concentration
                       0.8
                                                                        Temp: 100 °C
                       0.7

                       0.6                                                  Acid Conc
Furfural conc. (g/l)




                                                                                %
                       0.5

                       0.4
                                                                        ●       2
                       0.3                                              ■       4
                       0.2                                              ▲       6
                       0.1
                                                                        ○       8
                        0
                             0   20   40                60   80   100   □      10
                                           Time (min)
                     1st stage hydrolysis: Xylose concentration
                              Effect of solid/liquor ratio

                     50
                                                               Temp: 100 °C
                     40                                        Acid conc: 8%
Xylose conc. (g/l)




                     30
                                                                    Solid/liqu
                     20                                              or ratio

                     10
                                                                ●      1:8
                                                                ■      1:5
                     0
                          0   20   40          60   80   100    ▲      1:4
                                     Time (min)
                      1st stage hydrolysis: Glucose concentration
                               Effect of solid/liquor ratio
                      3.5
                                                                       Temp: 100 °C
                       3
                                                                       Acid conc: 8%
Glucose conc. (g/l)




                      2.5

                       2                                                    Solid/liquor
                      1.5
                                                                                ratio
                       1
                                                                        ●       1:8
                      0.5                                               ■       1:5
                       0                                                ▲       1:4
                            0   20   40                60   80   100
                                          Time (min)
                       1st stage hydrolysis: Furfural concentration
                                Effect of solid/liquor ratio
                       0.7
                                                                 Temp: 100 °C
                       0.6                                       Acid conc: 8%
Furfural conc. (g/l)




                       0.5
                                                                     Solid/liquor
                       0.4
                                                                         ratio
                       0.3
                                                                 ●       1:8
                       0.2
                                                                 ■       1:5
                       0.1
                                                                 ▲       1:4
                        0
                             0   20   40         60   80   100
                                        Time (min)
                     2nd stage hydrolysis: Xylose concentration
                            Effect of acid concentration
                     10
                                                             Temp: 80 °C
                      9
                                                                   Acid
                      8
                                                                  Conc %
Xylose conc. (g/l)




                      7
                      6                                      ●     18
                      5
                      4
                                                             ■     25
                      3                                      ▲     32
                      2
                      1                                      ○     40
                      0
                          0   20   40        60   80   100
                                    Time (min)
            2nd stage hydrolysis: Glucose concentration
                    Effect of acid concentration
                      80                                              Temp: 80 °C
                      70
                                                                          Acid Conc
                      60
                                                                              %
Glucose conc. (g/l)




                      50
                                                                      ●      18
                      40
                                                                      ■      25
                      30
                                                                      ▲      32
                      20

                      10
                                                                      ○      40
                       0
                           0   20   40                60   80   100
                                         Time (min)
               2nd stage hydrolysis: Furfural concentration
                       Effect of acid concentration
                        0.3                                           Temp: 80 °C

                       0.25                                               Acid Conc
                                                                              %
Furfural conc. (g/l)




                        0.2
                                                                      ●      18
                       0.15                                           ■      25
                        0.1                                           ▲      32
                                                                      ○      40
                       0.05


                         0
                              0   20   40             60   80   100
                                            Time (min)
  Hydrolysis of Sugarcane Bagasse
• Week acid predominantly hydrolyzes hemicelluloses to
  xylose and strong acid cellulose to glucose.
• Hydrolysis to furfural is low for both week as well as
  strong acids.
• Maximum recovery of xylose was obtained at acid
  concentration of 8 % and solid to liquid ratio of 1:4.
• Maximum recovery of glucose was obtained at acid
  concentration of 40 %.
• About 92 % of total sugars present in bagasse could be
  recovered in the two-stage acid hydrolysis.
 Sugar recovery from the hydrolyzate
• Sugars and the acid from hydrolysis mixture
  could be separated by ion-exchange
  technique.
• About 95 % acid free sugars were recovered
  with strong anion and weak anion mixture in
  the ratio of 5:2 and residence time 44
  minutes, and
• About 95 % acid was recovered in the
  regeneration step.
          Fermentation of 1st stage hydrolysate by yeast KS
                             7 g/l glucose and 18 g/l xylose
                                                                                   Temp: 50 °C
                    30                                 12
                                                                                    Concentration
                    25                                 10                             ■ Sugar




                                                            Ethanol conc. (g/L)
                                                             Xylitol conc. (g/L)
Sugar conc. (g/L)




                    20                                 8                              ∆ Xylitol
   DCW (g/L)




                    15                                 6                              ● Ethanol
                                                                                     ▲ Dry cell
                    10                                 4

                     5                                 2                           Sugar present in
                                                                                   hydrolysate was
                     0                                 0
                                                                                   consumed
                         0    2       4       6    8
                                   Time (h)
                                                                                   within 8 h
  Batch fermentation of dilute acid bagasse
                hydrolysate
                                    Ethanol     Xylitol
Maximum concentration, g/l             5.4       11.7
Yield, % of theoretical yield on      42.3       46.8
the basis of total sugars
Yield, % on the basis of                           65
Xylose, %
Productivity, g.l-1.h-1               0.73        1.46

Ethanol yield was low due to the low percentage of
glucose in hydrolysate. No inhibition was observed
during fermentation by the inhibitors such as furfural.
                     Fermentation of 2nd stage glucose rich hydrolysate
                                       by yeast KS
                    40                                                      16                         Temp: 50 °C
                    35                                                      14
                                                                                                       Concentration
                    30                                                      12




                                                                                 Ethanol conc. (g/l)
Sugar conc. (g/l)




                    25                                                      10                         ● Sugar




                                                                                     DCW (g/l)
                    20                                                      8                          ■ Ethanol
                    15                                                      6
                                                                                                       ▲ Dry cell wt.
                    10                                                      4

                     5                                                      2

                     0                                                      0
                         0   2   4   6   8      10 12   14   16   18   20
                                             Time (h)
        Batch fermentation of 2nd stage
                 hydrolysate
Maximum ethanol conc. g/l                              14.8
Ethanol yield in 20 h, % of theoretical yield          83.44
Average sugar consumption rate, g.l-1.h-1              1.74
volumetric productivity, g.l-1.h-1                     0.74
specific productivity, g.g-1.h-1                       0.3

The results are better than reported in literature: Ballesteros et
al. (44) and Tomás-Pejó et al. (25) with different strain and
biomass
    Batch fermentation of 2nd stage
             hydrolysate
• The dry cell weight was almost constant
  throughout the process.
• The final ethanol concentration in broth was
  low due to low initial sugar concentration in
  the hydrolysate, which could be overcome
  either by improvement of saccharification of
  sugarcane bagasse or by mixing other high
  sugar containing feed-stocks.
              CONCLUSIONS
1. A thermophilic strain was isolated from the
   soil samples collected from the dumping site
   of sugarcane bagasse.
2. The strain was characterized as yeast
   Kluyveromyces sp. IIPE453 (deposited in
   ‘Microbial Type Culture Collection, Institute
   of Microbial Technology, Chandigarh (India)’
   with deposition no. MTCC 5314).
             CONCLUSIONS
3. The yeast strain showed growth and
   fermentation on glucose, mannose,
   galactose, xylose, sucrose, cellobiose and
   lactose.
4. The optimum temperature and pH for growth
   and fermentation were observed to be 50 C
   and 5.0 respectively.
               CONCLUSIONS
5. About 92 % of the sugars present in the
   bagasse biomass could be recovered by acid
   hydrolysis in two steps, first with dilute acid
   to hydrolyze hemicelluloses and then with
   concentrated acid to hydrolyze cellulose.
6. Bagasse hydrolysate could be fermented to
   ethanol using the yeast Kluyveromyces sp.
   IIPE453 either in batch process or in
   continuous process.
              CONCLUSIONS
7. No inhibition during fermentation by the
   presence of inhibitors like furfural in the
   hydrolysate was observed.
8. The ethanol yield was, however, low due to low
   fermentation of xylose to ethanol and low
   percentage of glucose present in the bagasse
   hydrolysate.
9. The yield could be increases by increasing the
   glucose concentration in the hydrolysate
   through addition of molasses, sugarcane juice or
   some similar material.
   Thank you
for your kind attention

				
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