 Lipid extraction for biodiesel production

from municipal sewage water sludge

APPARATUS TO BE USE:

PROCEDURES: -Centrifuge
-Fume hood
The wastewater generated in the city flows across three main
-Oven
storm water channels in the three catchments where -Mortar and pestle
wastewater samples were periodically assessed in two seasons for -Freezer
lipid extraction. -Gas Chromatograph
- Flame Ionization Detector (FID)
Sample collection and preparation: The municipal primary and - stainless steel column, dimension 10 X 1/8, packed
secondary sludge samples were collected from with 5% DEGS-PS
three principle waste water treatment plants like Hebbala,
Nayandahalli and Vrishabavati located in Bangalore. Raw
sludge was allowed to settle for 24 h at 10°C. After discarding the METHODS:
supernatant liquid the resulting sludge was then
centrifuged at 3000 rpm for 10 min for further dewatering. - Bligh and Dyer
Dewatered sludge was spread on tray and put in a fume - A.O.A.C standard methods
hood for 4 days to dry under vacuum at ambient temperature. The - Duncan's new multiple range test
dried sludge contained approximately 94% solids - ANOVA
measured by drying the sludge at constant mass in an oven at
120°C. Dried sludge was crushed in a mortar and
pestle homogenized and then stored in a freezer prior to use. The
powdered sample was then washed with distilled
water in order to remove all the soluble impurities, then dried at
500C overnight prior to use as a dehydrating agent
for the extraction of total lipids (oil). Extraction from organic
solvents was carried out by following the method of
Bligh and Dyer, 1959 (10).

Determination of the physico chemical properties of the lipid:

A.O.A.C standard methods were used to
determine the properties like Lipid, Viscosity, Density, Acid
value, Iodine value and Value of saponification.

Determination of fatty acid composition: Analysis of FAME

was carried out on Gas Chromatograph (11). The GC
was equipped with Flame Ionization Detector (FID) and stainless
steel column, dimension 10 X 1/8, packed with 5
% DEGS-PS. The column was conditioned at 1800C about 2
hours for attaining thermal stability before use. The
operating condition was programmed at oven temperature 150 0C
(hold time 5min) with increasing rate 80C/min
to1900C (hold time 0 min), 20C/min to 2000C (hold time 10min),
injection temperature 2500C and detector
temperature 2500 C. About 10μL of sample dissolved in hexane
was loaded onto the column. Nitrogen was used as a
carrier gas with flow rate of 20 ml/min.
The concentration of individual fatty acids in the test samples
were determined by comparing the peaks obtained
from the GC analysis with the peaks of authentic standards and n-
heptane was used as an internal standard. All tests
were performed in triplicate the experimental design was
completely randomized with three replicates. All data were
expressed as mean values ± SE. The comparison between the
mean values were tested using Duncan's new multiple
range test and the ANOVA was also performed to find out the
LSD (p = 0.05).
  Electricity Generation from Biowaste Based
Microbial Fuel Cells

PROCEDURES:

A salt solution, in our case, of pure NaCl, would be added to

each of the biowaste samples to make the mixture electrically
conductive. This mixture would be placed in a sealed chamber
to stop entering of oxygen, thus forcing the microorganism to
use anaerobic respiration. An electrode would then be placed
in the solution that would act as the anode. In the second
chamber of the MFC there would be placed another solution
and another electrode. This electrode, called the cathode
would be positively charged and would be the equivalent of
the oxygen sink at the end of the electron transport chain, only
now it would be external to the biological cell.
The solution would be an oxidizing agent that would pick up
the electrons at the cathode. In our case, we shall use
potassium ferricyanide as the oxidizing agent. Potassium
ferricyanide is added to the cathode to accept electrons. It is
very reactive with the graphite electrode. Ferricyanide has a
fairly positive potential compared to the organic matter in the
anode and helps to drive the flow of electrons. With the
addition of ferricyanide ions, the power can be increased 50-
80% over a MFC with dissolved oxygen .
The Experimental Findings Table 1 below depicts the
micro voltmeter readings in millivolts when i. Cow dung,
ii. Drain water, iii. Rice washing water, iv. Cow dung +
Slurry, v. Drain water + Slurry, vi. Rice washing water +
Slurry, vii. Cow dung + Slurry + Vermicompost, viii. Drain
water + Slurry + Vermicompost, ix. Rice washing water +
Slurry + Vermicompost, x. Slurry, and xi. Slurry +
Vermicompost, were used in the cells. These findings
have been included in a monograph by Barua [50]. After
every experiment was set up, the first datum was taken
after 1 complete day. In this way, after 1 complete day
every consecutive datum was taken. The values noted
were the peak values observed during the process of
measurement of the data. This was so done because the
electricity produced was intermittent probably due to
coarseness of the cells

Connecting the two electrodes would be a wire and

completing the circuit and connecting the two chambers there
would be a salt bridge. In place of commercially available
electrodes, graphite rods extracted from dry cells would be
utilized, the tips of which had been soldered to copper wires
traveling from one chamber to the other. The soldering was
performed at a melting temperature of 391o C. For preparation
of salt bridges, a water solution containing concentrations of
3% NaCl and 1.6% agar was allowed to boil inside a
microwave oven for nearly 3 minutes. The hot solution was
poured into sawed PVC pipe sections each of length 4 inches
by sealing one end with polythene. The setup was thereafter
allowed to cool for nearly 2 hours inside a High Efficiency
Performance Air Filter. The salt bridges were thus ready for
use.

Shewanella putrefaciens
Introduction
Shewanella putrefaciens is a bacteria that is found mainly in
marine environments. It is a gram negative bacteria,
meaning it does not dye during gram staining, which usually
indicates a stronger antibiotic resistance. It is also a
facultative anaerobe, meaning it can undergo aerobic
respiration when oxygen is present, and can reduce iron
and magnesium metabolically. Because of this Shewanella
putrefaciens can reduce Uranium and create uranium
deposits (fredrickson). Sheweanella putrefaciens grows
quickly on both solid and liquid media and is recognizable
for its pink color. Shewanella putrefaciens was first isolated
from dairy products in 1931 by Derby and Hammer. It is
classified as an Achromobacter and later named by
MacDonell and Colwell in 1985 (McNair).
Scanning electomicrograph image of Shewanella Putrefaciens
CN32 cells taken by the Department of Energy.
Effect on Marine Life
Shewanella putrefaciens can be found in freshwater,
brackish, and salt water ecosystems. Many healthy marines
animals are contaminated with Shewanella putrefaciens
only to have it be realized when food caught by seafood
industries spoils due to the bacterias presence. In
freshwater animals, and in particular fish species of trout,
the bacteria has been shown to cause disease. The effect
of the bacteria is seen through external lesions and visible
bacterial colonies. Fatality from Shewanella Putrefaciens
usually only occurs if the fish are already in poor health,
under environmental stress or a whole body inner infection
occurs that impedes organ function (Fisheries). Most
research done on Shewanella putrefaciens in relation to
marine life concentrates on the prevention of bacterial
outbreaks in fisheries. Much of the problem in prevention
comes from Shewanella's tendency to become a
contaminant or saprophyte, meaning it is often found living
among other bacterial infections on previously damaged
organs, as well as the bacteria's ability to survive at
extreme low temperatures and respiratory diversity (Hau).
These things combined make the bacteria hard to detect
until after the death of an organism, and hard to kill without
the use of antibiotics. Shewanella putrefaciens is also
known to cause the rotting smell associated with dead fish
because of its production of trimethylamines (McNair)
Open lesion of Shewanella putrefaciens on a carp taken by
Environment Agency, UK.
Impact on Humans
Shewanella putrefaciens as a humans pathogen is very
rare. If it does effect human health it is typically only seen to
show effects in combination eith other bacterial infections
such as E.coli, pneumonia, and streptococcus. Infections
form Shewanella putrfaciens mainly occur in soft tissue
such as skin, intra-abdominal areas, or in the blood
(Pagani) (McNair).
Shewanella putrefaciens is a main food spoilage bacteria in
marine fish, which in turn can effect human health, but also
creates a problem for the food industry. It also posses an
even larger problem for the food industry because of its
ability to form film on the food processing equipment that is
mainly made of stainless steal. Studies done by Applied
and Environmental Microbiology society looked deeper into
this problem and the possibility that Shewanella
Putrefaciens colonies on the equipment may be the source
of further bacterial pollution while also causing corrosion of
the equipment itself. The persistence of Shewanella
putrefaciens colonies on the equipment, even after
dissenfection, is partially due to the fact that it is a gram
negative bacterium that has a higher resistance to
antibiotics. The results of this study showed that although
disinfection did not have a significant impact on Shewanella
putrefaciens growth on the equipment, the presence of the
bacteria P. fluorescens did inhibit growth. This is due to the
fact Shewanella putrefaciens tends to grow in "microbial
communities" where other bacteria is part of the film
formed. When P. fluoresens is present in microbial film with
Shewanella putrefaciens it outcompetes and limits its
growth (Bagge).

Conclusion
Shewanella putrefaciens is also well known for its use in
biotechnology. Shewanella putrefaciens is a metal
reducing, facilitate anaerobe and this quality contributes to
its ability to be used in biotechnology. It is used as many
things from a bioremediate of chlorinated compounds to a
radionuclide and a biocatalyst. Its ability to be a biocatalyst
and to reduce iron has lead to interesting research done
with Shewanella putrefaciens being used in fuel cells. This
research is being done at the Korean Institute of Science
and Technologyand has shown that Shewanella has the
ability to be used in a fuel cell as part of a biosensor for
lactate. This means with the presence of Shewanella
putrefaciens as a electronon acceptor and metal reducer of
iron their was a change of charge detected in the fuel cell
when lactate was added. This presence of an
electrochemical reaction could mean a lot of things for the
use of Shewanella putrefaciens in fuel cells later on (Kim).
The bacteria has also been shown to derive energy by
reducing uranium, manganese, Vanadium, and Technetium
(Min).
  Electrical Energy Production Through Microbial
Fuel Cell using Industrial Wastewater Fisheries

Material and Equipment

The materials that used include activated sludge, fish

waste and chemicals to artificial wastewater analysis.
Tools that used among a set of tools comprising MFC
digital multimeter, vessel 500 ml, copper wires, carbon
electrode, and thermometer as well as the tools used to
manufacture and wastewater analysis.

Methods

The research begins with manufacture of fishery

wastewater. Waste is made by counting the fish then
mix water with a ratio of fish: water is 1: 5. Set of tools
that will be used MFC prepared the electrode chamber
7x10x10 cm and 7x1x1 cm. Liquid waste that has been
made is inserted into a vessel of 600 ml. After that, add
microorganisms or bacteria according to treatment, and
measured that electricity. Electricity measured for 5
days. The flow chart of working procedure is shown in
Fig. 1.
MICROBIAL FUEL CELL WITH A SIDE OF
BETTA FISH

"MUCK" is actually the correct term for soil from

Wetlands .

Muck has all kinds of awesome bacteria, one of

which is Geobacter, it produces electrons as part of
its cellular digestion!

Step 2: Prepare The Proton Exchange Membrane

Fuel cell that harvests electrons from the bacteria in mud! And
ELECTRons mean ELECTRicity.
The cell also makes a great habitat for a beta fish... The
bacteria decompose the fish poop adding to the cell's fuel and
keeping the water clean!
Step 1: Get Some MUCK!
75g salt. 200ml water. 5 grams agar or gelatin. Bring
to boil. Petri dish. Fridge. Done.
The bacteria produce hydrogen IONS (an ion is an
atom that has lost electrons)...a hydrogen ATOM is
nothing but a proton and an electron buzzing
around it...its like if the Earth was a proton and the
moon was an electron...take away the moon....er I
mean the electron and all you are left with is a
single PROTON aka a hydrogen ION
The PEM as they say allows protons through it and
recombine with oxygen and the electrons to form a
circuit and H20 as a byproduct.
 copper wire's "dendrites" or "feelers" as I like to call
them.
Step 3: Make Electrodes

Step 4: Put It All Together

PREPARE THE ANODE (-)

Cut up strips of window screen and fold them into a

square.

Google how to make "char cloth"....normally its

made by survivalists to act as tinder for fire making
but the high carbon content will make the bacteria
grow and exchange their electrons to the window
screen wire! Wrap the screen around a thick mat of
carbon cloth. You can also just strip the insulation off
the black wire and wrap the copper inner wires
around a thick mat of char cloth if you don't have
window screen or alligator clips.

PREPARE THE CATHODE (+)

Strip the red wire's insulation back to expose the

 Fill the Beaker with MUCK and the anode (make sure the
anode is covered on top and bottom)

 Put the PEM gel mold on top

 Fill up gently with water
 Put cathode in water (half in half out..see pix)

! After a few days you can watch the voltage increase as more
bacteria grow on the anode!

Right now I'm experimenting with "charge pump circuits" to

bring the voltage up from 500-600mv to 3 volts!
  Improvement of power generation using
Shewanella putrefaciens mediated
bioanode in a single chambered microbial
fuel cell: Effect of different anodic
operating conditions
Methods
1. Microbial strain, media and growth conditions
S. putrefaciens (ATCC BAA1097™) was used as
biocatalyst in the anode chamber. Colonies of S.
putrefaciens were grown on LB agar (HiMedia
Laboratories Pvt. Ltd., India) at 37
C for 24 h. Single colonies were incubated in 50 mL
LB broth (HiMedia Laboratories Pvt. Ltd., India) in
100 mL conical flasks, shaken continuously on a
rotary shaker (180 RPM) for at least 24 h under
aerobic conditions. 10 mL of the culture was used as
inoculum for the MFCs. For experiment purposes,
lactate based medium was used as anolyte in MFCs;
for 1000 mL anolyte preparation- 3 mL Na-lactate
(60% v/v); 0.425 g KCl; 2.028 g anhydride Na2SO4;
0.19 g CaCl22H2O; 0.105 g MgSO47H2O and 1.004
g yeast extract (Difco) were taken. The anolyte pH
was adjusted to 7 (unless stated otherwise) (Khilari et
al., 2014) with 1.0 M carbonate/bicarbonate buffer.
Desirable chemical oxygen demand (COD) was
maintained by altering the Na-lactate concentration in
the medium. Since the unmodified lactate medium
contained 0.19 g CaCl2, all concentrations of CaCl2
added for Taguchi optimization process were
normalized to the un-modified medium.
2. Synthesis of nano Fe2O3
Fe2O3 nano structure was prepared by a simple
solvothermal method. In a typical synthesis, 30 mL
0.05 M ethanolic solution of FeCl3 was taken in a
beaker and 360 mg urea was added to this solution
followed by stirring for 10 min. After stirring a clear
solution was formed which was transferred to a 50
mL Teflon-line stainless steel autoclave.
Hydrothermal dwell temperature was set at 150
C for 12 h. Then the autoclave was allowed to cool
down naturally to room temperature and precipitate
formed inside Teflon line container was collected by
centrifugation. Subsequently the product was washed
with distilled water followed by ethanol for several
times. Finally the washed precipitate was dried in an
oven at 60
C for 6 h.
3. Physical characterizations of nano Fe2O3
The crystallographic phase and structure were
evaluated from X-ray diffractogram. Powder X-ray
diffraction was carried out in a Rigaku Ultima III
using Cu Ka (40 kV, 40 mA) radiation over a range
of
20
80
. The surface morphology and composition was
studied with a Zeiss Supra scanning electron
microscope at an accelerating voltage of 5 kV.
4. Nano Fe2O3 decorated anode preparation
The anode was modified with nano Fe2O3 containing
electrode modifier ink. In a typical ink preparation,
requisite amount of nano Fe2O3 (0.2, 0.4, 0.8
mg/cm2 ) was dispersed in acetone-isopropyl alcohol
(1:1) solution with 5 wt% polytetrafluoroethylene
(PTFE; 60 wt%; Sigma Aldrich). The ink was
subsequently sprayed on a carbon cloth by a gravity
spray gun. Then the modified carbon cloth was
allowed to dry at 70
C in an oven for 6 h. Finally the dried Fe2O3 loaded
carbon cloth was used as anode in MFCs.
5. MFC assembly construction
Nine identical sMFCs were used for the experiments.
The MFC consisted of an anode compartment and a
membrane cathode assembly (MCA) placed on
opposite sides. The cuboidal anode chamber is made
from transparent polyacrylic material with outer
dimensions 7 8 3.5 cm3 of 110 mL capacity. The
anode chamber had two ports at the top, one for
electrode terminal and the other for reference
electrode (Ag/AgCl, saturated KCl; +197 mV,
Equiptronics, India) and sampling. The anode
consisted of a carbon cloth of working surface area
12 cm2 with a stainless steel wire welded to form the
terminal. A low cost custom made KOH doped
polyvinyl alcohol (PVA) -
polydiallyldimethylammonium chloride (PDDA)
anion exchange membrane (AEM) was used as
separator in sMFCs. The thickness of anion exchange
membrane was found to be 110 ± 05 lm. Membrane
cathode assembly (MCA) was prepared by coating
membrane with catalyst loaded cathode. In order to
prepare cathode, conductive ink containing cathode
catalyst [0.15 mg/cm2 Manganese cobaltite nanorods
(MnCo2O4-NRs) dust as reported in our earlier work
and carbon black Vulcan XC-72 (0.35 mg/cm2 ;
Cabot Corp.; India)] was taken in 20 mL 1:1 452 S.
Pandit et al. / Bioresource Technology 166 (2014)
451–457
 Efficient production of
glutathione using
hydrolyzate of banana peel
as novel substrate

Fermentative production of glutathione with hydrolyzate

of banana peels pretreated by dilute acid

CHEN Xue-dong,WANG Da-hui,DONG Ying-ying,WEI

Gong-yuan(School of Biology and Basic Medical
Sciences,Soochow University,Suzhou 215123)
The preparation of fermentable sugars from banana peel
s by dilute acid and its application in the fermentative producti
on of glutathione was investigated in this work,and it is feasibl
e that glutathione can be biosynthesized with Candida utilis us
ing the syrup from banana peel hydrolyzate as the substrate.Si
ngle factor experiments and uniform design was applied to opt
imize the key parameters influencing the hydrolysis of banana
peels by dilute sulfuric acid.Second order quadratic equations
were established based on the yield of reducing sugars and the
yield of glutathione,respectively.The validation of the models
showed that the maximum yield of reducing sugars and the ma
ximum yield of glutathione required different reaction conditio
ns for banana peels hydrolysis.Under the maximal conditions f
or each parameter,the maximum yield of reducing sugars and t
he maximum yield of glutathione were achieved as 287.6 mg/
g and 5.40 mg/g,respectively.The application of banana waste
for the production of glutathione will also give a good referenc
e for other useful chemical compounds using lignocellulosic m
aterial as substrate.