Figure 2. Major type of linkages found in lignin Bugg et al. 2011
2.3 Polysaccharide Degrading Microorganisms
The biodegradation of polysaccharide structures involves the concerted action of a variety of hydrolytic enzymes. Some microorganisms have been
identified having hydrolytic enzymes for cleavage of almost chemical bonds that found in plant structures.
Streptomyces coalicolor A32, Bacillus halodurans C-125, Bacillus
subtilis 168, Cellvibrio japonicus, Clostridium acetobutylicum ATT 824,
Bifidobacterium longum NCC2705, Xanthomonas axonopodis pv. Citri str. 306,
Xanthomonas campestris pv campestris str. ATTC33913, Geobacillus
stearothermophillus T-6, Geobacillus stearothermophillus 21 are known as
bacteria having ability to produced hemicellulolytic enzymes Shallom Shoham 2003.
Bacillus circulans, Bacillus subtilis, Clostridium cellulolytic, Clostridium thermocellum, Cellulomonas fimi, Pseudomonas fluorescens,
and Vibrio sp. can produce mannanase to hydrolyze mannans, which is the biggest content of NSPs
in PKM Moreira Filho 2008; Sundu Dingle 2003. Mannan degrading bacteria also distribute in aquatic environment. Araki and Kitamikado 1978
found 8 genera of bacteria, i.e Pseudomonas, Alcaligenes, Klebsiella,
Enterobacter, Vibrio, Aeromonas, Moraxella, and Bacillus. These bacteria were
collected from sea, freshwater, and land environments. Mannan–degrading enzyme is also produced by some eukaryotic
organisms, such as Trichoderma reesei, Aspergillus aculeatus, Agaricus bisporus Moreira Filho 2008; Sundu Dingle 2003. Cellulolytic fungus such as
Trichoderma koningii has been used to reduce fiber and increase protein content
of PKM by solid-state fermentation Ng 2003. To decrease fiber and increase protein content of palm kernel, Illuyemi et al. 2006 also tried to ferment palm
kernel cake using Sclerotium rolfsii, Trichoderma harzianum, Trichoderma longiobrachiatium, Trichoderma koningii,
and Aspergillus niger. Yopi et al. 2006 found that Streptomyces limpanii and Saccharopolyspora flava have a
capability to degrade mannan of palm kernel cake. Pseudomonas
spp., P. acidovorans, P. cepacia, P. cruciviae, P. flourescens, P. multivorans, P. ovalis, P. paucimobilis, P. pseudoalcaligenes, P.
putida ,
Xanthomonas, Acinetobacter, Flavobacterium, Achromobacter, Agrobacterium, Beijerinckia, Aeromonas, Erwinia, Actinomadura, Arthrobacter
spp., A. simplex, Corynebacterium, Nocardia, Rhodococcus, Streptomyces spp., S. badius, S. cyaneus, S. rimosus, S. viridosporus, B. megaterium, B. subtilis
are known as bacteria that have ability to degrade lignin Zimmermann 1990.
2.4 Bioconversion using
Hermetia illucens larvae
The bioconversion or known as biotransformation is the process that uses live organisms, often microorganisms, to convert a substrate into chemically
modified form. One of the types of bioconversion is conversion of organic refuse by saprophages. In this system, live organisms feed on organic matter to reduce
and convert organic waste in to live feedstock for livestock farming.
One of organisms that already used in bioconversion process is H. illucens larvae. This larvae has been widely recognized as a putrescent waste i.e. already
decomposed by bacteria management agent. The larvae could be used in reducing some organic waste such as farming industry waste; food industry waste, kitchen
waste institutional, restaurant, and domestic, and human waste Olivier 2001. Lardé 1990 reported that the coffee pulp lost its 29.8 of the initial dry matter
after digestion by the larvae in 13 days. Sheppard et al. 1994 reported that the
larvae reduced at least 50 in depth the manure accumulation in a caged layer hen manure management system. Hem et al. 2008 used these larvae to reduce
PKM. Larvae H. illucens that already used to reduce by product or manure is
useful as feed for poultry. Newton et al. 1977 used these larvae as feed for poultry and pig. In aquaculture, these larvae already used as feed for tilapia Hem
et al . 2008, catfish Bondari Sheppard 1981, and botia Septivia 2008.
Prepupae of these species can replace fish meal up to 25 in feed formulation for rainbow fish St-Hilaire et al. 2007.
2.5 Fermentation Fermentation is catabolism of organic compound, generally carbohydrate,
that carried out by microorganism Gandjar et al. 2006. Some aspects that influence the fermentation including physiochemical and biochemical parameter
are particle size, initial moisture, pH, pre-treatment of the substrate, relative humidity, temperature on incubation, agitation and aeration, age and size of
inoculums. The temperature of the substrate is affects the growth of microorganism, spore formation, germination, and product formation. Water
content in the fermentation system influences microbial activity. High moistures results in decreased substrate porosity, which in turn prevents oxygen penetration.
Otherwise, low moisture may lead poor accessibility of nutrients resulting poor microbial growth Pandey 2003.
The biggest content in by product of agro-industry is polysaccharides. The polysaccharides in this fermentation process will be converted into simple
monomeric or dimeric sugars. Cellulose is degraded into glucose by exo-1,4- -D- glucanases or cellobiohydrolases EC 3.2.1.91, endo-1,4- -D-glucanases EC
3.2.1.4 and 1,4- -D-glucosidases EC 3.2.1.21 Figure 3 Mussatto Teixeira 2010.
Figure 3. Schematic representation of cellulase enzymes over the cellulose structure Mussatto Teixeira 2010
Hemicellulose is degraded into variety of monosaccharide, including galactose, arabinose, xylose, mannose, and glucose. Mannan, as a biggest content
of hemicelluloses in PKM, is degraded by some enzymes i.e. endo- -mannanase 1,4- -D-mannan mannohydrolase, EC 3.2.178, exo- -mannosidase 1,4- -D-
mannopyranoside hydrolase, EC 3.2.1.25, and exo- -glucosidase 1,4- -D- glucoside glucohydrolase, EC 3.2.1.21. Endo- -mannanase
cleavage -1,4-linked internal linkages of the mannan backbone randomly to produce new chain ends.
Exo- -mannosidase cleava ge -1,4-linked mannosides and produce mannose from
the nonreducing end of mannans and manno-oligosaccharides. Exo- -glucosidase hydrolyze 1,4- -D-glucopyranose at the nonreducing end of the oligosaccharides
released from glucomannan and galacto- glucomannan by -mannanase Figure 4
Moreira Filho 2008.
Figure 4. Schematic hydrolyzed of mannans by some enzyme Moreira Filho 2008
-glucoside -1,4-endoglucanase
-1,4-exoglucanase cellulose
cellobiose glucose
1,4
1,4 1,4
1,4
As a complex aromatic heteropolymer, degradation of lignin needs various enzymes. Lignin consists of some components. The catabolic pathway for
breakdown lignin components have been identified and characteristics using model substrate. The pathway of degradation lignin components are shows in
Figure 5. The pathway of -aryl ether lignin dimer compound is by oxidation of
the α-hydroxyl group to the corresponding ketone which catalysed by the NAD-
dependent dehydrogenase LigD. Then, an unusual reductive ether cleavage reaction takes place, via a novel glutathione dependent -etherase enzyme. The
LigEFG gene products are responsible for this transformation. In Pseudomonas putida
and Rhodococcus jostii RHA1, the ketone product has been detected by- product of lignocellulose breakdown via the -etherase cleavage reaction, or via a
-elimination reaction. The ketone product is then metabolised to vanillic acid, probably via oxidation of the -hydroxyl group to the carboxylic acid. In S.
paucimobili s SYK-6 demethylation of vanillic acid to protocatechuic acid is
catalysed by a tetrahydrofolate dependent demethylase LigM. The demethylation reaction in Pseudomonas and Acinetobacter is catalysed by a non heme iron
dependent demethylase enzyme. The product, protocatechuic acid, is then a substrate for oxidative ring cleavage. In white-
rot fungi, metabolism of -aryl ether model compounds is predominantly via C
α
-C oxidative cleavage, to give vanillin as a product. Vanillin is then oxidised to vanillic acid by vanillate
dehydrogenase Figure 5 Bugg et al. 2011.
Figure 5. Aroma tic degradation pathway of -Aryl ether lignin compound
Bugg et al. 2011
Degradation of the biphenyl compound is via oxidation 2,3- dihydroxybiphenyl, followed by oxidative meta-cleavage. The pathway for the
degradation of the biphenyl compound of lignin in S. paucimobilis SYK-6 is shown in Figure 6. Demethylation of one methoxy group is catalysed by a non-
heme iron-dependent demethylase enzyme LigX. The catecholic product of LigX is then a substrate for oxidative meta-cleavage by the extra dioldioxygenase LigZ,
and the ring fission product is then cleaved by C–C hydrolase LigY, to form 5-
carboxyvanillic acid and 4-carboxy-2-hydroxypentadienoic acid. Decarboxylase enzymes LigW convert 5-carboxyvanillic acid into the central intermediate
vanillic acid. By analogy with other aromatic degradation pathways, where the product of hydrolytic C–C cleavage is often the substrate for a hydratase enzyme,
the by product of LigY-catalysed cleavage, 4-carboxy-2-hydroxypentadienoic acid, is probably hydrated to form 4-hydroxy-4-methyl-2-oxoglutarate, for which
an aldolase enzyme. Aldolase-catalysed cleavage would then generate two molecules of pyruvic acid Bugg et al. 2011.
Figure 6. Aromatic degradation pathway of biphenyl lignin compound Bugg et al. 2011
The degradation of diarylpropane lignin compounds is shown in Figure 7. In P. chysosporium, the process is by oxidation the cleavage of the C
α
–C bond occurs in, to give aromatic aldehyde products. This reaction is catalysed by lignin
peroxidase. In Pseudomonas paucimobilis TMY1009, the degradation process is catalyzed by an enzyme activity which catalyses a novel fragmentation reaction,
namely elimination of formaldehyde and water. The product of this reaction is
metabolised by lignostilbene. Lignostilbene dioxygenase is a non-heme iron- dependent dioxygenase enzyme that catalyses the oxidative cleavage of
lignostilbene to give two molecules of vanillin Bugg et al. 2011.
Figure 7. Aromatic degradation pathway of diarypropane lignin compound Bugg et al. 2011
Breakdown of the phenylcoumarane compound of lignin has been studied in fungus Phanerochaete chrysposporium and Fusarium solani Figure 8.
Degradation of the alkylated phenylcoumarane by P. chrysosporium was initially by oxidation of the sidechain, then oxidation of the heterocyclic ring to a furan,
and finall y oxidative C
α
–C bond cleavage. In Fusarium solani M-13-1, breakdown of the phenolic phenycoumarane proceeded via direct C
α
–C bond to give 5-acetylvanillone. Loss of the -hydroxyl group could be rationalised by the
formation of an epoxide intermediate, followed by hydrolytic cleavage of the C
α
– C bond Bugg et al. 2011.
Figure 8. Aromatic degradation pathway of phenylcomarane lignin compound Bugg et al. 2011
The pathway for breakdown of the pinoresinol compound of lignin has also been studied in Fusarium solani M-13-1 Figure 9. Oxidation of the benzylic
position was observed, leading to C–O bond cleavage, to form a monocyclic ketone intermediate. Further aryl–alkyl oxidation yielded a carboxylic acid
product, and the corresponding lactone. In bacteria, S. paucimobilis SYK-6 is reported to degrade pinoresinol lignin model compounds, but the genes
responsible have not yet been identi fied. The catabolic pathways for both
heterocyclic lignin compounds appear to involve α-hydroxylation as an initial step
Bugg et al. 2011.
Figure 9. Aromatic degradation pathway of pinoresinol lignin compound Bugg et al. 2011
III. MATERIALS AND METHODS
3.1 Site of Research
The research was conducted at Balai Riset Budidaya Ikan Hias BRBIH, Depok from March to September 2011.
3.2 Condition of Fermentation
PKM supply has been ordered from PT. Perkebunan Nusantara VII, Bandar Lampung. The fermentation was made by mixing 42 kg of PKM with 84
L of water 1:2 and put it in the big drum height 81 cm; diameter 48 cm. The fermentation height in drum is 80 cm. The sample was taken from the depth of
±40 cm using pipe at some point. At 12 h intervals during 7 days take the sample for analysis of pH, total acid, temperature, and microbial analysis. Analysis of D-
mannose was conducted at 0, 36, 48, 72, 96, and 120 hours. Analysis of concentration of volatile organic acid was conducted at 0, 36, 72 and 168 hour.
Analysis of fiber was conducted at 0, 36, and 168 hours. Analysis of protein content was conducted at the beginning and at the end of fermentation Figure
10.
3.3 Microbial analysis
For all determination, 10 grams of fermented sample dispersed into 90 ml of NaCl 0.9 sterile, and then submitted to a mechanical stirrer for 5 minutes to
extract cells from the substrate. This corresponds to 10
-1
dilution. The extraction step followed by serial dilution, ranging from 10
-2
to 10
-8
. From each dilution, 0.1 mL was surface plated for determination of microbial count in each sample of
respective agar media, followed by incubation at 30 C. Total aerobic bacteria
were determined on Trypticase Soy Agar TSA Fluka incubated at 30 C for 48
hours. Total anaerobic bacteria were determined on Trypticase Soy Agar TSA Fluka incubated at 30
C for 48 hours in anaerobic condition. Enumeration of lactic acid bacteria was carried out using De Man, Rogosa and Sharpe MRS
Sigma medium after incubation at 30 C for 4 days. Enumeration of clostridial
bacteria was carried out using Reinforced Clostridial Agar RCA Pronadisa after incubation at 30
C for 48 hours in anaerobic condition. Typical
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