microalgae biotechnology

workkinkajouBiotechnology

Dec 5, 2012 (4 years and 8 months ago)

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BIOTEKNOLOGI MIKROORGANISME LAUT


Marine micro


represent vast and rich biomedical resource



algae, bacteria, fungi : can be found in all parts of the ocean, cosmopolitan, many exist as symbionts
of marine plants & animals, many unculturable



pharmacological resource potential:

Unidentified deep
-
sea bacterium


produce new chemical class of compounds


inhibit tumor cell
proliferation & replication of HIV/AIDS

Several marine fungi


produce antibiotics and neuroactive substances



Industrial r
esource:

Bacteria living in very high temperature water at deep
-
sea hydrothermal vent


manipulate &
genetically engineered


heat resistant enzymes


commercial applications, eg. Stratagene
(California): enzyme for PCR

Bacteria of hydrothermal vent


anot
her potentially attribute to oxydize H2S and use the enrgy to fix
CO2


organic compounds

Other bacteria


reproduce extremely rapidly and produce methane


biomass conversion into
methane more efficiently


Uncultured Micro

Genomic and bioinformatic method
s: powerful tools to access the gene products of these uncultured
micro

Eg. Total DNA from environmental sample can be purified without culturing the micro

Environment DNA


sequenced analogously to a genome and allows access not only to the protein
produc
ts of uncultured bacterial species but also to the genomic potential of the environment
(“ecological genomic”)

Eg. Bioinformatic analysis


cloning and expression of selected genes from the uncultured bacteria will
likely lead to the discovery of novel bio
active molecules

HTC (High Through
-
put Culturing)


produces to isolate cells in very low nutrient media


microbial
metabolic processes

Use of microtiter dishes and a newly developed procedure in which cell arrays are made on microscope
slides and
screened by fluorescence in situ hybridization (FISH)


Biomedical Resource



problem: antibiotic resistant microorganisms


easy to transmit, high prevalence, multidrug
resistant (= transferable resistance genes), fast resistance development, malpractice o
f antibiotics, etc.


big problem in developing countries

Eg. Pathogenic resistant bacteria


to human:

Staphylococcus aureus (MRSA), vancomycin
-
resistant Enterococcus faecium (VREF)

Gentamicin
-
resistant E. faecalis

Trovafloxacin & Ciprofloxacin
-
resistants

S. aureus

Pseudomonas, Salmonella, Shigella, Campylobacter, Mycobacterium

Eg. Less available of anti
-
micotyc drugs:

Candida albicans, Apergillus fumigatus, Cryptocarrion neoformans, Pneumicystis carinii

Marine microbes


alternative antibiotics!

Perbedaan komposisi kimia: highly halogenated


dipengaruhi oleh lingkungan laut


a. Proses
biokimia dan fisiologis yang kahs dan b. beragam habitat: shoreline, sediment, deep
-
sea, tropics, etc.


Latar Belakang

ZoBell

(1946)


mikrobia laut memiliki kemam
puan/aktivitas penting bagi kelangsungan proses
-
proses
biologi, fisiko
-
kimia, dan geologi (biogeokimia) lautan

Mikrob laut


jaman modern: industri besar, dengan nilai ekonomi jutaan US$

Mikrob laut


mampu mendiami seluruh bagian laut

Sifat
-
sifat:

1. morf
ologi sel:

Umumnya Gram negatif, bentuk batang, spirillum (vibrio), helicoid (kumparan), bergerak aktif


flagella
(adaptasi), tp pembentuk spora jarang

2. fisiologi:

Mampu mencerna semua senyawa organik dan anorganik (sebagian besar), garam (Na Cl) adala
h
keharusan


air laut diencerkan sampai 50%


pertumbuhan terhambat

3. kecenderungan melekat:

Mikrob laut cenderung menempel pada suatu permukaan


membentuk biofilm, biofouling

4. penyebaran/agihan:

Faktor yang memengaruhi


gerakan air laut, jarak dari pantai, kedalaman, cahaya matahari, iklim, dan
faktor biotik


Pengaruh Faktor Fisik dan Kimia (abiotik)

1. Suhu:

37 oC


42% terbunuh

45 oC


15% (sisa)

Optimum= 18


22 oC

2. Tekanan:

Tidak langsung


kelarutan senyawa kimia,

tahan hingga 3.000 atm!

3. Salinitas:

Ion Na+ dan Cl
-



penting

4. Konsentrasi ion
-
ion: K, Mg, Ca, Mn, Fe, Ni, Co, dst


kesetimbangan ion

Metabolisme: beda dengan mikrob darat

5. Senyawa organik:

Bahan organik terlarut (BOT), bahan organik partikel (BOP), bahan organik


CO2 + KH + Asam amino +
senyawa organik sederhana


organisme heterotrof


Keragaman Mikrobia Laut

Semua organisme bersel tunggal:

Autotrofik




prokaryot: bakteri, cyanobakteri

He
terotrofik



ekaryot: alga, protista

Virus

Ukuran: 0,3


150 um

3 kelompok besar: bacteria, archaea, eukarya





Prokaryot

Ekaryot

Perbedaan berdasar komposisi genetik


molekul ribosom RNA (rRNA): 16S dan 18S

a. Prokaryot Laut:

morfologi sederhana


rod
s (batang), sferis (membulat), filamen (benang), ukuran < 1


2 um, kecuali
bakteri Epulopiscium fishlesonii: Gram positif, panjang: 200


800 um, hidup dalam usus ikan di laut
merah

sangat beragam dalam hal taksonomi dan metabolisme, seperti: fototrof, ke
motrof, litotrof, organotrof
atau kombinasinya: kemoautotrof, kemolitotrof, dst


b. Jamur Laut:

ekaryot heterotrofik


contoh: jamur benang, kapang (yeast)


penting di daerah pantai sebagai
dekomposer (pengurai), tapi yeast merupakan parasit di laut terbuka (pada kopepoda)

ekaryot osmotrofik atau sapotrofik


Jejaring Makanan Mikrobia Laut

Produktivitas primer


fitoplankton (alga, diatom, dinoflagellata)


ko
pepoda


ikan. Ini teori klasik

Dikatakan “klasik” karena peran bakteri heterotrof sebagai sumber makanan bagi hewan benthos belum
disebutkan/dilibatkan di dalamnya

Percobaan botol gelap: O2 berkurang


respirasi oleh mikrobia (bukan kopepoda)


Pomeroy (1
974)


perubahan cara pandang!

3 hal poko dalam tulisan Pomeroy:

1. produsen primer utama bukan hanya fitoplankton tapi juga nanoplankton atau sel
-
sel fototrofik
(ukuran < 60 um)

2. mikrobia (atau organisme <366 um) yang merupakan bagian dari plankton


su
mber respirasi dan
metabolisme dalam air laut

3. bahan organik tidak
-
hidup (terlarut dan partikel)


sumber utama makanan bagi mikrobia heterotrof

Konsep yang terkait dengan “hipotesis” Pomeroy adalah: lingkaran mikrobia (microbial loop) oleh Azam
et al. (1983)


mikrob heterotrof (bakteri, bakterivor, protista)


peran sebagai jalur regenerasi
nitrogen organik, fosfor, unsur
-
unsur bioaktif dan mewak
ili mekanisme pengubahan C dan enerji dari
jaring makanan yang berdasar/bersumber
pada
fitoplankton.


Mikrobia Laut Patogen

Semula diduga mikrob laut tidak patogen! Contoh: Vibrio cholerae


sumber: saluran air limbah.
Penyakit yang ditimbulkan: tifus, kol
era, diare, gastroenteritis, dll. Sanitasi: masalah kesehatan, misal
kolam renang di pantai.


Manfaat Ekonomi Mikrobia Laut

Bakteri


pencemaran


merugikan: biofouling




menguntungkan: bioremediasi



pembusuk makanan: ikan, kerang, udang, dst. Beberapate
rhadap suhu pembekuan es, konsentrasi
garam laut. Beberapa jenis (kelompok dinoflagellata, diatom)


menghasilkkan racun/toksin dalam
kerang.



produk fermentasi (menguntungkan): ganggang coklat, ikan, cumi


oleh: Alteromonas, Aspergillus,
Penicillium, Clostridium, Rhodotorula, dst.


Bioteknologi Mikrobia Laut

Sumber metabolit sekunder

Simbiosis dengan organisme laut lain


organisme manakah yang menghasilkan metabolit sekunder?


misal: antibiotik, racun (TT
X, STX)

1947: Rosenfeld & ZoBell


bakteri laut mempunyai aktivitas antibiotik

1966: Burkholder et al.


berhasil memurnikan senyawa antibiotik dari bakteri laut

1966: Lovell


struktur kimia


highly brominated pyrrole


senyawa metabolit sekunder = antib
iotik


sangat berbeda dengan senyawa antibiorik yang dihasilkan oleh mikrobia darat


Tabel 1. Sumber bakteri penghasil antibakteri

Sumber

Jumlah strain dari Genus

Pseudomonas/

Alteromonas

Vibrio

Flavobacterium

Air laut

12

-

1

sedimen

1

-

-

fitoplankton

5

5

-

zooplankton

10

1

-

sponge

2

-

-

Total %
-
ase

81

16,3

2,7

Sumber: Nair & Simidu, 1987


Tabel 2. Agihan bakteri anti terhadap V. Vulnificus RIMD2219009 dalam usus ikan pesisir

Strain Uji

<1,0

1,0


9,9

10,0


19,9

20,0


29,9

> 30

Acinetobacter

10

0

0

0

0

Bacillus

41

0

0

0

1

Coryneforms

52

1

1

2

1

Enterobacteriaceae

125

1

5

0

0

Favobacterium

60

0

0

0

0

Micrococcus

3

0

0

0

0

Moraxella

24

0

0

0

0

Pseudomonas

100

0

1

0

0

Staphylococcus

12

0

0

0

0

Vibrionaceae

600

1

3

0

11

Total

1027

3

10

2

13

Sumber: Sugita et al. 1998


Tabel 3. Jenis Bakteri laut Penghasil antibiotik

Bakteri

Antibiotik

Pustaka

Bacillus cereus QN03323

Tiopeptide

Nagai et al. 2003

Bacillus sp. MK
-
PNG
-
276A

Cyclic decapetide

(Loloatin A, B, C, D)

Gerard et
al. 1999

Bacillus sp. PP19
-
H3

macrolactins

Sano et al. 2001

Bacillus sp strain NM12

Siderophore (MW < 5 kDa)

Sugita et al. 1998

Actinomycete isolat CNB
-
632

Marinone, debromo
-
marinone,
sesquiterpenoid, naphthoquinone

Pathirana et al. 1992

Actinomycete
(Chainia
purpurogena SS
-
228)

benzanthraquinone

Kitahara et al. 1975; Okazaki
et al. 1975

Myxobacterium (Haliangium
luteum)

haliangicin

Yamanaka et al. 2001;
Kundim et al. 2003

Streptomyces sp

Δ
-
楮domy捩cone

䉩Bb慮椠整⁡氮 1997

Streptomyces sp MT
-
MA 190

lerneomides

Capon et al. 2000

Streptomycete

Dihydrophencomycin methyl esther

Pusecker et al. 1997

S. rishiriensies MJ 773
-
88K4

lactonamycin

Matsumoto et al. 1999

S. tenjimariensis SS
-
939

Istamycins A & B

Okami et al. 1979

S. maritimus

Enterosin,
wailupemycins

Piel et al. 2000

Sumber: Isnansetyo, 2007


Tabel 4. Marine bacteria that are proved to produce pharmaco
-
medical substances

Species/Strain

Source

Biomedical Activity

Chainia purpurogena

sediment

Antibiotic, anticancer

Streptomyces
tenjimariensis

sediment

antibiotic

S. griseus

sediment

antibiotic

S. sioyaensis

sediment

anticancer

Maduromycete

sediment

antibiotic

Streptomyces sp

sediment

antibiotic

Actinomycete

sediment

antibiotic

Actinomycete

Gorgonian soft coral

Anticancer,
antibiotic

Streptomyces sp

jellyfish

Antibiotic, antiinflammatory

Streptomyces sp

Sponge

antibiotic

Pseudomonas bromoutilis/

Alteromonas sp

Seagrass

antibiotic

Chromobacterium sp

seawater

antibiotic

Alteromonas rubra

Not reported

bronchodilator

Alteromonas sp

Sponge

anticancer

Alteromonas haloplanktis

Deep
-
sea sediment

annticancer

Alteromonas sp

Fish

Enzyme inhibition

Pseudomonas fluorescens

Ascidian

antimicrobial

Thermococcus

Hydrothermal vents

antifungal

Bacillus

Deep
-
sea sediment

anticancer

Source: Austin 1988, Jensen & Fenical 1994








MICROALGAE BIOTECHNOLOGY

Algae:

Net primary production: 52 billion ton organic carbon per yyear: 50% total organiic carbon on earth

+ 40,000 species, heterogenous group, broad spectrum phenotype

Mostly eukaryotes, live in aquatic environment or extreme environment


eg. Dunaliella salina: nearly
saturated with NaCl, Chlamydomonas nivalis
: adapted to low temperature, poor nutrition, permanent
freeze
-
thaw cycles, high irradiation

Symbiotic relation
ship: eg. Symbiodinium microadriaticum: in the gastrodermis of reef
-
building corals,
Chlorella: in jelly fish
-
related hydra


Utilization of Algae

In History


Food source: Nostoc, Spirulina, Aphanizomenon sp


nutrient dense food


Asia, Africa, Mexico


eg
.
In China: Nostoc: to survive during famine

Live feeds: 1940’s


microalgae in aquaculture (shellfish or fish farming)

Nutrient source: protein, fat

Health food

Renewable fuels

Fertelizers

Wastewater treatment

Others: carotenoid ataxanthin


pharmaceutica
l, nutraceuticals, agriculture, animan nutrition. In
1990’s


Haematococcus pluvialis



Present Time

About 10
7

tons of algae harvested each yyear


algal biotechnology industries: food, feed, food and
feed additives, cosmetics, pigments

Human food: Chlorel
la and Spirulina


high protein content, nutritive value, easy to grow

Animal feed: Spirulina and Chlorella


cats, dogs, aquarium fish, birds, horses, poultry, cows, bulls

Aquaculture: bivalve molluscs (oyster, scallops, clams, mussels), larva & juvenile
stages of abalone,
crustaceans, fish


eg. Nitzschia closterium, Chaetoceros spp, Thalassiosira pseudonana, Dunaliella,
Nannochloris, Pyramimonas, Pavlova

Chemicals and Pharmaceuticals: novel & valuable compounds


fatty acids, pigments, vitamins, other
bi
oactive compounds


polyunnsaturated fatty acids
ω
-
3. Eg. Crypthecodinium cohnii (
ω
-
3), Spirulina
(gamma
-
linoleic acid/GLA), Porphyridium

arachidonic acid (ARA), Nannochloropsis


eicosapenttaenoic acid (EPA), Chlorella


polysaccharides (dietary supplements, cosmetics, nutrition),
Lyngbya majuscula


immune modulators, nutrition management

Pigments: not only chlorophylls, but other pigments


to improve the efficiency of light energy
utilization and protection from damage by sunlight. Eg. Carotenoids & phycobiliproteins, carotenoids


pro
-
vitamin A, antioxidants


Dunaliella spp. Phycobiliproteins


phycoerithrin & phycocyanin


Spirulina, Por
phyridium. Phycobiliproteins


health food, pharmaceuticals, cosmetics


The Basis of Algal Transgenic

Ease of cultivating

Short life cycle in liquid, axenic culture

Grow in high densities

Previous molecular, biochemical, physiological, ecological of target

species


desired


Production of Transgenic Algae

25 algal species


stable transformation, transformation efficiency: species
-
dependent. Eg.
Cyanidioschyzon: + 200 transformants per ug plasmid
-
DNA appeared when 3
-
4 x 108 cells were spread
on algal plate


Selectable Marker Genes

Eg. Antibiotic resistance genes


Chlamydomonas reinhardtii


the R100.1 plasmid/bacteriophage T4/synthetic aminoglycoside
adenyltransferase gene aadA confers resistance to spectinomycin & strepromycin

Streptoalloteichus hindustanu
s: ble gene to zeomycin & phleomycin


Methods to Introduce DNA into Algal Cells

To cause temporal permeabilizaion of the cell membrane, enabling DNA molecules to enter the cell

Most popular method: micro
-
particle bombardment, micro
-
projectile bombardment,
particle gun
transformation, gene gun transformation or biolistics

Less complex and less expensive: preparation of a suspension of microalgae


agitated in the presence
of micro
-

or macro
-
particles, polyethylene gycol and DNA


Problems in Algal Genetic Eng
ineering

Gene silencing: through methylation


caused by positional effects and epigenetic mechanisms

DNA constructs with a heterologous origin


the bias in codon usage

Introns occurence: they will likely not be spliced correctly

Heterologous flanking sequences: promoters


inadequate recognition of the heterologous promoter
region and lack of adequate regulation

Insufficient DNA delivery: failure to integrate into the genome, improper transport into the chloroplast
or through the
plasma membrane into the extra
-
cellular compartment