The aim of present study was to investigate the production of rhamnolipid
biosurfactant produced by five strains of Pseudomonas aeruginosa originated
from hydrocarbon rich environments (soil polluted ...with petroleum and
petroleum derivates and industrial waste - mineral mettal cutting oil). The
first part of the study was optimization of production of rhamnolipids by
strain P. aeruginosa NCAIM (P) B 001380, a producer of several microbial
methabolites with potential industrial application. With regard to carbon and
nitrogen source several media were tested to enhance production of
rhamnolipids. Phosphatelimited proteose peptone-ammonium salt (PPAS) medium
supplemented with sunflower oil as a source of carbon and mineral ammonium
chloride and peptone as a nitrogen source greatly improved rhamnolipid
production, from 0.15 on basic PPAS (C/N ratio 4.0), to 3.00 g/l, on
optimized PPAS medium (C/N ratio 7.7). Further, the effect of three factors:
temperature, concentration of carbon and nitrogen source in optimized PPAS
medium on rhamnolipid production was analyzed by Response Surface
Methodology. It was found that the environmental isolates of P. aeruginosa
had a significant potential for improved production of rhamnolipids on
different types of substrates, including cheap, renewable sources like
sunflower oil from deep fryer and sunflower oil mill effluent. MS analysis
indicated that rhamnolipid preparations were mixtures of
mono-rhamno-mono-lipidic, mono-rhamno-di-lipidic- and dirhamno- di-lipidic
congeners. High througout screening of rhamnolipid mixtures produced by
different environmental P. aeruginosa strains (NCAIM (P) B 001380, D1, D2,
D3, 67) and referent clinical strain ATCC 27853 on optimised medium was
performed by MALDI-TOF analysis. Observed differences between rhamnolipid
profiles indicated a possibility to define rhamnolipid fingerprint of
analyzed P. aeruginosa strains. Detailed comparative analysis of rhamnolipids
from environmental isolates of P. aeruginosa was undertaken to evaluate
strain specific rhamnolipid fingerprints obtained on different growth
conditions. Rhamnolipids were monitored by HPLC-ESI mass spectrometry, which
allowed fast and reliable identification and quantification of present
congeners. The highest concentration of total rhamnolipids of 3.33 g/l was
obtained by the strain P. aeruginosa 67, recovered from petroleum
contaminated soil, and strains D1 (1.73 g/l) and D2 (1.70 g/l), recovered
from natural microbial consortia originated from mazut contaminated soil,
grown on sunflower oil as a carbon source. Di- to mono-rhamnolipids ratios
were in range of 0.90 to 5.39 for different media composition and from 1.12
to 4.17 for different producing strains. Rhamnolipid profiles of purified
mixtures of all tested strains are similar with chain length from C8-C12,
pronounced abundance of Rha-C10-C10 and Rha-Rha-C10-C10 congeners, and a low
content of hydroxyalkanoyloxy-alkanoic acid Concentrations of major congeners
of rhamnolipids were found to slightly vary, depending on strain and growth
conditions, while variations in minor congeners were more pronounced.
Statistically significant increase of CMC values was observed with lowering
the ratio of total mono- to di-rhamnolipids ratio indicating that
mono-rhamnolipids start to form micelles at lower concentration than
di-rhamnolipids. Rhamnolipid mixtures showed antimicrobial activity on
Gram-positive strains, while Gram-negative becterial strains and fungi had
low sensitivity or were insensitive. Additionaly, the tolerance of P.
aeruginosa NCAIM (P) B 00138 to heavy metals (Cd, Cu and Hg) was determined,
uptake of cadmium was monitored and Cd effect on protein profile expression
was analyzed.
Cilj ove studije je bio da se ispita proizvodnja ramnolipida dobijenih pomoću
pet sojeva Pseudomonas aeruginosa, izolovanih iz sredina bogatih
ugljovodonicima (zemljišta zagađenog naftom i naftnim derivatima i
industrijskog otpada – mineralnog ulja za sečenje metala). Prvi deo
istraživanja je bila optimizacija proizvodnje ramnolipida pomoću soja P.
aeruginosa NCAIM (P) B 001380, producenta nekoliko mikrobioloških metabolita
koji imaju potencijal za industrijsku primenu. Podloge sa različitim sastavom
(izvor ugljenika i azota) su testirane kako bi se poboljšala produkciju
ramnolipida. Phosphate-limited proteose peptone-ammonium salt (PPAS) podloga
sa suncokretovim uljem, kao izvorom ugljenika, i amonijum hloridom i
peptonom, kao izvorom azota, značajno je povećala proizvodnju ramnolipida od
0,15 na osnovnoj PPAS (C/N odnos 4,0), na 3,00 g/l, na optimizovanoj PPAS
podlozi (C/N odnos 7,7). U nastavku je primenjena statistička analiza
Response Surface Methodology za testiranje efekata tri faktora: temperature,
koncentracije izvora ugljenika i azota (v/v) u optimizovanoj PPAS podlozi, na
proizvodnju ramnolipida. Utvrđeno je da ekološki izolati P. aeruginosa imaju
značajan potencijal za poboljšavanje produkcije ramnolipida na različitim
tipovima supstrata, uključujući ekonomične, obnovljive izvore kao što su
suncokretovo ulje iz friteze i otpadne frakcije tokom rafinisanja
suncokretovog ulja. MS analize su pokazale da su izolovani ramnolipidni
biosurfaktanti smeše monoramno- mono-lipidnih, mono-ramno-di-lipidnih- i
di-ramno-di-lipidnih jedinjenja. “High througout skrining“ ramnolipidnih
smeša dobijenih pomoću različitih sojeva P. aeruginosa (NCAIM (P) B 001380,
D1, D2, D3, 67) i referentnog kliničkog soja ATCC 27853 na optimizovanoj
podlozi urađen je pomoću MALDI-TOF analize. Razlike između ramnolipidnih
profila koje su specifične za soj su ukazale na mogućnost da se definiše
ramnolipid “fingerprint” analiziranih izolata P. aeruginosa. Detaljna
komparativna analiza ramnolipida je urađena kako bi se video efekat uslova
kultivacije na soj - specifični “fingerprint”ramnolipida. Ramnolipidi su
praćeni pomoću HPLC-ESI MS, što je omogućilo brzu i pouzdanu identifikacije i
kvantifikaciju prisutnih struktura. Najveća koncentracija ukupnih ramnolipida
od 3,33 g/l dobijena je za soj P. aeruginosa 67, koji je izolovan iz
zemljišta zagađenog naftom i sojeve D1 (1,73 g/l) i D2 (1,70 g/l), iz
prirodnog mikrobnog konzorcijumima poreklom iz zemljišta zagađenog mazutom,
gajenih na suncokretovom ulju, kao izvoru ugljenika. Odnos ukupnih
di-/mono-ramnolipida su bili u rasponu od 0,90 do 5,39 za različite izvore
ugljenika i od 1,12 do 4,17 za različite produkcione sojeve. Za sve profile
ramnolipidnih smeša tipični su bili: lanci hidroksi-masnih kiselina dužina
C8-C12, najzastupljenije strukture su bile Rha-C10-C10 i Rha-Rha- C10-C10 i
nizak sadržaj hidroksi-alkanoiloksi karboksilnih kiselina. Takođe,
koncentracije glavnih ramnolipidnih struktura su se neznatno razlikovale u
zavisnosti od soja i uslova rasta, dok su razlike u sporednim strukturama
bile izraženije. Statistički značajno povećanje vrednosti CMC je primećeno sa
snižavanjem odnosa ukupnih mono-/di-ramnolipida, što je pokazalo da
monoramnolipidi počinju da formiraju micele pri nižoj koncentraciji od di -
ramnolipida. Izolovane ramnolipidne smeše su pokazale antimikrobnu aktivnost
na Gram-pozitivne bakterije, dok su Gram-negativni bakterijski sojevi i
gljive imale ili nisku osetljivost ili su bili neosetljivi. Dodatno, u ovoj
studiji je ispitivana tolerancija P. aeruginosa NCAIM (P) B 00138 na teške
metale (Cd, Cu i Hg), praćeno usvajanje kadmijuma i efekat kadmijuma na
proteinsku ekspresiju.
Produção de biotensoativos a partir de resíduos de óleos e gorduras Costa, Siddhartha Georges Valadares Almeida de Oliveira(Universidade Estadual Paulista Instituto de Biociências de Rio Claro Departamento de Bioquímica e Microbiologia); Nitschke, Márcia(Universidade de São Paulo Instituto de Química de São Carlos); Contiero, Jonas(Universidade Estadual Paulista Instituto de Biociências de Rio Claro Departamento de Bioquímica e Microbiologia)
Ciência e tecnologia de alimentos,
01/2008, Letnik:
28, Številka:
1
Journal Article
Recenzirano
Odprti dostop
O presente trabalho visou a seleção de microrganismos com capacidade de produzir biotensoativos a partir de resíduos de óleos e gorduras gerados em restaurantes e indústrias alimentícias. Borra de ...soja, gordura de frango, gordura vegetal hidrogenada e óleo de soja usado em frituras foram estudados como fonte de carbono. Os isolados LMI 6c e LMI 7a, ambos pertencentes ao gênero Pseudomonas, foram selecionados como potenciais produtores de biotensoativos. Dentre os resíduos propostos, a borra de soja foi considerada o melhor substrato, gerando 9,69 g.L-1 de ramnolipídios e uma tensão superficial de 31 mN/m.
The purpose of this study was to select microorganisms that were able to produce biosurfactants from fats and oils wastes generated by the food industry and restaurants. Soybean soapstock, chicken fat, hydrogenated vegetable fat and soybean frying oil were evaluated as alternative substrates. Pseudomonas sp. isolates LMI 6c and LMI 7a showed a capacity to utilize the substrates and to produce rhamnolipid surfactants. Soybean soapstock was considered the best substrate, generating 9.69 g.L-1 of rhamnolipids and a surface tension of 31 mN/m.
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