In situ bioremediation using biosurfactant produced by solid state fermentation

The discovery that certain microorganisms, living within a marine environment, can actually degrade components of oil, has made possible the utilization of biological methods for the treatment of oil spills. A biosurfactant accelerates the process of degradation of pollutant composites. The objective of this work was to study the bioremediation in situ of a diesel oil spill by utilizing a biosurfactant produced through fermentation and then compare it with chemical remediation. The quantification and identification of hydrocarbons were carried out by the process of gas chromatography. The soil indigenous microorganisms were monitored. The experiment with biosurfactant reached reductions of 99% of the aliphatic hydrocarbons, while that of the chemical disperser experiment reached a maximum of 90% reduction in 180 days. In 15 days the biosurfactant removed 77% of the aliphatic hydrocarbons, the diesel oil experiment 8.7% and the chemical disperser only 5%. The biosurfactant was 99% effective for the removal of aromatic polycyclic hydrocarbons, up to 3 rings.     

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.     

Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains

Methanol is regarded as an attractive substrate for biotechnological production of value-added bulk products, such as amino acids and polyamines. In the present study, the methylotrophic and thermophilic bacterium Bacillus methanolicus was engineered into a microbial cell factory for the production of the platform chemical 1,5-diaminopentane (cadaverine) from methanol. This was achieved by the heterologous expression of the Escherichia coli genes cadA and ldcC encoding two different lysine decarboxylase enzymes, and by increasing the overall L-lysine production levels in this host. Both CadA and LdcC were functional in B. methanolicus cultivated at 50°C and expression of cadA resulted in cadaverine production levels up to 500 mg l⁻¹ during shake flask conditions. A volume-corrected concentration of 11.3 g l⁻¹ of cadaverine was obtained by high-cell density fed-batch methanol fermentation. Our results demonstrated that efficient conversion of L-lysine into cadaverine presumably has severe effects on feedback regulation of the L-lysine biosynthetic pathway in B. methanolicus. By also investigating the cadaverine tolerance level, B. methanolicus proved to be an exciting alternative host and comparable to the well-known bacterial hosts E. coli and Corynebacterium glutamicum. This study represents the first demonstration of microbial production of cadaverine from methanol.     

Fermentative production of chiral acetoin by wild-type Bacillus strains

Abstract

In recent years, there have been many studies on producing acetoin by microbial fermentation, while only a few studies have focused on chiral acetoin biosynthesis. The weight assignment method was first applied to balance the chiral purity (expressed as the enantiomeric excess value) and the titer of acetoin. Bacillus sp. H-18W, a thermophile, was selected from seven Bacillus strains for chiral acetoin production. To lower the cost of the fermentation medium, soybean meal was used as a feedstock. Four kinds of frequently used commercial proteinases with different active sites were tested for the hydrolyzation of the soybean meal, and the combination of the acidic proteinase and the neutral proteinase showed the best results. In a fermentation medium containing 100?g L^?1 glucose and 200?g L^?1 hydrolysate, Bacillus sp. H-18W produced 21.84?g L^?1 acetoin with an ee value of 96.25% at 60?h. This is the first report of using a thermophilic strain to produce chiral acetoin by microbial fermentation. Thermophilic fermentation can reduce the risk of bacterial contamination and can save cooling water. Using soybean meal hydrolysate and glucose as feedstocks, this work provides an economical and alternative method for the production of chiral pure acetoin.     

Continuous production of the lipopeptide biosurfactant of Bacillus licheniformis JF-2

Bacillus licheniformis JF-2 synthesizes a surfactin-like lipopeptide that is the most effective biosurfactant known. In shake-flask cultures the biosurfactant is produced by actively growing cells (mid-linear phase), but subsequently it becomes rapidly internalized by the cells as soon as the culture enters the stationary phase. This deactivation phenomenon is a major hurdle in the efficient production of the biosurfactant. We have shown that the synthesis of the JF-2 lipopeptide is strongly dependent on O₂ concentration with substantial production observed only in cultures grown under O₂-limiting conditions. In continuous cultures the biosurfactant was produced only within a narrow window of low dilution rates. At a dilution rate of 0.12 h^–1 and low dissolved O₂, the biosurfactant concentration was maintained at 33 mg/l, which is virtually the same as the maximum concentration obtained in optimized batch fermentations.     

Oil biodegradation by Bacillus strains isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil

Sixteen spore forming Gram-positive bacteria were isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil. These strains were identified as belonging to the genus Bacillus using classical biochemical techniques and API 50CH kits, and their identity was confirmed by sequencing of part of the 16S rRNA gene. All strains were tested for oil degradation ability in microplates using Arabian Light and Marlin oils and only seven strains showed positive results in both kinds of oils. They were also able to grow in the presence of carbazole, n-hexadecane and polyalphaolefin (PAO), but not in toluene, as the only carbon sources. The production of key enzymes involved with aromatic hydrocarbons biodegradation process by Bacillus strains (catechol 1,2-dioxygenase and catechol 2,3-dioxygenase) was verified spectrophotometrically by detection of cis,cis-muconic acid and 2-hydroxymuconic semialdehyde, and results indicated that the ortho ring cleavage pathway is preferential. Furthermore, polymerase chain reaction (PCR) products were obtained when the DNA of seven Bacillus strains were screened for the presence of catabolic genes encoding alkane monooxygenase, catechol 1,2-dioxygenase, and/or catechol 2,3-dioxygenase. This is the first study on Bacillus strains isolated from an oil reservoir in Brazil.     

Clarified cashew apple juice as alternative raw material for biosurfactant production by Bacillus subtilis in a batch bioreactor

Clarified cashew apple juice was evaluated as carbon source for surfactin production by Bacillus subtilis LAMI005 isolated from the tank of chlorination at the Wastewater Treatment Plant on Campus do Pici (WWTP-PICI) in the Federal University of Ceará, Brazil. The highest surfactin concentration using clarified cashew apple juice (CCAJ) supplemented with mineral medium (MM-CCAJ) was 123 mg/L, achieved after 48 h of fermentation. Almost 2-fold less than the amount produced using mineral medium supplemented with 10 g/L of glucose and 8.7 g/L of fructose (MM-GF). However, critical micelle concentration of the biosurfactants produced using MM-CCAJ was 2.5-fold lower than the one produced using MM-GF, which indicates it is a more efficient biosurfactant. Surface tension decreased from 38.50 ± 0.0 to 29.00 ± 0.0 dyne/cm when B. subtilis was grown on MM-CCAJ media (24.68% of reduction on surface tension) and remained constant up to 72 h. Emulsification index was 51.15 and 66.70% using soybean oil and kerosene, respectively. Surfactin produced in MM-CCAJ showed an emulsifying activity of, respectively, 1.75 and 2.3 U when n-hexadecane or soybean oil was tested. However, when mineral medium supplemented with 10 g/L of glucose (MM-G) was used an emulsifying activity of 2.0 and 1.75 U, with n-hexadecane and soybean oil, respectively, was obtained. These results indicate that it is feasible to produce surfactin from CCAJ, a renewable and low-cost carbon source.     

Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51

The nutritional medium requirement for biosurfactant production by Bacillus licheniformis K51 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were H3PO4, CaCl2, H3BO3, and Na-EDTA. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were (g/l): glucose, 1.1; NaNO3, 4.4; MgSO4 x 7H2O, 0.8; KCl, 0.4; CaCl2, 0.27; H3PO4, 1.0 ml/l; and trace elements (mg/l): H3BO3, 0.25; CuSO4, 0.6; MnSO4, 2.2; Na2MoO4, 0.5; ZnSO4, 6.0; FeSO4, 8.0; CoCl2, 1.0; and Na-EDTA, 30.0. Using this statistical optimization method, the relative biosurfactant yield as critical micelle dilution (CMD) was increased from 10x to 105x, which is ten times higher than the non-optimized rich medium.     

Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species

 A glycolipid-producing bacterium, Pseudomonas aeruginosa GL1, was isolated from the soil contaminated with polycyclic aromatic hydrocarbons (PAH) from a manufactured gas plant. The glycolipid produced was characterized in detail by chromatographic procedures as a mixture of four rhamnolipids, consisting of different associations of rhamnose and hydroxy fatty acids: the main component was monorhamnosyl di-3-hydroxydecanoic acid. The rhamnolipid composition presented marked analogies with a defined part of P. aeruginosa outer membrane lipopolysaccharides (lipopolysaccharide band A). Rhamnolipid production was stimulated under conditions of nitrogen limitation. Glycerol yielded higher productions than did hydrophobic carbon sources. Cell hydrophobicity decreased during growth on glycerol and on n-hexadecane whereas glycolipid production increased. P. aeruginosa GL1 was found to be unable to grow on a variety of 2, 3 and 4 cycle PAH. However, it was shown to persist after at least 12 subcultures in a bacterial population growing on a mixture of pure PAH, suggesting a physiological role for rhamnolipid as a means to enhance PAH availability in a mutualistic PAH-degrading bacterial community. Received: 4 July 1995/Received revision: 7 September 1995/Accepted: 13 September 1995     

Substrate dependent production of extracellular biosurfactant by a marine bacterium

The potential of a marine microorganism to utilize different carbon substrates for the production of an extracellular biosurfactant was evaluated. Among the several carbon substrates tested for this purpose, production of the crude biosurfactant was found to be highest with glycerol (2.9+/-0.11 g L(-1)) followed by starch (2.5+/-0.11 g L(-1)), glucose (1.16+/-0.11 g L(-1)) and sucrose (0.94+/-0.07 g L(-1)). The crude biosurfactant obtained from glycerol, starch and sucrose media had significantly higher antimicrobial action than those obtained from glucose containing medium. RP-HPLC resolved the crude biosurfactants into several fractions one of which had significant antimicrobial action. The antimicrobial fraction was found in higher concentrations in biosurfactant obtained using glycerol, starch and sucrose as compared to the biosurfactants from glucose medium, thereby explaining higher antimicrobial activity. The carbon substrate was thus found to affect biosurfactant production both in a qualitative and quantitative manner.     

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides

Abstract Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis -based insecticides (Bactimos^TM, DiPel^TM, Florbac^TM FC, Foray^TM 48B, Novodor^TM FC, Turex^TM, VecTobac^TM, XenTari^TM). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPel^TM the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVP^TM which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis .     

Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis

Strains of Bacillus cereus can produce a heat-stable toxin (cereulide). In this study, 101 Bacillus strains representing 7 Bacillus species were tested for production of heat-stable toxins. Strains of B. megaterium, B. firmus and B. simplex were found to produce novel heat-stable toxins, which showed varying levels of toxicity. B. cereus strains (18 out of 54) were positive for toxin production. Thirteen were of serovar H1, and it was of interest that some were of clinical origin. Two were of serovars 17B and 20, which are not usually implicated in the emetic syndrome. Partial purification of the novel B. megaterium, B. simplex and B. firmus toxins showed they had similar physical characteristics to the B. cereus emetic toxin, cereulide.     

The isolation of a thermophilic biosurfactant producing Bacillus SP

Summary A thermophilic Bacillus strain has been isolated on a hydrocarbon containing medium and grew at up to 50°C. This strain produced biosurfactant and its 20h old culture broth had low surface and interfacial tension (27–29 and 1.5 mN/m, respectively). It emulsified Kerosene and other hydrocarbons efficiently (E–24 = 95 %) and was able to recover more than 95 % of the residual oil from sandpack columns. Potential uses in oil industries are discussed.     

Enhanced biosurfactant production by a mutant Bacillus subtilis strain

Summary Ultraviolet mutation of Bacillus subtilis ATCC 21332 yielded a stable mutant that produced over three times more of the biosurfactant, surfactin, than the parent strain. By protoplast fusing the mutant (Suf-1) with the marker strain, B. subtilis BGSC strain IA28, the mutation was located between argC4 and hisA1 on the genetic map.NRCC 30549     

Naphthalene degradation and biosurfactant activity by Bacillus cereus 28BN

Biosurfactant activity and naphthalene degradation by a new strain identified as Bacillus cereus 28BN were studied. The strain grew well and produced effective biosurfactants in the presence of n-alkanes, naphthalene, crude oil and vegetable oils. The biosurfactants were detected by the surface tension lowering of the medium, thin layer chromatography and infrared spectra analysis. With (2%) naphthalene as the sole carbon source, high levels of rhamnolipids at a concentration of 2.3 g 1(-1) were determined in the stationary growth. After 20 d of incubation 72 +/- 4% of the initial naphthalene was degraded. This is the first report for a Bacillus cereus rhamnolipid producing strain that utilized naphthalene under aerobic conditions. The strain looks promising for application in environmental technologies.     

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H₂O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4—used as an indicator strain, B. licheniformis T6-5, and B. firmus H₂O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H₂O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H₂O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H₂O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.     

In situ determination of primary production by means of a new incubator ISIS

Summary 1. A new sampler-incubator, ISIS, is described which, by feeding a solution of carbon-14-carbonate into the sample at the moment of sampling, permits a true in situ determination of primary production in a water body.2. Several experiments, which exemplify the ISIS technique, are described in detail.3. The advantages and disadvantages of the new and current standard techniques for measuring primary production are discussed.

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In-situ-Bestimmung der Primärproduktion mittels eines neuen Inkubators (ISIS)

Kurzfassung Die Notwendigkeit, Störungen des physikalischen, chemischen und biologischen Milieus während der Messung biologischer Vorgänge möglichst gering zu halten, hat zu der Konstruktion eines neuen, ISIS genannten und einfach zu handhabenden Inkubators zur Bestimmung der Primärproduktion in situ geführt. Eine ISIS-Einheit besteht aus einem Paar zylindrischer Kammern von 1 l Volumen, die an einem zentralen Schaft befestigt werden, der die Führungen für Auslöser und Befestigung am hydrographischen Draht enthält. Die eine Kammer ist transparent, die andere ist lichtundurchlässig. Die Einheit wird mit geöffneten Kammern in das Wasser herabgelassen und nach einer Anpassungszeit durch Fallgewichte ausgelöst. Hierbei werden beide Kammern gleichzeitig durch Deckel von oben und unten verschlossen. Kurz bevor die Deckel die O-Ringdichtung der Gefäße erreichen, wird eine am oberen Deckel befestigte Ampulle mit dem¹⁴C markierten Carbonat durch einen am unteren Deckel befestigten Stift zerbrochen; dabei wird die Lösung mit dem Wasser in den Behältern schnell vermischt. Der Draht mit den in verschiedenen Wassertiefen hängenden Inkubatoren wird dann an einer Boje oder unter einer Plattform befestigt; das Schiff ist während der Inkubationszeit für andere Zwecke frei. Nach der gewünschten Inkubationszeit wird das Gerät möglichst schnell an Bord geholt. Kleinere Proben werden in vorbereitete Flaschen mit Photosynthese-Inhibitoren abgefüllt und zur Filtration vorbereitet. Die Kohlenstofffixierung wird dann in der gewohnten Weise durchgeführt. Diese neue In-situ-Methode hat unter verschiedenen Versuchsbedingungen sehr präzise und reproduzierbare Resultate erbracht. Versuche, welche die Anwendbarkeit des Verfahrens im offenen Meer sowie in tropischen Aestuarien demonstrieren, werden beschrieben und diskutiert.

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Contribution No. 399, Hawaii Institute of Marine Biology.     

Analysis of alkane-dependent methanogenic community derived from production water of a high-temperature petroleum reservoir

Microbial assemblage in an n-alkanes-dependent thermophilic methanogenic enrichment cultures derived from production waters of a high-temperature petroleum reservoir was investigated in this study. Substantially higher amounts of methane were generated from the enrichment cultures incubated at 55 °C for 528 days with a mixture of long-chain n-alkanes (C₁₅–C₂₀). Stoichiometric estimation showed that alkanes-dependent methanogenesis accounted for about 19.8% of the total amount of methane expected. Hydrogen was occasionally detected together with methane in the gas phase of the cultures. Chemical analysis of the liquid cultures resulted only in low concentrations of acetate and formate. Phylogenetic analysis of the enrichment revealed the presence of several bacterial taxa related to Firmicutes, Thermodesulfobiaceae, Thermotogaceae, Nitrospiraceae, Dictyoglomaceae, Candidate division OP8 and others without close cultured representatives, and Archaea predominantly related to uncultured members in the order Archaeoglobales and CO₂-reducing methanogens. Screening of genomic DNA retrieved from the alkanes-amended enrichment cultures also suggested the presence of new alkylsuccinate synthase alpha-subunit (assA) homologues. These findings suggest the presence of poorly characterized (putative) anaerobic n-alkanes degraders in the thermophilic methanogenic enrichment cultures. Our results indicate that methanogenesis of alkanes under thermophilic condition is likely to proceed via syntrophic acetate and/or formate oxidation linked with hydrogenotrophic methanogenesis.     

Evaluation of in situ valine production by Bacillus subtilis in young pigs

Mutants of Bacillus subtilis can be developed to overproduce Val in vitro. It was hypothesized that addition of Bacillus subtilis mutants to pig diets can be a strategy to supply the animal with Val. The objective was to investigate the effect of Bacillus subtilis mutants on growth performance and blood amino acid (AA) concentrations when fed to piglets. Experiment 1 included 18 pigs (15.0±1.1 kg) fed one of three diets containing either 0.63 or 0.69 standardized ileal digestible (SID) Val: Lys, or 0.63 SID Val: Lys supplemented with a Bacillus subtilis mutant (mutant 1). Blood samples were obtained 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding and analyzed for AAs. In Experiment 2, 80 piglets (9.1±1.1 kg) were fed one of four diets containing 0.63 or 0.67 SID Val: Lys, or 0.63 SID Val: Lys supplemented with another Bacillus subtilis mutant (mutant 2) or its parent wild type. Average daily feed intake, daily weight gain and feed conversion ratio were measured on days 7, 14 and 21. On day 17, blood samples were taken and analyzed for AAs. On days 24 to 26, six pigs from each dietary treatment were fitted with a permanent jugular vein catheter, and blood samples were taken for AA analysis 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding. In experiment 1, Bacillus subtilis mutant 1 tended (P<0.10) to increase the plasma levels of Val at 2 and 3 h post-feeding, but this was not confirmed in Experiment 2. In Experiment 2, Bacillus subtilis mutant 2 and the wild type did not result in a growth performance different from the negative and positive controls. In conclusion, results obtained with the mutant strains of Bacillus subtilis were not better than results obtained with the wild-type strain, and for both strains, the results were not different than the negative control.