Process for producing rhamnolipids from corn chaff

Abstract

Process for the production of rhamnolipids by fermentation comprising the use of a rhamnolipid producing microorganism in a fermentation medium containing corn chaff and at least one compound selected from fatty acids, mono-, di- or triglycerides of fatty acids, alkyl esters of fatty acids, glycerol and any mixture thereof, responding to the comprehensive need of finding effective uses for some agricultural wastes generated by the transformation of cereals and enabling the production of surfactants obtained from renewable, biological raw materials and completely vegetal sources.

Description

[0001] PROCESS FOR PRODUCING RHAMNOLIPIDS FROM CORN CHAFF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a process for producing biosurfactants, and, in particular, rhamnolipids, by fermentation. The process is characterized by the use, as one of the carbon sources used by microorganisms which can produce biosurfactants, of corn chaff. Corn chaff is obtained by the mechanical shelling process to which corncobs are subjected after harvesting. This process responds to the comprehensive need of finding effective uses for some agricultural wastes generated by the transformation of cereals, currently not served by an adequate management chain. The process enables the production of completely biobased surfactants, i.e. of surfactants obtained from renewable, biological raw materials and completely vegetal sources.

[0004] BACKGROUND OF THE ART

[0005] The production chains of the agricultural sector generate by-products and waste. The quantities of the latter are often marginally considered by the official statistics of the sector, causing underestimations and uneven data, which do not provide a clear and exhaustive picture of the situation. The latest available data on the production of vegetable waste of agricultural origin refers to 1997, the year in which only the Italian production exceeded 20 million t / year of dry matter, of which about 13 million t / year from cereal production. This corresponds to 2-4% of the product obtained, which is mainly sent to anaerobic digestion plants for producing biogas.

[0006] It is known that agricultural waste can be used as substrate to produce molecules of microbiological origin, allowing the growth of microorganisms and the related biosynthesis of molecules with high added value, such as biosurfactants, which have characteristics like those of the synthetic surfactants.

[0007] Indeed, biosurfactants are in all their chemical-physical characteristics, surfactants, i.e. amphipathic molecules, able to accumulate at the interface between two immiscible phases because of their both hydrophilic and hydrophobic nature. Biosurfactants reduce the surface and interfacial tension (IFT) in liquids with different phases of matter, such as gas, liquid and solid, thus increasing the miscibility of substances that are not compatible with each other, typically giving rise to stable foams and oil and water emulsions. This peculiarity makes biosurfactants excellent and completely biobased alternatives to conventional synthetic surfactants in any domestic and industrial field, for example in detergents, textiles, paints, polymers, pharmaceuticals, agrochemicals, paper, personal care products, and in the oil and gas industry and remediation field.

[0008] However, the market of biosurfactants in general is still small compared to that of synthetic surfactants, mainly due to higher production costs. Three main factors make the commercialization of biosurfactants difficult: i) high costs of raw materials employed for their production; ii) the high recovery and purification costs; and (iii) the low yields in the production processes. Several techniques and approaches have been adopted worldwide to reduce the costs of biosurfactant production and make it more efficient. The use of cheaper substrates, optimized culture conditions in processes carried out in bioreactors, cost effective recovery processes and strain improvements, all of them have been investigated to improve biosurfactant productivity. The use of agricultural waste in biosurfactant production is considered one of the promising strategies to make these products more competitive and favorably combines with the need of increasing the value of the agricultural production. Biosurfactants are produced by bacteria, yeasts and filamentous fungi. At present, members of the genera Pseudomonas, Bacillus, Rhodococcus and Candida are the most widely implicated in the industrial production of these biomolecules. Biosurfactants are generally classified into glycolipids, fatty acid phospholipids, lipopeptides and lipoproteins, polymeric surfactants and particulate surfactants. Some anionic biosurfactants, such as rhamnolipids (RLs), represent a valid alternative to ethoxylated surfactants, responding to the increasing demand for natural, preferably vegetable-based, ingredients that are sustainably sourced, and which have no impact on the environment once released post-use.

[0009] Rhamnolipids are long known glycolipids and were described as early as in 1946. They have two moieties: the rhamnose moiety (also known as glycan part) and the lipid moiety (also known as aglycone part) linked via glycosidic linkage. The glycan moiety is hydrophilic and made of one or two rhamnose units; the lipid moiety is hydrophobic and comprises one or more saturated / unsaturated [3-hydroxy fatty acids chains of C8-C24 length, linked together by an ester bond. By using different sugars, polyols, hydrocarbons and oils, different organisms can produce approximately sixty congeners (or homologs) of rhamnolipids. Purified rhamnolipids can lower the surface tension of water to around 50 mN / m with a CMC of around 20 mg / L, as reported in Biomolecules 2019, 9, 885 by Shreve, G.S. and Makula, R. Rhamnolipids are widely studied for their surfactant properties, but also for their antibacterial, antifungal and antiviral activities and have been widely used in the fields of bioremediation and biodegradation. Rhamnolipids are also used in various biotechnological and industrial applications, such as the synthesis and stabilization of nanoparticles, the preparation of microemulsions; they are also used as anticaking agents and as a source of rhamnose.

[0010] Production of rhamnolipids is usually performed with natural isolates of bacteria of the genus Pseudomonas a nd Burkholderia (US 4,933,281, US 2011 / 0306569, US 7,202,063), but cases of use of recombinant strains have also been reported (US 9,854,799; US 10,174,353; US 2014 / 0235561; US 2017 / 0096695; US 2013 / 0130319). The Gram-negative bacterium Pseudomonas aeruginosa (PA) is the best studied rhamnolipid producer. It produces mainly 3-[3-(2-O-a-L-Rhamnopyranosyl-a-L- rhamnopyranosyloxy)decanoyloxy]decanoic acid (Rha2-C10-C10), 3-[(6-Deoxy-a- L-mannopyranosyl)oxy]decanoic acid (Rha-C10), 3-[3-(a-L- Rhamnopyranosyloxy)decanoyloxy]decanoic acid (Rha-C10-C10) and 3-[(2-O-a-L- Rhamnopyranosyl-a-L-rhamnopyranosyl)oxy] decanoic acid (Rha2-C10) [1,6], Genetically modified strain of the genus Pseudomonas can produce up to 100 g / L from batch or fed batch cultures supplemented with 160 g / L of soybean oil. However, recombinant strains are often undesired by the final consumers.

[0011] Producer microbial strains other than Pseudomonas have been reported but those species are of significance in very specific applications.

[0012] Besides the microorganism properties, the production strategies for rhamnolipids rely on the use of different carbon sources (US 11,142,782, WO2016179249), usually a mixture of carbohydrates and lipids with salts that provide nitrogen, phosphorous, magnesium and other elements. Furthermore, several studies were performed in the production of rhamnolipids from food and / or agricultural waste.

[0013] Among the patent literature, US 2021 / 0079436 relates to a method for preparing rhamnolipids using anaerobic digestate prepared by anaerobic digestion of organic waste, preferably from food waste.

[0014] CN 104498566 describes the preparation of rhamnolipids by the semi-solid fermentation in the presence of agricultural waste comprising rapeseed meal, cottonseed meal, bran, straw, peanut shell, and rice husk.

[0015] CN 106801075 provides a production method of rhamnolipid based on the synchronous treatment of manioc waste with a cellulase and Pseudomonas aeruginosa (synchronous saccharification and fermentation).

[0016] US 2022 / 0364128 provides a production process that makes use of lignocellulosic substrates, such as cornstalks, in a method for preparing biosurfactants.

[0017] WO 2014 / 039940 describes a combination of bacteria that can generate rhamnolipids when grown on hemicellulose, cellulose, or lignin that have been produced because of agricultural activity; examples include, but are not limited to, lawn trimmings, food waste, corn stover, and forestry residues.

[0018] Substrates like fermented distillery & whey waste, soybean oil refinery waste, frying oil waste, corn oil processing by-products, olive oil mill effluents, have been also mentioned, mainly in academic papers.

[0019] Lignocellulosic waste from the cereal supply chain is an excellent growth substrate for various kinds of microorganisms; they are rich in complex sugars, which can be used by numerous microorganisms as a source of nourishment. As reported in the literature, several microorganisms possess an enzymatic set suitable for the degradation of lignocellulosic components.

[0020] However, to produce biosurfactants, one of the drawbacks of using lignocellulosic material, such as corn stover, vegetable stalks, forestry residues, cereal waste, is the fact that normally the lignocellulosic material needs to be pre-treated chemically or enzymatically, to make its polymeric constituents bioavailable.

[0021] The object of the present invention is the development of an efficient process for producing rhamnolipids by fermentation, starting from a specific lignocellulosic waste biomass that can be used even without preliminary chemical or enzymatic degradation pretreatment.

[0022] It has now surprisingly been found that this object may be achieved by using corn chaff as such, or mechanically shredded corn chaff, as culturing nutrient in a fermentation process for obtaining rhamnolipids.

[0023] Of course, this is extremely advantageous in terms of time, sustainability, energy saving and costs.

[0024] DRAWINGS

[0025] Fig. 1: graph of the growth curves of Pseudomonas aeruginosa on corn chaff and other agricultural wastes.

[0026] Fig. 2: photographic reproduction of the result of the qualitative evaluation of the production of biosurfactants, by Pseudomonas aeruginosa grown on corn chaff, by oil displacement activity test (ODA).

[0027] Fig. 3: thin layer chromatography of Rhamnolipids obtained after the growth of Pseudomonas aeruginosa on corn chaff (A and B are replicas of the same sample, standard on the right). Fig. 4: graph of the Rhamnolipids (RLs) production in Examples AA (on the right) and BB (on the left).

[0028] Fig. 5: graph of the production of Rhamnolipids (RLs) in fermentation medium inoculated at different incubation times of the seed culture.

[0029] Fig. 6: graph of rhamnolipids (RLs) production decrease after the addition of a- amylase to the fermentative medium

[0030] SUMMARY

[0031] The process for producing rhamnolipids by fermentation comprises the steps of 1) inserting a rhamnolipid-producing microorganism into a culture medium suitable for the growth (seed inoculum); 2) inoculating a fermentation medium with the seed inoculum, the fermentation medium containing i) corn chaff, ii) at least one of fatty acids, mono-, di- or triglycerides of fatty acids, alkyl esters of fatty acids, glycerol or any mixtures thereof; 3) culturing the rhamnolipid producing microorganism in the fermentation medium, to obtain a fermentation medium containing one or more rhamnolipids and the rhamnolipid producing microorganism; 4) isolating the rhamnolipids.

[0032] DETAILED DESCRIPTION

[0033] The rhamnolipid-producing microorganism may be any natural or genetically modified microorganism that is able to produce rhamnolipids, even though natural microorganisms are preferred, and among these are Pseudomonas putida, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas clemancea,

[0034] Pseudomonas collierea, Pseudomonas luteola, Pseudomonas stutzeri,

[0035] Pseudomonas teessidea, Reni bacterium salmoninarum, Cellulomonas cellulans, Tetragenococcus koreensis, Burkholderia glumae, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia plantarii, Burkholderia thailandensis, Acinetobacter calcoaceticus, Enterobacter asburiae, Enterobacter hormaechei, Pantoea stewartii and Pantoea ananatis and the li ke; the microorganism is more preferably Pseudomonas aeruginosa or a Burkholderia, most preferably it is Pseudomonas aeruginosa.

[0036] Pseudomonas aeruginosa is a Gram negative, facultatively aerobic, opportunistic pathogenic bacterium capable of growing through aerobic respiration and through anaerobic respiration using nitrate as the final electron acceptor. Suitable strain to be used in this method is any of those intrinsically able to produce rhamnolipids and any recombinant strains carrying heterologous genes to synthesize rhamnolipids. There are no specific limitations in the preparation of the seed inoculum, having the seed the role of supplying enough bacteria, in a suitable physiological state to allow growth in the production medium (or fermentation medium) which is used in the process. Preferably, the inoculum is grown on Luria Bertani medium (LB medium) and dosed in the fermentation medium at 1-5% by weight of the fermentation medium used for production.

[0037] The fermentation medium typically contains, by weight percentage (wt%), 1-20 % of corn chaff, 1-20 % of at least one of fatty acids, mono-, di- or triglycerides of fatty acids, alkyl esters of fatty acids, glycerol or any mixtures thereof, and preferably 0.1-1 wt% of a nitrogen source, 0.05-1 wt% of a phosphorous source, and 0.1-1 wt% of other essential elements sources which can typically, without limitation, being supplied in the form of trace element solutions as described in (Sharma et al. 3 Biotech (2018) 8(1), 20; Sun et al. Biotechnol progress, 2021, 37(4), e3155), the balance being substantially water.

[0038] The characterising carbon source of the process is thus made of corn chaff mixed with at least one of fatty acids, mono-, di- or triglycerides of fatty acids, alkyl esters of fatty acids and / or glycerol.

[0039] Chaff is the dry, scaly protective casing of the seeds of cereal grains or similar plant material. Corn chaff is mainly made of the beeswings, the usually brownish or reddish outermost material that holds the grains and is released from the corn cob during shelling and of a part of chaff that remains on the corn cob after the grains have been removed. The corn chaff that is useful for the process (herein also called "loose corn chaff") is made of the beeswings together with few fragments of grain that are gathered during shelling in small pieces together with the beeswings.

[0040] Loose corn chaff is a light weight small sized material (typically max. 5-10 mm2, 0.1-1 mm thick with a density of 100-500 g / L, preferably 200-400 g / L).

[0041] Advantageously, the corn chaff that is used in the process of the invention may be untreated, i.e. it may be, and it is preferably used, as such in the process without chemical or enzymatic treatments, optionally only shredded to a smaller size. When shredded, the untreated corn chaff preferably passes through a 20-mesh sieve or more preferably through a 40-mesh sieve.

[0042] By "untreated corn chaff" we mean both loose corn chaff and shredded loose corn chaff, which have not undergone chemical or enzymatic hydrolysis of their polymeric constituents. The fact that untreated corn chaff can be directly used in the process to produce rhamnolipids represents a relevant advantage.

[0043] In fact, cereal waste mainly consists of a lignocellulosic material that is normally difficult to be degraded by microorganisms (especially due to the macromorphology of the material). For this reason, these materials are not known to be ideal fermentation substrates unless after suitable treatments. Numerous chemical, enzymatic and / or thermal treatments have been described for the preparation of the lignocellulosic material suitable for microbial digestion.

[0044] Surprisingly it has been found that untreated corn chaff can be used as such in the process of the present invention. It has also been found that using in the process untreated corn chaff shredded with a cereal mill further improves rhamnolipids yield.

[0045] It should be noted that shredding is by far to be preferred to chemical, enzymatic or thermal treatments due to its simplicity, eco-compatibility and to the possibility of being carried out directly at the source in the cereal treatment plants. In the process of this invention, more drastic treatments (such as maceration in the presence of strong acids at high temperatures) did not show any advantages compared to what was obtained with shredding.

[0046] By shredding it is to be understood any mechanical treatment which reduces the size of the corn chaff as it is produced by any mechanical shelling process.

[0047] Suitable fatty acids that may be included in the fermentation medium are C8-C22 alkyl or alkenyl, possibly hydroxyl substituted, preferably linear, carboxylic acids.

[0048] C12-C18 alkyl or alkenyl linear carboxylic acids are preferred among fatty acids. Mixtures of such fatty acids, conveniently found in natural vegetable oils, can be used.

[0049] Suitable mono-, di- or triglycerides of fatty acids that may be included in the fermentation medium are the mono-, di- and triglycerides of the above-mentioned fatty acids and mixtures thereof. Natural triglycerides of the above fatty acids, i.e. vegetable oils, such as soybean oil, olive oil, palm oil, coconut oil, castor oil, sunflower oil, rapeseed oil, and the like are the preferred component of the fermentation medium, especially in combination with glycerol.

[0050] Suitable components are also waste vegetable oils ("waste cooking oils") that beside triglycerides also contain minor portions of monoglycerides, diglycerides and variable quantities of fatty acids (5-20 wt%).

[0051] Suitable alkyl esters of fatty acids that may be included in the fermentation medium are the alkyl esters, such as the methyl esters, of the above-mentioned fatty acids or of mixtures thereof.

[0052] Alternatively, or advantageously in mixture with the above-mentioned fatty acids or fatty acids derivatives and especially in mixture with a vegetable oil or a waste cooking oil, the fermentation medium may contain glycerol.

[0053] Advantageously, the carbon source in the fermentation medium may essentially consist of (or consists of) corn chaff, vegetable oil or waste cooking oils and glycerol. Besides the above described carbon source, to the fermentation medium usually (but not mandatory) at least a nitrogen source, at least a phosphorous source and other essential elements sources are added.

[0054] The nitrogen source may be sodium nitrate, when present typically accounting for

[0055] 0.1 wt%-0.4 wt% of the fermentation medium. A phosphorous source, such as potassium dihydrogen phosphate, is typically included and may also account for 0.1wt%-0.4wt% of the fermentation medium. Other essential elements sources that may be added are iron sulphate (0.01 wt%- 0.15 wt%), potassium chloride (0.09 wt%-0.15 wt%), magnesium chloride (0.06 wt%- 0.08 wt%), calcium chloride (0.01 wt%-0.03 wt%), manganese sulphate (0.015 wt%- 0.018wt%), zinc sulphate (O.O2wt%-O.O3wt%), sodium molybdate 0.001wt%- 0.002wt% (percentage are referred to the fermentation medium total weight). The fermentation medium is typically a pourable viscous aqueous fluid in which the corn chaff is dispersed. The fermentation medium can eventually change its viscosity during sterilization and growth of the microorganism.

[0056] The amount of water in it ranges between 75 and 90 wt%.

[0057] According to a preferred embodiment, the fermentation medium contains from 10 to 200 g / L of corn chaff and from 20 to 60 g / L of at least one of triglycerides of fatty acids and / or glycerol.

[0058] During the culturing phase, the fermentation medium is stirred at about 150-250 rotations per minute (rpm). An antifoam agent, such as an alcohol like ethanol, isopropanol, 2-ethylhexanol, a silicone based defoamer or other defoamer may be added to the fermentation medium before or during culturing. Advantageously, if an antifoam agent is present, it is an alcohol or a silicon antifoam.

[0059] In the process, the rhamnolipid producing microorganism is typically fermented for from about 1 day to about 14 days.

[0060] The fermentation medium may also be a pasty, non-pourable medium, provided that enough aeration of it is dispensed with a suitable mixing apparatus or other means. The average dissolved oxygen level in the fermentation medium is preferably from 5 to 50% during the culturing phase.

[0061] During culturing, the fermentation medium has pH from 2.5 to 8.0, preferably from 3.0 to 7.0, more preferably from 3.5 to 6.0 and temperature from about 15°C to about 70°C, preferably from 20 to 50°C, more preferably from 25 to 40°C.

[0062] The culturing stage may be performed in batch or with continuous or stepwise addition (fed-batch) of nutrients (carbon source and other nutrients) of the fermentation medium. The batch culture is performed by adding in one step all the nutrients, sterilizing the medium and performing the fermentation as described above. The continuous fermentation is performed by adding some volume of the nutrients to the medium in a continuous manner and by removing some volume of the fermentation culture at the same time; stepwise addition (fed-batch) is performed by adding some of the nutrients to the fermentation culture according to specifical signals given by the fermentation parameters.

[0063] The recovery of the rhamnolipids may be made according to any methodology known in the art.

[0064] Experimental part

[0065] PRELIMINARY TESTS

[0066] For sake of standardisation, in all tests untreated corn chaff sieved through a 40 mesh sieve is used. The same is for the other agricultural wastes.

[0067] Evaluation of the growth of the microorganisms on corn chaff.

[0068] To preliminarily evaluate the usefulness of corn chaff as a growth substrate for microorganisms, the growth of Pseudomonas aeruginosa on media composed of corn chaff or other agricultural wastes in water was evaluated. The indicators of microbial growth, such as the vital count and the associated physiology (by determining in example the pH and consumption of sugars), were monitored.

[0069] For these growth tests, the corn chaff and some other agricultural waste, supplied as the only nutrient source (concentration 100 g / L) were used, suspended in 100 mL of ultrapure water in 500 mL flasks with baffle, sterilized in autoclave at 123°C for 20 minutes:

[0070] * The control is a common medium for the production of rhamnolipids, i.e. 20g / L soybean meal.

[0071] The growth of Pseudomonas aeruginosa was performed and evaluated according to the following protocol.

[0072] The strain was revitalized in 9 cm Petri dishes containing the selected agarized- medium by seeding the culture stored at -80°C (working cell bank, WCB) using a sterile loop. The prepared plates were then incubated at 24-28°C for 24-48 hours. The cultivation was then continued in 500 mL baffled-flasks, containing 100 mL of vegetative medium (Luria Bertani Broth, LB), sterilized in an autoclave at 121°C for 20 minutes. The inoculation of the vegetative medium is carried out by suspending a loop of bacterial cells in Luria Bertani broth, taking a volume (1 to 5 ml) of the above suspension, and placing it in 500 ml baffled-flasks, containing 100 mL of vegetative medium. The volume of the bacterial cell suspension is set in a way to obtain an OD600 (measured in LB medium as the reference) in the range 0.1-0.3 in the 500 ml flask. Alternatively, the inoculation is carried out directly from the WCB into the vegetative medium according to the OD600 parameters set above.

[0073] From the vegetative grown culture, 10 ml are taken and inoculated in 100 mL of the fermentation medium (containing the corn chaff and / or other nutrients depending on the test) dispensed in 500 ml baffled flasks and sterilized at 123°C for 20 min. Incubation is performed as above.

[0074] In all cases, the cultures are placed in the 500 mL baffled-flasks at 28 °C and stirred at 200-250 rpm.

[0075] For the viable counts, media solidified with the addition of Agar were used, in order to be able to observe the characteristics of the single microbial colonies. Physiological solution (0.9% w / w), was used to prepare the dilutions of microorganisms and to carry out the viable counts. Physiological solution was prepared by dissolving 9 g / L of sodium chloride (NaCI) in deionized water (H2O). Once dissolved, the solution was sterilized in an autoclave at a temperature of 121°C for 20 minutes.

[0076] The viable Petri dish plate count allowed to evaluate the concentration of viable cells expressed as Colony Forming Units per unit volume (CFU / mL). This method is used, as an indication of vitality, in all the phases of the fermentation in which a passage from one medium to another is involved, or as an indication of the growth of a microorganism on the substrate under analysis. For the microbial count, serial dilutions are made starting from 1 mL of broth culture (either vegetative culture or fermentative culture according to the experimental requirements), with subsequent plating on agar medium; 100 pL of the diluted suspension described above, are deposited on the plate, and are then spread over until the liquid is dried. After 24-48 hours of incubation at 28°C or 37°C (depending on the strain under examination) it is possible to count the colonies (Colony Forming Units) and determine the titre expressed in CFU / mL of broth culture. To have a statistically reliable data, the number of colonies grown on each plate was between 30 and 300.

[0077] The results (titres) obtained for fermentation cultures (those cultures run with corn chaff and / or other nutrients relevant for rhamnolipid production) are reported in Fig 1. The values obtained are the average of three distinct measurements, with a standard deviation of less than 5%.

[0078] The growth tests carried out on Pseudomonas aeruginosa show the best growth on corn chaff, while oat and emmer chaff and pea peel reach lower, but still interesting, values.

[0079] From these growth tests, the relevance of the use of corn chaff as a growth medium for microorganisms emerged.

[0080] The rhamnolipid productivity of Pseudomonas aeruginosa was qualitatively evaluated by the oil displacement activity test (ODA) and analysis on thin layer chromatography, and subsequently confirmed and quantified by means of emulsification index ( E 124%) (Cooper and Goldenberg. 1987, Appl. Environ.

[0081] Microbiol. 53: 224-229). Qualitative evaluation through ODA test of the production of rhamnolipids by Pseudomonas aeruginosa grown on corn chaff.

[0082] The oil dispersion test in water, or oil displacement assay, represents a rapid and effective qualitative method for evaluating the surfactant activity of the molecule under investigation. It also offers the advantage of being able to allow testing of many samples in a short time. The assay is commonly performed in Petri dishes with a diameter of 5 cm, to which 30 pL of light crude oil, 3 mL of demineralised water and finally 3 pL of the sample to be analysed have been added. The diameter of the halo obtained on the oil deposited on the surface of water, after dropping the sample, represents a qualitative indication of the surfactant power of the substance under examination. This assay, developed by Morikawa in 2000, (BBA-Molecular and Cell Biology of Lipids. 1488:211), exploits the ability of biosurfactants to create circular areas in which the fluid is displaced once added to an apolar liquid such as oil. The size of these zones can be correlated to the activity of the biosurfactant.

[0083] The ODA test result is considered positive if oil displacement is observed for a sample after growth on the tested substrates.

[0084] The ODA test made possible to identify the production of surfactant substances in the culture broths of Pseudomonas aeruginosa that have grown only on corn chaff (Fig. 2 shows ODA test on aliquots taken from the supernatant coming from culture broths of P. aeruginosa; the test turns positive 72 hours of growth of the strain in the suitable medium). Oat and emmer chaff and pea peel did not show positive results in this test (no biosurfactants production).

[0085] Negative controls were performed, using as sample an aliquot of supernatant from abiotic media, prepared with the same method. The positive results obtained with corn chaff were confirmed by TLC (thin layer chromatography) (Fig. 3, where A and B are replicas of the same sample and on the right is the rhamnolipid standard).

[0086] To perform the TLC analysis of rhamnolipids, a mixture of chloroform (CHCI3), methanol (MeOH) and ultrapure water (H2O) in a ratio of 65:15:1 is used as the mobile phase.

[0087] The development of the TLC takes place using the orcinol reagent. The orcinol reagent is prepared by dissolving 0.2 g of orcinol in 89.6 mL of deionized water (H2O) and 10 mL of 96% sulfuric acid (H2SO4). The sulfuric acid is added slowly to limit the exothermic reaction.

[0088] The produced rhamnolipids were compared with a standard of rhamnolipids from Sigma-Aldrich.

[0089] Evaluation of the production of rhamnolipids by Pseudomonas aeruginosa by use of the Emulsification Index.

[0090] The production of surfactant substances was also evaluated by determining the emulsification index; the test shows, in a simple and immediate way, the surfaceactive capacity of the compounds present in the culture broths.

[0091] The emulsification index (El 24%) represents a parameter for determining the emulsifying power of a surfactant molecule. The protocol is reported in the literature (Cooper and Goldenberg 1987, Appl. Environ. Microbiol. 53: 224-229). In detail, the measurement was performed by mixing equivalent volumes, equal to 2 mL, of the solution (eventually after dilution) containing the biosurfactant under analysis and n-hexadecane or light crude oil. The mixture was stirred with the aid of a vortex mixer for exactly 2 minutes and then incubated at 25°C for 24 hours. The emulsification index (expressed as a percentage) is calculated as the ratio between the height of the emulsified phase and the total height of the liquid column.

[0092] Semi-quantitative evaluation of rhamnolipid production by Pseudomonas aeruginosa was performed by determining the emulsification of n-hexadecane and petroleum with water.

[0093] The determination with n-hexadecane, and with supernatant from broth culture at 144 hours, determined an emulsification index of 100%.

[0094] The supernatant of the abiotic culture alone instead determined a 50% emulsification (control).

[0095] The results obtained with the control, show a surfactant effect, probably due to some component of the corn chaff, which could partially hinder the effect of rhamnolipids, thus making the test with n-hexadecane not suitable for a quantitative determination; however, the test remains a proof of the production of surfactant substances by Pseudomonas aeruginosa.

[0096] The test was replicated using light crude oil instead of n-hexadecane.

[0097] The result of the test showed an emulsification index of 90%, with an emulsifying index of 75% in the abiotic broth.

[0098] Tests with corn chaff and / or other waste mixtures

[0099] Two mixtures composed of corn chaff and / or other agricultural wastes were also tested as possible nutrients; 100 mL of ultrapure water in 500 mL baffled-flasks were added to the dry nutrient and the suspension was sterilized at 123 °C for 20 minutes:

[0100]

[0101] The oil displacement activity (ODA) was tested with aliquots of broth culture from the above fermented mixtures. The only mixture that gave a positive result was that with oat and emmer chaff / corn chaff, while a negative result was obtained with oat and emmer chaff / pea peel, effectively confirming the data obtained with individual agricultural waste and the peculiarity of corn chaff in stimulating biosurfactant production.

[0102] EXAMPLES

[0103] Production trials by adding vegetable oil in the fermentation medium

[0104] Trials were carried out with the goal to increase the biosurfactant production, by adding oil to the fermentation medium.

[0105] 20 g / L of oil, both soybean oil and exhausted sunflower oil (a waste cooking oil of considerable interest from a circular economy perspective), were added individually to corn chaff dosed at the same concentration used in the previous tests.

[0106] To the dry corn chaff and 20 g / L of oil, ultrapure water was added up to a total volume of 100 mL in 500 mL baffled-flasks. The resulting medium was sterilized at

[0107] 123°C for 20 minutes:

[0108] Both cultivation conditions ( / '.e. with the two types of oily liquid phase) gave a positive result in the ODA test.

[0109] In this case too, chromatographic analyses were performed with the aid of thin layer chromatography (TLC). The samples to which soybean oil and waste oil were added were analysed. A very well-defined spot is visible at the rhamnolipids (both monorhamnolipids and di-rhamnolipids) retention time. Comparison was performed by use of a rhamnolipid standard (Sigma-Aldrich).

[0110] Additionally, the amount of rhamnolipids produced by Pseudomonas aeruginosa in Examples AA and BB was determined by titration of rhamnose using an HPLC method:

[0111] Instrument: Agilent Technologies 1260 Infinity

[0112] Column: Aminex HPX-87H (BioRad) 300x7.8 mm cation exchange

[0113] Mobile phase: 5 mM sulfuric acid

[0114] Flow: 0.6mL / min

[0115] Elution system: isocratic

[0116] Injection volume: 10 pl

[0117] Oven temperature: 30 °C

[0118] Detectors: Refractive Index (RI D) UV (X=210 nm)

[0119] Temperature of the detectors: 30 °C

[0120] Analysis time: 30 minutes

[0121] For the quantitative analysis, the concentration in g / L of rhamnolipids was determined using a calibration curve constructed with reference rhamnose standards.

[0122] Before injection into the instrument, the samples to be analysed were prepared according to the following protocol.

[0123] • sampling of 1 g of culture broth

[0124] • Acid hydrolysis of the sample with 160 pL of HCI at 37% (v / v) carried out at 95 °C for 4 hours in a thermomixer

[0125] • centrifugation at 16100 x g for 10 min

[0126] • recovery of 900 pL of supernatant (if the sample is too concentrated, it can be diluted with demineralized water)

[0127] • Addition of 100 pL of a 35% HCIO4 solution (v / v) (mix with vortex)

[0128] • Incubation for 10 min at -20 °C

[0129] • Addition of 55 pL of 7 M KOH solution (w / v) (mix with vortex)

[0130] • Centrifugation at 16100 x g for 2 minutes

[0131] • Filtration through PES membranes with porosity of 0.22 pm

[0132] • Dispensing in vials for HPLC

[0133] • HPLC analysis

[0134] The protocol foresees (whenever required) a preliminary treatment with chloroform for the complete removal of the residual oil from the fermentation sample. This procedure was performed by adding 900 pL of post-hydrolysis sample supernatant to 900 pL of chloroform (CHCI3), mixing on vortex and centrifugation for 5 minutes at 16100 x g. The separation of the two phases allows then the collection of the oil- free supernatant required for the analysis.

[0135] The results obtained were graphically represented in the histogram of Fig. 4.

[0136] The amount of RLs (g / L) is calculated by multiplying the amount of rhamnose (titrated on HPLC), for a correction factor that takes into account the sugar content in the mono- and di-RL molecule (Mohammad et al., 2011, Biosurfactants, Microbiology Monographs 20).

[0137] Production tests with the addition of glycerol and inorganic salts

[0138] To improve the production of surfactants in the tested strains, different compositions of the growth medium were tested.

[0139] All media were prepared using ultrapure water and sterilized at 123°C for 20 minutes. Trials were carried out with potentially industrial media, carbon sources were glycerol (40 g / L) and / or vegetable oil (20 g / L), while sodium nitrate (NaNCL) was used as the nitrogen source (2 g / L) and potassium dihydrogen phosphate (KH2PO4) (1 g / L) was the phosphate source.

[0140] The media components were dissolved in ultrapure water and dispensed into 500 mL baffled-flasks and sterilized at 123 °C for 20 minutes.

[0141] H, G and I are negative controls.

[0142] Media formulated with the addition of salts, maintained pH values between 5.5 and 6.5 ± 0.2, thanks to the buffering action of potassium dihydrogen phosphate (present in all conditions tested). Alkaline pH values were observed with medium H, probably due to the lysis of the microbial culture.

[0143] *control

[0144] **less than 0.4 g / L (1-2 g / L of RLs), trace visible by TLC;

[0145] *** no trace by TLC wco: waste cooking oil

[0146] In this case too, the oil displacement assay test (ODA test) was performed, with positive results for all conditions besides the negative controls.

[0147] Tests were performed also with the TLC (thin layer chromatography) method, to verify the actual presence of rhamnolipids; the trials had a positive outcome for all the mixtures tested (excluding the negative controls), showing two spots corresponding to mono-rhamnolipids and di-rhamnolipids.

[0148] Finally, a titration of the rhamnolipids present in the culture broth at the time of harvest (144 hours) was performed by HPLC. Under the conditions tested, the best results were obtained with trial C (below also identified as BCS388), and trial D. Production implementation: preliminary study of the seed inoculum (Example C).

[0149] Vegetative medium is the medium with an optimal nutrient concentration for maximizing growth. The purpose of the vegetative medium is to prepare microorganismsfor better growth and production of metabolites (rhamnolipids in this case) in the production medium.

[0150] The growth in vegetative medium is recognized as a fundamental step for optimizing the yield of the production of secondary metabolites; a growth step in vegetative medium was therefore verified to increase the production of rhamnolipids.

[0151] In the case of Pseudomonas aeruginosa, vegetative growth in Luria-Bertani broth was performed; the inoculum in vegetative medium was prepared starting from seeding of the microorganism from the WCB (working cell bank) to a suitable agar medium on Petri dishes. The Petri dish was grown overnight in an incubator at 28°C.

[0152] After growth on Petri dishes, a portion of the solid-grown culture is collected using a sterile loop and suspended in fresh Luria-Bertani broth medium in an amount sufficient to give a starting OD600 of 0.3 ± 0.01. The amount of inoculated biomass was verified by measuring the optical density at 600 nm using uninoculated Luria- Bertani broth medium as blank.

[0153] Different times were chosen for the transfer from vegetative medium to fermentation medium; in detail, the transfer took place in different stages of growth of the microbial culture. The different stages of growth were determined by measuring the optical density at 600 nm and by identifying the growth phases (lag, log (exponential and late exponential) and stationary) by graphing the OD600 measured values.

[0154] Microbial growth was confirmed by viable count at the time of transfer to evaluate the accuracy of the optical density measurement, which also includes non-viable cells.

[0155] Finally, titrations of the productive cultures inoculated with seeds from different growth phases were performed at different times, to evaluate the maximum concentration of rhamnolipids produced.

[0156] Fig. 5 reports the production of rhamnolipids in P. aeruginosa cultures on BCS388 medium inoculated from vegetative cultures grown for different incubation times (exponential, late exponential-stationary phase of growth).

[0157] The data show that the best condition for the transfer of the vegetative culture to production medium is the exponential phase of growth.

[0158] Pre-treatment of the medium containing corn chaff with a-amylase. Effects on the production of rhamnolipids

[0159] In order to make the culture medium more fluid and to make available in the early stages of microbial growth a greater quantity of glucose, the enzyme a-amylase was used; a-amylase is able to hydrolyse the a-1,4 glycosidic bonds inside starch molecules. This gives low molecular weight molecules such as glucose, maltose and maltotriose.

[0160] The enzyme was added to the BCS388 medium a few minutes before sterilization in the autoclave; in fact, the enzyme is activated by the high temperatures reached, making the procedure standardisable.

[0161] Different quantities of a-amylase, 50 pL and 100 pL (12.5 and 25 U / L of medium) were tested; a seed culture was prepared in vegetative medium (as described above) before transfer to the amylase-treated medium.

[0162] Fig. 6 shows the maximum production of rhamnolipids obtained under these conditions at 144 hours of fermentation in medium containing a-amylase (HPLC titration). The production of rhamnolipids seems to be reduced by the pretreatment with increasing amounts of the enzyme.

Claims

CLAIMS1. Process for producing rhamnolipids by fermentation that comprises the steps of 1) inserting a rhamnolipid-producing microorganism into a culture medium to obtain a seed culture 2) inoculating a fermentation medium with the seed culture, the fermentation medium containing i) corn chaff, ii) at least one of fatty acids, mono-, di- or triglycerides of fatty acids, alkyl esters of fatty acids, glycerol or any mixtures thereof; 3) culturing the rhamnolipid producing microorganism in the fermentation medium, to obtain a fermentation medium containing one or more rhamnolipids and at least one rhamnolipid producing microorganism; 4) isolating the one or more rhamnolipid.

2. Process of claim 1 wherein the rhamnolipid-producing microorganism is a Pseudomonas, or a Burkholderia.

3. Process of claim 2 wherein the rhamnolipid-producing microorganism is a Pseudomonas.

4. Process of claim 3 wherein the corn chaff is untreated corn chaff.

5. Process of claim 4 wherein the untreated corn chaff is shredded.

6. Process of claim 1 or 4 wherein the fermentation medium contains from 10 to 200 g / L of i) of corn chaff and ii) from 20 to 60 g / L of at least one of triglycerides of fatty acids and / or glycerol.

7. Process of claim 6 wherein the fermentation medium contains triglycerides of fatty acids and glycerol.

8. Process of claim 7 wherein the triglycerides of fatty acids are vegetable oil or vegetable waste oils.

9. Process of claim 8 wherein the fermentation medium further contains at least one source of nitrogen and at least one source of phosphorous.

10. Process of claim 1 wherein the rhamnolipid producing microorganism is cultured at pH comprised between 2.5 and 8.0 for from about 1 day to about 14 days at a temperature of from 15°C to 70°C.

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