Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library.

Xylanases are key biocatalysts in the degradation of the ß-1,4-glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-ß-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. KEY POINTS: • A GH10 endo-1,4-ß-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB's in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis.

Epilactose as a Promising Butyrate-Promoter Prebiotic via Microbiota Modulation.

Epilactose is a disaccharide composed of galactose and mannose, and it is currently considered an "under development" prebiotic. In this study, we described the prebiotic potential of epilactose by in vitro fermentation using human fecal inocula from individuals following a Mediterranean diet (DM) or a Vegan diet (DV). The prebiotic effect of epilactose was also compared with lactulose and raffinose, and interesting correlations were established between metabolites and microbiota modulation. The production of several metabolites (lactate, short-chain fatty acids, and gases) confirmed the prebiotic properties of epilactose. For both donors, the microbiota analysis showed that epilactose significantly stimulated the butyrate-producing bacteria, suggesting that its prebiotic effect could be independent of the donor diet. Butyrate is one of the current golden metabolites due to its benefits for the gut and systemic health. In the presence of epilactose, the production of butyrate was 70- and 63-fold higher for the DM donor, when compared to lactulose and raffinose, respectively. For the DV donor, an increase of 29- and 89-fold in the butyrate production was obtained when compared to lactulose and raffinose, respectively. In conclusion, this study suggests that epilactose holds potential functional properties for human health, especially towards the modulation of butyrate-producing strains.

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples.

The renewable, abundant , and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples' microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited ß-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples. KEY POINTS: • Sequence- and function-based metagenomics were used to find CAZymes in compost samples. • Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes. • Compost-derived fosmid libraries are enriched in clones with ß-glucosidase activity.

Tailoring fructooligosaccharides composition with engineered Zymomonas mobilis ZM4.

Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated ß-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1-sacB that was able to produce 73.4 ± 1.6 g L-1 of FOS, with a productivity of 1.53 ± 0.03 g L-1 h-1 and a yield of 0.31 ± 0.03 gFOS gsucrose-1. This is the first report on the FOS production using a mutant Z. mobilis ZM4 strain in a one-step process. KEY POINTS: • Zymomonas mobilis was engineered to produce FOS in a one-step fermentation process. • Mutant strains produced FOS mixtures with high concentration of 6-kestose. • A new route to produce tailor-made FOS mixtures was presented.

Hydrolysates containing xylooligosaccharides produced by different strategies: Structural characterization, antioxidant and prebiotic activities.

This study explores the structural characterization, antioxidant and prebiotic activities of hydrolysates containing xylooligosaccharides (XOS) produced by different strategies: direct fermentation of beechwood xylan (FermBX) and enzymatic treatment of beechwood (EnzBX) and rice husk (EnzRH) xylans. EnzBX and EnzRH showed XOS with a backbone of (1 â†’ 4)-linked-xylopyranosyl residues and branches of arabinose, galactose, and uronic acids. FermBX presented the highest content of total phenolic compounds (14 mg GAE/g) and flavonoids (0.6 mg QE/g), which may contribute to its antioxidant capacity -39.1 µmol TE/g (DPPH), 45.7 µmol TE/g (ABTS), and 79.9 µmol Fe II/g (FRAP). The fermentation of hydrolysates decreased the abundance of microorganisms associated with intestinal diseases from Eubacteriales, Desulfovibrionales and Methanobacteriales orders, while stimulating the growth of organisms belonging to Bacteroides, Megamonas and Limosilactobacillus genera. The production of short-chain fatty acids, ammonia, and CO2 suggested the prebiotic potential. In conclusion, hydrolysates without previous purification and obtained from non-chemical approaches demonstrated promising biological activities for further food applications.

Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (> 99% of humic acids effectively removed) and sufficient quantity (10.5-13.8 µg g-1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory. KEY POINTS/HIGHLIGHTS: • Metagenomic DNA was successfully extracted from compost samples rich in humic acids • The improved protocol was established by optimizing the cell lysis method and buffer • Complete removal of humic acids was achieved through the use of activated charcoal • The suitability of the DNA was proven by the construction of a metagenomic library.

Novel and emerging prebiotics: Advances and opportunities.

Consumers are conscientiously changing their eating preferences toward healthier options, such as functional foods enriched with pre- and probiotics. Prebiotics are attractive bioactive compounds with multidimensional beneficial action on both human and animal health, namely on the gastrointestinal tract, cardiometabolism, bones or mental health. Conventionally, prebiotics are non-digestible carbohydrates which generally present favorable organoleptic properties, temperature and acidic stability, and are considered interesting food ingredients. However, according to the current definition of prebiotics, application categories other than food are accepted, as well as non-carbohydrate substrates and bioactivity at extra-intestinal sites. Regulatory issues are considered a major concern for prebiotics since a clear understanding and application of these compounds among the consumers, regulators, scientists, suppliers or manufacturers, health-care providers and standards or recommendation-setting organizations are of utmost importance. Prebiotics can be divided in several categories according to their development and regulatory status. Inulin, galactooligosaccharides, fructooligosaccharides and lactulose are generally classified as well established prebiotics. Xylooligosaccharides, isomaltooligosaccharides, chitooligosaccharides and lactosucrose are classified as "emerging" prebiotics, while raffinose, neoagaro-oligosaccharides and epilactose are "under development." Other substances, such as human milk oligosaccharides, polyphenols, polyunsaturated fatty acids, proteins, protein hydrolysates and peptides are considered "new candidates." This chapter will encompass actual information about the non-established prebiotics, mainly their physicochemical properties, market, legislation, biological activity and possible applications. Generally, there is a lack of clear demonstrations about the effective health benefits associated with all the non-established prebiotics. Overcoming this limitation will undoubtedly increase the demand for these compounds and their market size will follow the consumer's trend.

Sustainable Lipase Production by Diutina rugosa NRRL Y-95 Through a Combined Use of Agro-Industrial Residues as Feedstock.

The potential use of alternative culture media towards the development of a sustainable bioprocess to produce lipases by Diutina rugosa is clearly demonstrated. First, a synthetic medium containing glucose, peptone, yeast extract, oleic acid, and ammonium sulfate was proposed, with lipase activity of 143 U/L. Then, alternative culture media formulated with agro-industrial residues, such as molasses, corn steep liquor (CSL), and olive mill waste (OMW), were investigated. An experimental design was conducted, and only CSL concentration was found to have a positive effect in lipase production. The highest lipase activity (561 U/L) was produced on a mixture of molasses (5 g/L), CSL (6 g/L), OMW (0.5% v/v), 0.5 g/L of ammonium sulfate, and 3 g/L of peptone at 24 h of cultivation. Lipase production was also carried out in a 1-L bioreactor leading to a slightly higher lipase activity at 24 h of cultivation. The semi-purified enzyme exhibits an optimum temperature and pH of 40 °C and 7.0, respectively. Finally, the media cost per unit of lipase produced (UPC) was influenced by the medium components, specially by the inducer used. The lowest UPC was obtained when the agro-industrial residues were combined and used at the improved concentrations.

Zymomonas mobilis as an emerging biotechnological chassis for the production of industrially relevant compounds.

Zymomonas mobilis is a well-recognized ethanologenic bacterium with outstanding characteristics which make it a promising platform for the biotechnological production of relevant building blocks and fine chemicals compounds. In the last years, research has been focused on the physiological, genetic, and metabolic engineering strategies aiming at expanding Z. mobilis ability to metabolize lignocellulosic substrates toward biofuel production. With the expansion of the Z. mobilis molecular and computational modeling toolbox, the potential of this bacterium as a cell factory has been thoroughly explored. The number of genomic, transcriptomic, proteomic, and fluxomic data that is becoming available for this bacterium has increased. For this reason, in the forthcoming years, systems biology is expected to continue driving the improvement of Z. mobilis for current and emergent biotechnological applications. While the existing molecular toolbox allowed the creation of stable Z. mobilis strains with improved traits for pinpointed biotechnological applications, the development of new and more flexible tools is crucial to boost the engineering capabilities of this bacterium. Novel genetic toolkits based on the CRISPR-Cas9 system and recombineering have been recently used for the metabolic engineering of Z. mobilis. However, they are mostly at the proof-of-concept stage and need to be further improved.

In vitro assessment of prebiotic properties of xylooligosaccharides produced by Bacillus subtilis 3610.

Xylooligosaccharides (XOS) are emergent prebiotics exhibiting high potential as food ingredients. In this work, in vitro studies were performed using human fecal inocula from two healthy donors (D 1 and D2) to evaluate the prebiotic effect of commercial lactulose and XOS produced in a single-step by recombinant Bacillus subtilis 3610. The fermentation of lactulose led to the highest production of lactate (D1: 33.7 ±â€¯0.5 mM; D2:19.7 ±â€¯0.3 mM) and acetate (D1: 77.5 ±â€¯0.6 mM; D2: 81.0 ±â€¯0.7 mM), while XOS led to the highest production of butyrate (D1: 9.0 ±â€¯0.6 mM; D2: 10.5 ±â€¯0.8 mM) and CO2 (D1: 8.92 ±â€¯0.02 mM; D2: 11.4 ±â€¯0.3 mM). Microbiota analysis showed a significant decrease in the relative abundance of Proteobacteria for both substrates and an increase in Bifidobacterium and Lactobacillus for lactulose, and Bacteroides for XOS.

From lignocellulosic residues to market: Production and commercial potential of xylooligosaccharides.

The updated definition of prebiotic expands the range of potential applications in which emerging xylooligosaccharides (XOS) can be used. It has been demonstrated that XOS exhibit prebiotic effects at lower amounts compared to others, making them competitively priced prebiotics. As a result, the industry is focused on developing alternative approaches to improve processes efficiency that can meet the increasing demand while reducing costs. Recent advances have been made towards greener and more efficient processes, by applying process integration strategies to produce XOS from costless lignocellulosic residues and using genetic engineering to create microorganisms that convert these residues to XOS. In addition, collecting more in vivo data on their performance will be key to achieve regulatory claims, greatly increasing XOS commercial value.

Downscale fermentation for xylooligosaccharides production by recombinant Bacillus subtilis 3610.

The global demand of prebiotics such as xylooligosaccharides (XOS) has been growing over the years, motivating the search for different production processes with increased efficiency. In this study, a cloned Bacillus subtilis 3610, containing the xylanase gene xyn2 of Trichoderma reesei coupled with an endogenous secretion tag, was selected for XOS production through direct fermentation of beechwood xylan. A mixture of XOS with a degree of polymerization ranging from 4 to 6 was obtained, presenting high stability after a static in vitro digestion (98.5 ± 0.2%). The maximum production yield expressed as total XOS per amount of xylan (306 ± 4 mg/g) was achieved after 8 h of fermentation operating under one-time impulse fed-batch. The optimal conditions found were pH 6.0 and 42.5 °C, using 2.5 g/L of initial concentration of xylan increased up to 5.0 g/L at 3 h. Xylopentaose was the major oligosaccharide produced, representing 47% of the total production yield.

One-step process for producing prebiotic arabino-xylooligosaccharides from brewer's spent grain employing Trichoderma species.

Xylooligosaccharides (XOS) are prebiotic nutraceuticals that can be sourced from lignocellulosic biomass, such as agro-residues. This study reports for the first time an optimization study of XOS production from agro-residues by direct fermentation using two Trichoderma species. A total of 13 residues were evaluated as potential substrates for single-step production. The best results were found for Trichoderma reesei using brewers' spent grain (BSG) as substrate. Under optimal conditions (3 days, pH 7.0, 30 °C and 20 g/L of BSG), a production yield of 38.3 ±â€¯1.8 mg/g (xylose equivalents/g of BSG) was achieved. The obtained oligosaccharides were identified as arabino-xylooligosacharides (AXOS) with degree of polymerization from 2 to 5. One-step fermentation proved to be a promising strategy for AXOS production from BSG, presenting a performance comparable with the use of commercial enzymes. This study provides new insights towards the bioprocess integration, enabling further developments of low-cost bioprocesses for the production of these valuable compounds.

Single-step production of arabino-xylooligosaccharides by recombinant Bacillus subtilis 3610 cultivated in brewers' spent grain.

Brewers' spent grain (BSG) is an inexpensive and abundant brewery by-product that can be used to produce prebiotic arabino-xylooligosaccharides (AXOS). In this study, Bacillus subtilis 3610 was used, for the first time, to produce AXOS through direct fermentation of BSG. Additionally, the microorganism was genetically modified to improve the AXOS production. The xylanase gene xyn2 from Trichoderma reesei coupled with a secretion tag endogenous to B. subtilis was cloned in pDR111 and integrated into its chromosome. After optimization by experimental design, AXOS with a degree of polymerization ranging from 2 to 6 were obtained. The maximum production yield expressed in xylose equivalents per amount of BSG (54.2 ± 1.1 mg/g) represents an increase of 33% comparing to the wild type. When compared with the enzymatic hydrolysis process, single-step fermentation with B. subtilis proved to be a very promising low-cost strategy for the simultaneous production of AXOS and valorization of BSG.

New ß-galactosidase producers with potential for prebiotic synthesis.

ß-Galactosidases (EC 3.2.1.23) are interesting enzymes with potential application in the pharmaceutical and food industry. In this work, a screening study was carried out to identify new fungal sources of ß-galactosidase. A total of 50 fungi were evaluated using a chromogenic test performed in agar plates. The most promising microorganisms were validated as effective ß-galactosidase producers under submerged fermentation conditions. The crude ß-galactosidases were characterized regarding their optimal pH (3.0-5.5) and temperature (45-65 °C). All enzymes showed ability to synthesize lactose-based prebiotics, namely lactulose (maximal yield 3.3%) and a galacto-oligosaccharide (GOS) (maximal yield 20%). Additionally, some enzymatic extracts with fructosyltransferase activity allowed to produce other type of prebiotics, namely fructo-oligosaccharides (FOS). This work, reports for the first time the simultaneous synthesis of different mixtures of GOS (2-15% yield and 0.07-0.5 g/L·h-1 productivity) and FOS (4-30% yield and 0.1-1 g/L·h-1 productivity) by crude extracts exhibiting dual enzymatic activity.

ß-galactosidase from Aspergillus lacticoffeatus: A promising biocatalyst for the synthesis of novel prebiotics.

ß-galactosidase (EC 3.2.1.23) are interesting enzymes able to catalyze lactose hydrolysis and transfer reactions to produce lactose-based prebiotics with potential application in the pharmaceutical and food industry. In this work, Aspergillus lacticoffeatus is described, for the first time, as an effective ß-galactosidase producer. The extracellular enzyme production was evaluated in synthetic and alternative media containing cheese whey and corn steep liquor. Although ß-galactosidase production occurred in all media (expect for the one composed solely by cheese whey), the highest enzymatic activity values (460U/mL) were obtained for the synthetic medium. Ochratoxin A production in synthetic medium was also evaluated and 9days of fermentation was identified as a suitable fermentation time to obtain a crude extract enzyme with mycotoxin concentration below the legal comparable value established for wine and grape juices (2ng/mL). The optimal pH and temperature for the crude extract enzyme was found in the range of 3.5-4.5 and 50-60°C, respectively. The ß-galactosidase activity was reduced in the presence of Ba2+, Fe2+, Li+, K+ and galactose, while additives (except for ascorbic acid) and detergents exhibited a positive effect on enzymatic activity. This enzyme was able to catalyze the synthesis of prebiotics, namely lactulose (2.5g/L) and a galacto-oligosaccharide (trisaccharide, 6.3g/L), either when whole cells or crude enzyme was used as biocatalyst. The lactulose production using fungal whole cells is herein reported for the first time. Additionally, A. lacticoffeatus was also found to produce an enzyme with fructosyltransferase activity and other prebiotics, namely fructo-oligosaccharide 1-kestose (2.4g/L).

Trypsin purification using magnetic particles of azocasein-iron composite.

This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources.

Purification and characterization of a collagenase from Penicillium sp. UCP 1286 by polyethylene glycol-phosphate aqueous two-phase system.

Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method.

Biocatalytic Approaches Using Lactulose: End Product Compared with Substrate.

Lactulose is a lactose-based carbohydrate with well-known prebiotic effect and recognized medical applications. Currently, the commercially available lactulose is chemically synthesized. Nevertheless, the process leads to low yields and high levels of by-products. Alternatively, lactulose can be produced by enzymatic synthesis, which provides a cleaner production under mild conditions. Two different enzymatic routes were reported for lactulose production. Lactulose can be obtained through hydrolysis and transfer reactions catalyzed by a glycosidase. Alternatively, lactulose can be produced by direct isomerization of lactose to lactulose catalyzed by cellobiose-2-epimerase. An interesting characteristic of lactulose is also its capacity to act as substrate in additional enzymatic synthesis which leads to the formation of attractive compounds, such as lactulose-based oligosaccharides and lactulose esters. Besides increasing the interest and potential of lactulose, these lactulose-based compounds can also offer new and promising functionalities and applications. Herein, we review the enzymes involved in the synthesis of lactulose, as well as the reaction conditions and yields. The potential of different enzymes is discussed and it is shown that reaction conditions and composition of products depend on the type of enzyme and its microbial source. The conversion of lactulose into lactulose-based compounds is also covered, describing in detail the biocatalysts involved, the reaction conditions used, and the potential of the final products obtained.

Recovery of Peniophora cinerea laccase using aqueous two-phase systems composed by ethylene oxide/propylene oxide copolymer and potassium phosphate salts.

Aqueous two-phase systems (ATPSs) composed by UCON (ethylene oxide/propylene oxide copolymer) and potassium phosphate salts were for the first time evaluated in the recovery of Peniophora cinerea laccase from complex fermented medium. The ATPSs were obtained by combining the random copolymer UCON with KH2PO4, potassium phosphate buffer pH 7 or K2HPO4. According to the results, protein partition occurred predominantly toward the saline phase (bottom phase) of the ATPSs, while some contaminants such as pigments partitioned mainly to the top phase. In preliminary tests, it was found that the salt with the lowest pH value (KH2PO4, pH 4.6) stimulated the enzyme activity, while the other salts (pH between 7.0 and 9.5) caused a strong inhibition. However, the salt inhibition was not observed in the equilibrium phases of the UCON-Potassium phosphate ATPSs. The laccase recovery was high for all the biphasic systems, but the highest value (134%) was obtained when using UCON combined with KH2PO4. When compared to conventional concentration and purification methods (lyophilization, ammonium sulfate precipitation, ultrafiltration, and ion exchange chromatography), ATPS was demonstrated to be an efficient alternative for P. cinerea laccase recovery from fermented medium.