Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cells in Dual Matrices

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.     

Screening of potential antibiotic action of cellulolytic fungi

Twenty different strains of filamentous fungi were initially selected for evaluation of cellulolytic activity using a single test in a simple mineral salts culture medium with filter paper as the only carbon source. Those fungi strains that were capable of completely breaking the filter paper strip within 4–8 d were assayed also for antimicrobial action, using Staphyloccocus a ureus ATCC 6538P according to the so-called agar piece method. We screened three different strains with both capacities: the production of cellulolytic activity and antibiotic action. The experimental results suggest that the fungi Pinicillium sp. FOPCO1, Aspergillus sp. F0Q001, and Cephalosporium sp. F03800 have both capabilities because they grew rapidly on cellulose as the only carbon source and were able to produce an area of growth inhibition in S. aureus of approx 2.04, 1.57, and 2.39 cm, respectively, on agar plates using the agar piece method. Subsequently, the antibiotic production obtained with those cellulolytic strains was evaluated by submerged fermentation at the flask level, in a simple culture medium containing lactose without biosynthesis precursor, obtaining 3670, 2830, and 4060 antibiotic units/mL, referred to as penicillin G, whereas for cellulolytic activity, the results were 1.34, 1.81 and 0.57 FPU/mL, respectively.     

Accumulation of Poly[(<Emphasis Type="Italic">R</Emphasis>)-3-hydroxyalkanoates] in <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> SU-1 During Growth with Two Different Carbon Sources in Batch Culture

Polyhydroxyalkanoates (PHAs) are polymers of hydroxyalkanoate, which are accumulated by many bacteria as food storage material under excess carbon source and limited nitrogen source. In our study, Enterobacter cloacae SU-1 isolated from the rhizospheric soil of Arachis hypogea was allowed to grow as batch culture in minimal media containing either glucose or lactose, and the pattern of PHA accumulation by E. cloacae SU-1 was studied. E. cloacae SU-1 was found to accumulate 94% of PHA/dry weight of the organism in 8 g/l lactose-containing medium. When the monomeric units of PHA of E. cloacae SU-1 was analyzed by gas chromatography, it was also found to accumulate medium chain length PHA 3-hydroxyoctanoate (3HO)/3-hydroxyhexanoate (3HH) in the presence of glucose and lactose, but the ratio of these monomers differed as 11:1 and 6:1, respectively.     

Isolation and characterization of microorganisms degrading nylon 4 in the composted soil

Two kinds of microorganisms, a bacterium (KT-1 strain) and a fungus (KT-2 strain), degrading nylon 4 (polyamide 4), which was easily prepared by the anionic ring-opening polymerization of 2-pyrrolidone, were isolated from the composted soil with the utilization of enrichment cultures and the culture using nylon 4 as a carbon source. KT-1 and KT-2 strains were identified as neighboring species to Stenotrophomonas sp. and Fusarium sp., respectively, by their morphological properties and the nucleotide sequences. These strains were confirmed to grow in the culture medium containing nylon 4 powders as a carbon source. In addition, nylon 4 film was decomposed in both mineral media containing KT-1 and KT-2 strains, respectively, and disappeared within two months. The MALDI TOF-MS analysis of nylon 4 recovered during the biodegradation test suggest that the isolated KT-2 strain recognize the acyllactam or carboxy chain end and degrade them or their neighboring amide bond.     

Lactic acid recovery from cheese whey fermentation broth using combined ultrafiltration and nanofiltration membranes

The separation of lactic acid from lactose in the ultrafiltration permeate of cheese whey broth was studied using a cross-flow nanofiltration membrane unit. Experiments to test lactic acid recovery were conducted at three levels of pressure (1.4, 2.1, and 2.8 MPa), two levels of initial lactic acid concentration (18.6 and 27 g/L), and two types of nanofiltration membranes (DS-5DK and DS-5HL). Higher pressure caused significantly higher permeate flux and higher lactose and lactic acid retention (p<0.0001). Higher initial lactic acid concentrations also caused significantly higher permeate flux, but significantly lower lactose and lactic acid retention (p<0.0001). The two tested membranes demonstrated significant differences on the permeate flux and lactose and lactic acid retention. Membrane DS-5DK was found to retain 100% of lactose at an initial lactic acid concentration of 18.6 g/L for all the tested pressures, and had a retention level of 99.5% of lactose at initial lactic acid concentration of 27 g/L when the pressure reached 2.8 MPa. For all the test when lactose retention reached 99–100%, as much as 64% of the lactic acid could be recovered in the permeate.     

Production of lactic acid from cheese whey by batch and repeated batch cultures of Lactobacillus sp. RKY2

The fermentative production of lactic acid from cheese whey and corn steep liquor (CSL) as cheap raw materials was investigated by using Lactobacillus sp. RKY2 in order to develop a cost-effective fermentation medium. Lactic acid yields based on consumed lactose were obtained at more than 0.98 g/g from the medium containing whey lactose. Lactic acid productivities and yields obtained from whey lactose medium were slightly higher than those obtained from pure lactose medium. The lactic acid productivity gradually decreased with increase in substrate concentration owing to substrate and product inhibitions. The fermentation efficiencies were improved by the addition of more CSL to the medium. Moreover, through the cell-recycle repeated batch fermentation, lactic acid productivity was maximized to 6.34 g/L/h, which was 6.2 times higher than that of the batch fermentation.     

Separate and Concentrate Lactic Acid Using Combination of Nanofiltration and Reverse Osmosis Membranes

The processes of lactic acid production include two key stages, which are (a) fermentation and (b) product recovery. In this study, free cell of Bifidobacterium longum was used to produce lactic acid from cheese whey. The produced lactic acid was then separated and purified from the fermentation broth using combination of nanofiltration and reverse osmosis membranes. Nanofiltration membrane with a molecular weight cutoff of 100–400 Da was used to separate lactic acid from lactose and cells in the cheese whey fermentation broth in the first step. The obtained permeate from the above nanofiltration is mainly composed of lactic acid and water, which was then concentrated with a reverse osmosis membrane in the second step. Among the tested nanofiltration membranes, HL membrane from GE Osmonics has the highest lactose retention (97 ± 1%). In the reverse osmosis process, the ADF membrane could retain 100% of lactic acid to obtain permeate with water only. The effect of membrane and pressure on permeate flux and retention of lactose/lactic acid was also reported in this paper.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Fermentation of milk permeate by proteolytic bacteria for protease production

Kinetics of cell growth and protease production by four proteolytic bacterial strains, namely,Bacillus subtilis EMCC 1020,Bacillus megaterium EMCC 1057,Serratia marcescens EMCC 1247, andPseudomonas fluorescens EMCC 1221, in milk permeate, compared with other fermentation media, were studied. The pH values, lactose utilization, and biochemical oxygen demand (BOD) reduction in milk permeate also were investigated. The four strains were able to grow in milk permeate, and to produce considerable amounts of protease, reaching 289 (EMCC 1020), 252 (EMCC 1057), 263 (EMCC 1247), and 212 (EMCC 1221) U/mL after 30 h of fermentation. The growth and enzyme activity of the four strains were greater in milk permeate than those in other fermentation media. The protease-producing bacteria were able to utilize lactose in milk permeate with values ranging from 37.62 to 54.97% and to reduce the BOD of milk permeate by 50.59-63.65%. Milk permeate proved to be the best medium for enzyme production by all organisms examined.     

Growth of lignocellulosic-fermenting fungi on different substrates under low oxygenation conditions

Four soil fungi able to grow under low oxygenation conditions were selected and used in studies to determine the production of enzymes that promote the degradation of lignocellulosic materials. The capacity of these fungi to ferment such materials was also investigated. The fungi were grown in sugarcane bagasse and sawdust at final concentrations of 4 and 10%, as the carbon sources. The strains were cultivated under microaerophilic and combined conditions of oxygenation (aerobic followed by microaerophilic conditions). The results obtained with the basidiomycete specie, Trichocladium canadense, Geotrichum sp., and Fusarium sp. suggest that they prefer lower oxygen concentration for growth and enzyme production. Lignocellulolytic activities were detected in all strains but varied with the carbon source used. The highest levels of these activities were produced by the Basidiomycete specie and Fusarium sp. Ethanol and other nongaseous fermentation products were detected following high-performance liquid chromatography analysis using a supelcogel C-610H column, demonstrating the fermentative capability of these strains. In view of their ability to produce enzymes necessary for the breakdown of lignocellulosic materials and to utilize most of the degradation products for growth, these strains have a great potential for biotechnological application.     

Polyhydroxyalkanoate (PHA) Biosynthesis from Whey Lactose

Summary: The potential of three different microbial wild type strains as polyhydroxyalkanoate (PHA) producers from whey lactose is compared. Homopolyester and co-polyester biosynthesis was investigated by the archaeon Haloferax mediterranei and the eubacterial strains Pseudomonas hydrogenovora and Hydrogenophaga pseudoflava. H. mediterranei accumulated 50 wt.-% of poly-3-(hydroxybutyrate-co-6%-hydroxyvalerate) in cell dry mass from hydrolyzed whey without addition of 3-hydroxyvalerate (3HV) precursors (specific productivity q_p: 2.9 mg/g h). Using P. hydrogenovora, the final percentage of poly-3-hydroxybutyrate (PHB) amounted to 12 wt.-% (q_p: 0.03 g/g h); co-feeding of valeric acid resulted in the production of 12 wt.-%. P-3(HB-co-21%-HV) (q_p: 0.02 g/g h). With H. pseudoflava, it was possible to reach 40 wt.-% P-3 (HB-co-5%-HV) on not-hydrolyzed whey lactose plus valeric acid as 3HV precursor (q_p: 9.1 mg/g h); on hydrolyzed whey lactose without addition of valeric acid, the strain produced 30 wt.-% of PHB (q_p: 0.16 g/g h). The characterization of the isolated biopolyesters completes the study.     

Mass cultivation of various algal species and their evaluation as a potential candidate for lipid production

Microalgae have been proposed as a promising source for biodiesel production. Focusing on algal strains for biodiesel production, efforts should be made to search new strains. Experiments were carried out to investigate the effects of growth parameters (nutrients, pH, light, aeration and temperature) and the oil percentage of eight algal strains (Chlorella sp., Cladophora sp., Hydrodictylium sp., Oedogonium sp., Oscillatoria sp., Spirogyra sp., Stigeocolonium sp., Ulothrix sp.). Results show that 6.5–7.5 is the optimum pH for the growth of all algal species. Temperature showed a greater variation (25°40°C). Ulothrix sp. gave more biomass productivity and is the most suitable strain for biodiesel production due to higher oil percentage (62%). Least biomass production was observed for Stigeocolonium sp. and least oil content was obtained from Hydrodictylium sp. It was observed that among these eight algal strains for biodiesel production, Ulothrix and Chlorella are the most promising algae species.     

Evaluation of the effects of crude glycerol on the production and properties of novel polyhydroxyalkanoate copolymers containing high 11‐hydroxyoctadecanoate by Cupriavidus necator IPT 029 and Bacillus megaterium IPT 429

Crude glycerol (CG), a by‐product from biodiesel production, is a carbon source with potential as feedstock for polyhydroxyalkanoate (PHA) production. PHAs are biological macromolecules synthesized by microorganisms as intracellular carbon and energy storage granules. PHA production and its properties were investigated using Cupriavidus necator IPT 029 and Bacillus megaterium IPT 429 cultivated with CGs from different origins. The highest PHA extraction percentage (71.56% [w/v]) occurred when C. necator IPT 029 metabolized CG 3 (from the processing of biodiesel from castor bean oil). The gas chromatography–mass spectrometry analyses revealed novel PHA constituents as building blocks of medium (3‐hydroxytetradecanoate) and long (11‐hydroxyoctadecanoate) chains. Molar mass distribution revealed range of 121–6900 kDa. The initial degradation temperature ranged from 181.83 to 287.50°C and the crystallinity ranged from 35.30 to 66.70%. The results obtained indicate that C. necator IPT 029 from CG 3 could produce copolymers with industrially applicable thermophysical properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Isolation and Characterization of Bacteria that Degrade Poly (Lactic Acid-Glycerol Ester)-Type Time-Release Electron Donors for Accelerated Biological Reductive Dechlorination

Hydrogen Release Compound (HRC^TM) is an important electron donor that has recently become available and is now becoming widely applied to the accelerated biological reductive dechlorination of chloroethenes such as tetrachloroethene (PCE) and trichloroethene (TCE). HRC is a benign poly(lactic acid-glycerol ester) specially formulated for the slow time-release of lactic acid. Lactic acid is then metabolized to hydrogen, which can be used in the reductive dechlorination of chloroethenes. To establish an advance diagnosis of the HRC addition effect for the bioremediation of polluted sites, 17 strains of HRC-degrading bacteria were isolated by liquid- and plate-culture methods. All these strains could grow on a basal medium containing purified HRC as the sole carbon source. The sequence analysis of the 16S rDNAs of 6 of the 17 strains shows that they all belong to the family β-Proteobacteria, which includes Burkholderia cepacia, Burkholderia vietnamiensis, Ralstonia sp., and Variovorax paradoxus. The time course of HRC degradaton by strains JM-11, JM-12 and JM-13 showed that the HRC degradation rates after 9 days of cultivation were 81.1%, 82.8% and 80.4%, respectively. Preliminary assay of the activities of the HRC-degrading enzyme indicated that HRC degradation may be specifically performed by specific lipases produced by HRC-degrading microorganisms.     

Immobilization ofLactobacillus bulgaricus in a hollowfiber bioreactor for production of lactic acid from acid whey permeate

Lactobacillus bulgaricus was immobilized in the shell side of an industrial hollow-fiber ultrafiltration module. Acid whey permeate, containing 46 g/L lactose supplemented with 10 g/L yeast extract, was pumped through the tube side at dilution rates of 0.2–2.5/h. At a cell concentration of 100 g/L, productivity was 1.5–5 g lactic acid/L/h.     

Isolation of PHA–Producing Bacteria from Date Syrup Waste

Since the major problem associated with the industrial production of Polyhydroxyalkanoates (PHAs) is their high production cost, this study was carried out using date syrup as the major carbon source to decrease the production cost and also help to supply other nutrient requirements. To isolate PHA–producing bacteria for this purpose, microorganisms were isolated from the syrup waste of a local date factory. These purified colonies were screened for intracellular granules by staining with Sudan Black. The positive-staining strains were grown for production of PHAs in 5% date syrup as carbon source supplemented with mineral salt medium. The culture was incubated at 30 °C with shaking at 140 rpm for 60 h. Among positively stained bacteria, the best PHA producers were selected on the basis of cell growth, cell dry weight, PHA content and the monomer composition of PHA. One of them could utilize date syrup for growth and produce the homopolymer of Polyhydroxybutyrate (PHB) with a cell density of about 5.1 g/L and maximum concentration of PHB equal to 3.6 g/L which is 71% of cell dry weight. Another one produces copolymer of Poly (hydroxybutyrate-hydroxyvalerate) in date syrup media with a maximum concentration of 2.2 g/L containing 10 wt % valerate in shake flask cultivation.     

Influence of reaction medium composition on enzymatic synthesis of galactooligosaccharides and lactulose from lactose concentrates prepared from whey permeate

The paper presents the results of investigations into the technological possibilities of controlling the transgalactosylation process of lactose in permeate after whey ultrafiltration in order to improve the efficiency of galactooligosaccharides or lactulose synthesis. The synthesis efficiency was influenced by the selection of a β-galactosidase preparation, substrate concentration and, in the synthesis of lactulose, also by the ratio of lactose and fructose added to the reaction mixture. The obtained synthesis efficiency of GOS and, most of all, of lactulose (65 g L⁻¹), may be found satisfactory. The study also resulted in a proposed GOS or lactulose concentrates (concentrated or dried) production technology using permeate after ultrafiltration of milk or whey as lactose sources. Presented at the 35th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 26–30 May 2008.     

Biohydrogen Production Through Dark Fermentation by a Microbial Consortium Using Whey Permeate as Substrate

Nowadays, hydrogen produced globally has been synthesized from fossil fuel with limited source. Therefore, research has been developed in order to explore biological H₂ production by dark fermentation. The purpose of this work was to evaluate the effect of initial pH and ferrous sulfate and ammonium sulfate concentrations on the production of biohydrogen by dark fermentation. The process was carried out in batch mode under anaerobic conditions, in the absence of light, and at standard room temperature and pressure. A microbial consortium provided by the effluent treatment plant of a local dairy company was inoculated into a synthetic medium supplemented with cheese whey permeate (20 g/L of lactose) as a carbon source. The influence of three variables was analyzed by a central composite design 2⁽³⁾, and the optimum results of hydrogen yield (4.13 mol H₂/mol lactose) and productivity (86.31 mmol H₂/L/day) were achieved at initial pH 7.0 and FeSO₄ and (NH₄)₂SO₄ concentrations of 0.6 and 1.5 g/L, respectively. Under these conditions, the kinetic parameters of fermentation were investigated by analyzing the profile of H₂ yield and productivity, metabolite concentrations, pH, and concentration of dissolved iron. In the kinetic analysis, the modified Gompertz equation described adequately the fermentative hydrogen production from cheese whey permeate (R ²?=?0.98). The profile of ethanol and volatile organic acids showed that lactic acid and butyric acid were the main metabolites produced, and the sum of both by-products corresponded to about 58 % of the total metabolites.     

Biosynthesis of High Quality Polyhydroxyalkanoate Co- and Terpolyesters for Potential Medical Application by the Archaeon Haloferax mediterranei

Summary : Haloferax mediterranei was investigated for the production of two different high-performance polyhydroxyalkanoates (PHAs). A copolyester containing 6 mol-% 3-hydroxyvalerate (3HV) was produced from whey sugars as sole carbon source. The maximum specific growth rate (µ_max.) and the maximum specific PHA production rate (q_{p max.}) were determined with 0.10 1/h and 0.15 1/h, respectively. The cells contained 72.8 wt.-% of P-(3HB-co-6%-3HV) which featured low melting points between 150 and 160 °C and narrow molecular mass distribution (polydispersity PDI = 1.5). Further, a PHA terpolyester with an increased 3HV fraction as well as 4-hydroxybutyrate (4HB) building blocks was accumulated by feeding of whey sugars plus 3HV - and 4HB precursors. Kinetic analysis of the process reveals a µ_max. of 0.14 1/h and a q_{p max.} of 0.23 1/h, respectively. The final percentage of P-(3HB-co-21.8%-3HV-co-5.1%-4HB) in biomass amounted to 87.5 wt.-%. Also this material showed a narrow molecular mass distribution (PDI = 1.5) and a high difference between the two melting endotherms of the material (between 140 and 150 °C) and the onset of decomposition at 236 °C. The accomplished work provides viable strategies to obtain different high-quality PHAs which might be potential candidates for application in the medical and pharmaceutical field.     

A simple model for the continuous production of butanol by immobilized Clostridia: II: Clostridium species DSM 2152 on glucose and whey permeate

The kinetic model developed for the continuous production of butanol by immobilized Clostridia (Chem. Eng. J., 32 (1986) B43) was tested for Clostridium species DSM 2152, immobilized in calcium alginate beads. The model described the butanol production rates adequately both on a model substrate, glucose, and on a technical substrate, whey permeate. For both substrates the r_max values were found to be the same (about 55 kg substrate m⁻³ alginate h⁻¹), but the maximal butanol concentration C_B,max was much lower on whey permeate media than on glucose media (4.7 and 7.4 kg m⁻³). The operational stability of the immobilized Clostridium species was good; experiments lasted for 1000 – 1800 h without loss of activity and without disruption of the calcium alginate beads.