Downstream protein separation by surfactant precipitation: a review.

In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein-surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted. Moreover, comparisons will be made to reverse micellar extraction, and the current drawbacks/challenges of surfactant precipitation will also be discussed. Finally, promising directions of future work with this separation technique will be highlighted.

Metabolic reduction of resazurin; location within the cell for cytotoxicity assays.

Resazurin is widely used as a metabolic indicator for living cells, however, there has been considerable debate in the literature with regards to the specific location in the cell where the non-fluorescent resazurin is reduced to the strongly fluorescent resorufin. This lack of clarity about the reduction site makes the use of resazurin reduction data in cytotoxicity studies difficult to interpret. In this study, E. faecalis, a Gram-positive and facultative anaerobic bacterial strain, and the most toxic chlorophenol, pentachlorophenol (PCP), were chosen as models for an anaerobe and toxicant, respectively. By studying the kinetics of resazurin reduction by E. faecalis after different treatments (cell disruption, bacterial filtration, and pre-exposure to toxicant), we confirmed that resazurin reduction to resorufin by live Gram-positive and facultative anaerobic bacterial cells can only happen intracellularly under anaerobic conditions, while resorufin reduction to dihydroresorufin can happen both intracellularly and extracellularly. Based on the understanding of these fundamental mechanisms, we suggest that resazurin reduction can be used as a quick bioassay for measuring cytotoxicity.

Novel approaches to purifying bacteriocin: A review.

Bacteriocin is a proteinaceous biomolecule produced by bacteria (both Gram-positive and Gram-negative) that exhibits antimicrobial activity against closely related species, and food-borne pathogens. It has recently gained importance and attracted the attention of several researchers looking to produce it from various substrates and bacterial strains. This ushers in a new era of food preservation where the use of bacteriocin in food products will be an alternative to chemical preservatives, and heat treatment which are understood to cause unwanted side effects, and reduce sensory and nutritional quality. However, this new market depends on the success of novel downstream separation schemes from various types of crude feedstocks which are both effective and economic. This review focuses on the downstream separation of bacteriocin from various sources using both conventional and novel techniques. Finally, recommendations for future interesting areas of research that need to be pursued are highlighted.

Global Profiling of Metabolite and Lipid Soluble Microbial Products in Anaerobic Wastewater Reactor Supernatant Using UPLC-MSE.

Identification of soluble microbial products (SMPs) released during bacterial metabolism in mixed cultures in bioreactors is essential to understanding fundamental mechanisms of their biological production. SMPs constitute one of the main foulants (together with colloids and bacterial flocs) in membrane bioreactors widely used to treat and ultimately recycle wastewater. More importantly, the composition and origin of potentially toxic, carcinogenic, or mutagenic SMPs in renewable/reused water supplies must be determined and controlled. Certain classes of SMPs have previously been studied by GC-MS, LC-MS, and MALDI-ToF MS; however, a more comprehensive LC-MS-based method for SMP identification is currently lacking. Here we develop a UPLC-MS approach to profile and identify metabolite SMPs in the supernatant of an anaerobic batch bioreactor. The small biomolecules were extracted into two fractions based on their polarity, and separate methods were then used for the polar and nonpolar metabolites in the aqueous and lipid fractions, respectively. SMPs that increased in the supernatant after feed addition were identified primarily as phospholipids, ceramides, with cardiolipins in the highest relative abundance, and these lipids have not been previously reported in wastewater effluent.

Compatible solute addition to biological systems treating waste/wastewater to counteract osmotic and other environmental stresses: a review.

This study reviews the addition of compatible solutes to biological systems as a strategy to counteract osmolarity and other environmental stresses. At high osmolarity many microorganisms accumulate organic solutes called "compatible solutes" in order to balance osmotic pressure between the cytoplasm and the environment. These organic compounds are called compatible solutes because they can function inside the cell without the need for special adaptation of the intracellular enzymes, and also serve as protein stabilizers in the presence of high ionic strength. Moreover, the compatible solutes strategy is regularly being employed by the cell, not only under osmotic stress at high salinity, but also under other extreme environmental conditions such as low temperature, freezing, heat, starvation, dryness, recalcitrant compounds and solvent stresses. The accumulation of these solutes from the environment has energetically a lower cost than de novo synthesis. Based on this cell mechanism several studies in the field of environmental biotechnology (most of them on biological wastewater treatment) employed this strategy by exogenously adding compatible solutes to the wastewater or medium in order to alleviate environmental stress. This current paper critically reviews and evaluates these studies, and examines the future potential of this approach. In addition to this, a strategy for the successful implementation of compatible solutes in biological systems is proposed.

Influence of Feed Composition on the Monomeric Structure of Free Bacterial Extracellular Polysaccharides in Anaerobic Digestion.

Six 5.0-L fill-and-draw batch reactors were used with different feed compositions containing a range of carbohydrates (glucose, sucrose, fructose) and nitrogen sources (urea, NH4Cl) at various concentrations to investigate free extracellular polysaccharide (EPS) production during anaerobic digestion (AD). This work analyzed not only their monosaccharide components, but also their specific linkage patterns and the change associated with different chemical nature in carbon substrates or nitrogen sources; all of these parameters can have profound biological consequences, and were correlated to macronutrients present in the feed. It is believed that feed composition is a major factor which determines the physicochemical characteristics of the free EPS. Our findings also suggest that the differences associated with the digestion of various carbon substrates and/or nitrogen sources could alter monomeric saccharide composition and concentrations of the free EPS. Such insights demonstrate that previous studies on feed C/N ratios tended to overestimate EPS production, while variations in the chemical nature of the nitrogen source were overlooked. Our results also link the physiochemical properties of free EPS with underlying biofouling mechanisms, and demonstrate that membrane fouling is to some extent dependent upon the prevailing nutritional environment and feed composition.

Chemical Characterization of Low Molecular Weight Soluble Microbial Products in an Anaerobic Membrane Bioreactor.

Effluents from wastewater treatment systems contain a variety of organic compounds, including end products from the degradation of influent substrates, nonbiodegradable feed compounds, and soluble microbial products (SMPs) produced by microbial metabolism. It is important to identify the major components of these SMPs to understand what is in wastewater effluents. In this study, physical pretreatments to extract and concentrate low molecular weight SMPs (MW< 580 Da) from effluents were optimized. Liquid-liquid extraction (LLE) of a 200 mL effluent sample showed the best performance using a mixture of n-hexane, chloroform, and dichloromethane (70 mL) for extraction. For solid phase extraction (SPE), two OasisHLB cartridges were connected in-line to optimize recovery, and the eluted samples from each cartridge were analyzed separately to avoid overlapping peaks. Four solvents varying from polar to nonpolar (methanol, acetone, dichloromethane, and n-hexane) were selected to maximize the number of compound peaks eluted. A combination of SPE (OasisHLB) followed by LLE was shown to maximize compound identification and quantification. However, the compounds identified accounted for only 2.1 mg of chemical oxygen demand (COD)/L (16% of total SMP as COD) because many SMPs have considerably higher MWs. Finally, the method was validated by analyzing a variety of different reactor effluents and feeds.

Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Activity kinetics, conformation, and energetics.

This study seeks to examine the ability of non-ionic/non-polar Colloidial Liquid Aphrons (CLAs) to preserve enzyme functionality upon immobilization and release. CLAs consisting of micron-sized oil droplets surrounded by a thin aqueous layer stabilized by a mixture of surfactants, were formulated by direct addition (pre-manufacture addition) using 1% Tween 80/mineral oil and 1% Tween 20 and the enzymes lipase, aprotinin and α-chymotrypsin. The results of activity assays for both lipase and α-chymotrypsin showed that kinetic activity increased upon immobilization by factors of 7 and 5.5, respectively, while aprotinin retained approximately 85% of its native activity. The conformation of the enzymes released through desorption showed no significant alterations compared to their native state. Changes in pH and temperature showed that optimum conditions did not change after immobilization, while analysis of activation energy for the immobilized enzyme showed an increase in activity at higher temperatures. Furthermore, the effect of bound water within the aphron structure allowed for some degree of enzyme hydration, and this hydration was needed for an active conformation with results showing a decrease in ΔH* for the immobilized system compared to its native counterpart.

"Protein" Measurement in Biological Wastewater Treatment Systems: A Critical Evaluation.

Five commercially available assay kits were tested on the same protein sample with the addition of 17 different types of interfering substances typically found in the biological wastewater treatment, and a comparison of the use of these assays with 22 different protein and peptide samples is also presented. It was shown that a wide variety of substances can interfere dramatically with these assays; the metachromatic response was also clearly influenced by different proteinaceous material. Measurement of the "protein" content in the effluent of an anaerobic membrane bioreactor was then carried out using these assay methods. Quantitative results of the "protein" concentration in the different effluent samples, with or without spiked additions of Bovine Serum Albumin (BSA), showed considerable disagreement. We concluded that the "protein" measured in wastewater samples using standard colorimetric assays often shows false positive results and has little correlation to their real value. A new analytical method needs to be developed in order to gain greater insight into the biological transformations occurring in anaerobic digestion, and how soluble microbial products (SMPs) are produced.

Stimulation and Inhibition of Anaerobic Digestion by Nickel and Cobalt: A Rapid Assessment Using the Resazurin Reduction Assay.

Stimulation of anaerobic digestion by essential trace metals is beneficial from a practical point of view to enhance the biodegradability and degradation rate of wastes. Hence, a quick method to determine which metal species, and at what concentration, can optimize anaerobic digestion is of great interest to both researchers and operators. In this present study, we investigated the effect of nickel(II), cobalt(II), and their mixture, on the anaerobic digestion of synthetic municipal wastewater. Using a volumetric method, that is, measuring methane production over time, revealed that anaerobic digestion was stimulated by the addition of 5 mg L-1 nickel(II), and cobalt(II), and their mixture in day(s). However, using a novel resazurin reduction assay, and based on its change in rate over time, we evaluated both inhibition at 250 mg L-1 nickel(II) and cobalt(II), and also the stimulatory effect of 5 mg L-1 nickel(II), and cobalt(II), and their mixture, in just 6 h. By investigating the dynamic distribution of these metals in the liquid phase of the anaerobic system and kinetics of resazurin reduction by nickel spiked anaerobic sludge, the concentration of nickel(II) on anaerobic digestion performance was profiled. Three critical concentrations were determined; stimulation starting (around 1 mg L-1), stimulation ending (around 100 mg L-1) and stimulation maximizing (around 10 mg L-1). Hence, we propose that the resazurin reduction assay is a novel and quick protocol for studying the stimulation of anaerobic bioprocesses by bioavailable essential trace metals.

Characterization and Significance of Sub-Visible Particles and Colloids in a Submerged Anaerobic Membrane Bioreactor (SAnMBR).

The distribution, composition and morphological structure of subvisible particles and colloids (0.01-10 µm) in the supernatant of a lab-scale submerged anaerobic membrane bioreactor (SAnMBR), and their role in membrane fouling, was investigated. Photometric analysis showed that the supernatant and membrane foulants were dominated by particles and colloids (0.45-10 µm), which accounted for over 90% of the total organics (proteins and polysaccharides). Excitation-emission matrix (EEM) fluorescence spectra and monosaccharide analysis showed that these particles and colloids were rich in fluorescent proteins, rhamnose, ribose and arabinose, all of which could be related to cellular and extracellular substances. Fluorescence and scanning electron microscopy confirmed the presence of bacterial cells in/on the subvisible particles and colloids. The microparticles (5-10 µm) were primarily composed of Streptobacilli and/or filamentous bacteria in the form of microcolonies, while the submicrometer particles and colloids (1-5 µm and 100 kDa-1 µm) had more free/single cocci and bacilli. The ratio of live/dead cells varied in different size-fractions, and the particles (1-10 µm) contained more live cells compared with the colloids (100 kDa-1 µm). Our findings suggest that bacterial cells in/on the particles and colloids could have an important effect on fouling in SAnMBRs as they represent pioneering species attaching to membranes to form fouling layers/biofilm. Such insights reveal that previous foulant-characterization studies in MBRs tended to overestimate organic fouling, while the biofouling induced by these bacteria in/on the particles and colloids was overlooked.

Effect of sparging rate on permeate quality in a submerged anaerobic membrane bioreactor (SAMBR) treating leachate from the organic fraction of municipal solid waste (OFMSW).

This paper focuses on the treatment of leachate from the organic fraction of municipal solid waste (OFMSW) in a submerged anaerobic membrane bioreactor (SAMBR). Operation of the SAMBR for this type of high strength wastewater was shown to be feasible at 5 days hydraulic retention time (HRT), 10 L min(-1) (LPM) biogas sparging rate and membrane fluxes in the range of 3-7 L m(-2) hr(-1) (LMH). Under these conditions, more than 90% COD removal was achieved during 4 months of operation without chemical cleaning the membrane. When the sparging rate was reduced to 2 LPM, the transmembrane pressure increased dramatically and the bulk soluble COD concentration increased due to a thicker fouling layer, while permeate soluble COD remained constant. Permeate soluble COD concentration increased by 20% when the sparging rate increased to 10 LPM.

Modeling and Application of a Rapid Fluorescence-Based Assay for Biotoxicity in Anaerobic Digestion.

The sensitivity of anaerobic digestion metabolism to a wide range of solutes makes it important to be able to monitor toxicants in the feed to anaerobic digesters to optimize their operation. In this study, a rapid fluorescence measurement technique based on resazurin reduction using a microplate reader was developed and applied for the detection of toxicants and/or inhibitors to digesters. A kinetic model was developed to describe the process of resazurin reduced to resorufin, and eventually to dihydroresorufin under anaerobic conditions. By modeling the assay results of resazurin (0.05, 0.1, 0.2, and 0.4 mM) reduction by a pure facultative anaerobic strain, Enterococcus faecalis, and fresh mixed anaerobic sludge, with or without 10 mg L(-1) spiked pentachlorophenol (PCP), we found it was clear that the pseudo-first-order rate constant for the reduction of resazurin to resorufin, k1, was a good measure of "toxicity". With lower biomass density and the optimal resazurin addition (0.1 mM), the toxicity of 10 mg L(-1) PCP for E. faecalis and fresh anaerobic sludge was detected in 10 min. By using this model, the toxicity differences among seven chlorophenols to E. faecalis and fresh mixed anaerobic sludge were elucidated within 30 min. The toxicity differences determined by this assay were comparable to toxicity sequences of various chlorophenols reported in the literature. These results suggest that the assay developed in this study not only can quickly detect toxicants for anaerobic digestion but also can efficiently detect the toxicity differences among a variety of similar toxicants.

Anaerobic toxicity assay of plasticisers.

Plasticisers are commonly found in landfill leachate and accumulate in the environment. Some of them are known as disruptive endocrine compound. This manuscript assessed the toxicity of three common plasticisers, including Bis(2-Ethylhexyl)phthalate (DEHP), o-hydroxybiphenyl (HBP) and 2,6-di-tert-butyl-4-(dimethylaminomethyl) phenol (MAMP) on the methanogens during the anaerobic process. It was found that DEHP and MAMP did not impede methanogenesis up to 200 mg/L, but no additional methane could be obtained from their degradation. In contrast, HBP severely inhibited methanogens at 200 mg/L, but after acclimatisation it could be metabolised resulting in a 25% increase in methane production compared to the control.

Resource recovery from food-processing wastewaters in a circular economy: a methodology for the future.

Food-processing wastewater (FPWW), as opposed to solid residues, occurs extensively throughout the world, and has considerable potential for resource recovery (RR), however, at present, it is severely underutilized. This paper serves as a suggested 'plan forward' to optimize integrated RR from FPWW. In order to optimize this potential and 'close the loop', there needs to be further development in in-depth analytical methods of the FPWW; axenic/mixed cultures or microbial communities capable of growing on FPWW and hence producing single-cell protein for animal feed and food additives; cost-effective methods for separating high-value-added solutes such as vitamins K2, B12, and B2; isoflavones and flavanones; integrated energy- and water-recovery flowsheets; and optimization methods to integrate RR and energy harvesting with minimal impact on the environment. Each of these areas is examined and future research directions are laid out.

Anaerobic membrane bioreactor performance with varying feed concentrations of ciprofloxacin.

The anaerobic membrane bioreactor (AnMBR) has considerable potential for treating wastewater, although there is very little data on the effect of antibiotics on AnMBR performance. This study examined the effect of Ciprofloxacin (CIP) - an antibiotic that can occur at high concentrations, and has a substantial impact on ecosystems, on AnMBR performance. The long-term (44 days) presence of 0.5 mg/L CIP in the feed did not have a strong effect on COD removal, volatile fatty acid (VFA) accumulation, or methane yield, but did affect the pH, soluble microbial products (SMPs) and suspended solids. However, at 4.7 mg/L CIP, a significant effect on all the parameters tested was seen. 16S rRNA gene-based community analysis demonstrated that CIP changed the phylogenetic structure and altered the species richness and diversity. The relative abundance of various genera was also changed, and this explained much of the change in AnMBR behavior.

Post-treatment of the permeate of a submerged anaerobic membrane bioreactor (SAMBR) treating landfill leachate.

In this study, various methods were compared to reduce the Chemical Oxygen Demand (COD) content of stabilised leachate from a Submerged Anaerobic Membrane Bioreactor (SAMBR). It was found that Powdered Activated Carbon (PAC) resulted in greater COD removals (84 %) than Granular Activated Carbon (GAC-80 %), an ultrafiltration membrane of 1kDa (75 %), coagulation-flocculation with FeCl(3) and polyelectrolyte (45 %), FeCl(3) alone (32 %), and polymeric adsorbents such as XAD7HP (46 %) and XAD4 (32 %). Results obtained on the <1 kDa fraction showed that PAC and GAC had a similar adsorption efficiency of about 60 % COD removal, followed by XAD7HP (48 %), XAD4 (27 %) and then FeCl(3) (23 %). The post-treatment sequence UF+GAC would result in a final effluent with less than 100 mg COD/L. Size Exclusion Chromatography (SEC) revealed that the extent of adsorption of low MW compounds onto PAC was limited due to low MW hydrophilic compounds, whereas the kinetics of PAC adsorption depended mainly on the adsorption of high MW aromatics.

Comparison of soluble microbial product (SMP) production in full-scale anaerobic/aerobic industrial wastewater treatment and a laboratory based synthetic feed anaerobic membrane system.

This study focused on the characterisation of soluble microbial products (SMPs) produced from a full-scale multi-stage (anaerobic/aerobic) industrial wastewater treatment plant, and contrasted them to the SMPs detected in the effluent of a lab-scale AnMBR treating synthetic wastewater to determine if there were any common solutes detected irrespective of the feed organics. Recently developed analytical methods using gas chromatography coupled mass spectrometry (GC-MS) and liquid chromatography coupled quadrupole-time-of-flight (LC-Q-ToF) for SMP characterisation in a wide molecular weight (MW) range of 30-2000 Da (Da) were applied. Samples collected from the Industrial Wastewater plant were the upflow anaerobic sludge blanket (UASB) influent and effluent, and aerobic membrane bioreactor (MBR) effluent before discharge. The GC-MS detected a spike in cyclooctasulphur in the UASB effluent, an indicator of shock-loading, which disappeared after the MBR process. Alkanes, acids and nitrogenous compounds were found to be the end-products from the GC-MS results, while LC-Q-ToF analysis revealed that eicosanoids, a group of cell-signalling molecules, were produced in the aerobic MBR, and made up 71% of its effluent. A comparison of the submerged anaerobic membrane bioreactor (SAMBR) and aerobic MBR effluents using GC-MS showed that there was only a small degree of similarity between the SMPs, comprising mainly long chain alkanes and phthalate. On the other hand, LC-Q-ToF showed a large contrast in compound composition, mostly having cell-signalling functions, which deepened our understanding of the different metabolic processes occurring in aerobic and anaerobic systems. These data could be useful for future work in various areas such as controlling quorum-sensing and biofilm formation, process optimisation and control, and microbial ecology.

Datasets on the optimization of alginate extraction from sargassum biomass using response surface methodology.

This article presents data associated with the extraction of sodium alginate from waste Sargassum seaweed in the Caribbean utilizing an optimization approach using Response Surface Methodology [1]. A Box-Behnken (BBD) Response Surface Methodology using Design Expert 10.0.3 software on the alkaline extraction process was used. Data consists of the effects of 4 process variables (temperature, extraction time, alkali concentration and excess volume of alkali: dried seaweed) on the yield of sodium alginate. The model was validated, and extracts were characterization using High Performance Liquid Chromatography (HPLC), Gel Permeation Chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). The data illustrates the applicability of our model in potentially valorizing this waste product into a valuable resource. Furthermore, our methodology can be applied to other macroalgae for efficient extraction of sodium alginate of commercial quality.

Current applications of Colloidal Liquid Aphrons: Predispersed solvent extraction, enzyme immobilization and drug delivery.

Colloidal Liquid Aphrons (CLAs) are micron sized discrete spherical solvent droplets formed by the dispersion of polyaphrons into a bulk aqueous phase at a low phase volume ratio where they can be kept homogenously suspended with only minimal agitation. CLAs have high stability due to the presence of a surfactant 'shell' surrounding the solvent core, and possess large surface areas per unit volume for mass transfer due to their small size. Therefore, CLAs are well suited for applications in pre-dispersed solvent extraction (PSE), enzyme immobilization, and have the potential to be used as a drug delivery system. Using PSE, CLAs have been used to remove metals such as Ni2+, Cu2+, Fe3+, Cr3+ and Mg2+ from dilute streams, separate organic dyes such as Yellow 1 from wastewater, extract succinic and lactic acid, reactively extract phenylalanine, and separate suspensions. CLAs have also been used to immobilize enzymes such as lipase, lysozyme and albumins with cases of superactivity being reported due to the influence of surfactant and solvent interactions with the enzyme. Furthermore, due to their similarity to current drug delivery systems such as microemulsions and hydrogels, and other advantages, CLA systems have the potential to be adapted for drug delivery systems also. This article provides a complete list of the current applications of Colloidal Liquid Aphrons (CLAs) in PSE and enzyme immobilization, and also presents insight into how CLAs can be utilized as a drug delivery method in the future. Finally, this review ends by summarizing potentially interesting research areas to pursue in this field.