Micro-fabricated polydimethyl siloxane (PDMS) surfaces regulate the development of marine microbial biofilm communities.

This study explored an antifouling (AF) concept based on deployment of microfabricated polydimethyl siloxane (PDMS) surfaces with 1-10 µm periodicity corrugated topographies in temperate marine waters. The effect of the surfaces on the development of microbial biofilms over 28 days and during different seasons, including both summer and winter, was examined using confocal laser scanning microscopy (CLSM) as well as terminal restriction fragment (T-RF) analysis for phylogenetic fingerprinting. The microscale topography significantly impacted biofilm development by altering the attachment pattern and reducing microcolony formation on the 1, 2 and 4 µm PDMS surfaces. Also, field deployments over 28 days showed a significant reduction in biovolume on the 4 and 10 µm PDMS surfaces despite altered environmental conditions. The microfabricated PDMS surfaces further significantly impacted on the community composition of the biofilms, as revealed by changes in T-RF profiles, at different stages of development. Moreover, altered biofilm resistance was demonstrated by exposing pre-established biofilms on 10 µm micro-fabricated surfaces to enhanced flagellate predation by a heterotrophic protist, Rhynchomonas nasuta. Pronounced changes in the overall marine microbial biofilm development as well as community composition warrant exploring substratum modification for marine AF applications.

ProxiMAX randomization: a new technology for non-degenerate saturation mutagenesis of contiguous codons.

Back in 2003, we published 'MAX' randomization, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. 'MAX' randomization saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an α-helix, as in zinc-finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple contiguous codons in a non-degenerate manner. We have now developed 'ProxiMAX' randomization, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomization uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialized chemistry, reagents or equipment, and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in predefined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomization is particularly relevant to antibody engineering.

The fouling hydroid Ectopleura larynx: a lack of effect of next generation antifouling technologies.

The hydroid Ectopleura larynx is one of the main fouling organisms on salmon aquaculture cages in Norway; this study investigated novel surface materials and microtopographies to deter its settlement. The settlement preferences of hydroid larvae for 12 materials with wettabilities ranging from hydrophobic (54°) to hydrophilic (112°) were tested in a no-choice bioassay. Although settlement differed between materials, with the highest average settlement on polytetrafluoroethylene (95%) and the lowest on untreated polyurethane (53%), no trend regarding the tested wettabilities could be found and none of the tested materials was able to reduce average settlement below 50%. Furthermore, nine high-density polyethylene (HDPE, 100-600 µm microtopographies) and seven polydimethylsiloxane (PDMS; 40-400 µm microtopographies) microtextured surfaces were tested. There was no systematic effect of microtopography on the settlement of E. larynx larvae. However, there was a preference for settlement in channels on PDMS microtopographies between 80 and 300 µm. Similarly, there were no preferences for any of the examined microtopographies in a 12-day field test using PDMS surfaces at a commercial fish farm. The study indicated that neither surface wettability (hydrophilicity-phobicity) nor microtopographies were effective at deterring the settlement of the hydroid E. larynx. The high plasticity of the aboral pole and the hydrorhiza of the hydroids may explain settlement even under unfavourable conditions, highlighting the successful colonisation traits of this dominant biofouling species.

Environmentally sustainable fibers from regenerated protein.

Concerns for the environment and consumer demand are driving research into environmentally friendly fibers as replacements for part of the 38 million tonnes of synthetic fiber produced annually. While much current research focuses on cellulosic fibers, we highlight that protein fibers regenerated from waste or byproduct sources should also be considered. Feather keratin and wheat gluten may both be suitable. They are annually renewable, commercially abundant, of consistent quality, and have guaranteed supply. They contain useful amino acids for fiber making, with interchain cross-linking possible via cysteine residues or through the metal-catalyzed photocrosslinking of tyrosine residues. Previous commercially produced fibers suffered from poor wet strength. Contemporary nanoparticle and cross-linking technology has the potential to overcome this, allowing commercial production to resume. This would bring together two existing large production and processing pipelines, agricultural protein production and textile processing, to divert potential waste streams into useful products.

A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations: implications for peptide-based analgesics.

Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor.

Mechanism of aerobic biological destabilisation of wool scour effluent emulsions.

Wool scouring effluent is a highly polluted industrial wastewater in which the main pollutant, wool wax, is held in a stable oil-in-water emulsion by non-ionic detergent. The use of microbial action to cause emulsion destabilisation has been proposed as a new treatment strategy for this effluent stream. This strategy aims at improving aerobic treatment performance by physically removing the high-COD, slowly bio-degradable wool wax from the system without bio-degradation. The mechanism by which an aerobic-mixed culture destabilises the wool scouring effluent emulsion was investigated. Our results show that destabilisation is due to partial bio-degradation of both the scouring detergent and the wool wax. Cleavage of the wool wax esters was the first stage in wax degradation, when 40-50% of wax was de-emulsified. Over the same period, detergent degradation was low, at 7-21%. With further incubation, detergent degradation increased, aiding further breakdown of the emulsion. The degradation of the detergent, a nonylphenol ethoxylate, resulted in both a reduction in molar concentration (of up to 82%) and a shortening of the ethoxylate chain length. The latter reduced the hydrophile-lipophile balance (HLB) from 12 to approximately 7, thereby reducing the ability of the residual detergent to stabilise the emulsion. Analysis of the emulsified and de-emulsified wax fractions could not identify a group of compounds that were preferentially de-emulsified based on molecular weight or polarity. These findings will assist in using a de-emulsification strategy in both existing and new treatment systems in order to save on aeration costs and treatment times for biological treatment of this highly polluted wastewater.

The effects of physical and chemical treatments on Na2S produced feather keratin films.

The industrial utilisation of feather keratin as a biopolymer has proven difficult due to the lack of a viable extraction technique and the poor mechanical properties of the regenerated products. Here, pure keratin films were produced from chicken feathers using sodium sulphide as sole extraction reagent in a scheme that allows films to be formed without residual chemicals. In a comparison to other films, those produced using Na2S extraction were found to be superior to other regenerated protein films and were similar to un-oriented commercial polymers. However, there was considerable variation in tensile properties between twenty repetitions of extracting and casting films which was attributed to variations in chain entanglement caused by the drying conditions. Chemical and physical treatments including crosslinking, dehydration and addition of nano-particles were investigated as means to enhance these properties. Significant increases were achieved by soaking films in isopropyl alcohol or weak acid (13 to 50% increases) or by formaldehyde or glutaraldehyde crosslinking (24 to 40% increases). The wide range of values across the pure keratin films indicates that the best route to further strength improvement may be from optimising self-assembly via controlling drying conditions, rather than from chemical treatment.

Larval settlement: the role of surface topography for sessile coral reef invertebrates.

For sessile marine invertebrates with complex life cycles, habitat choice is directed by the larval phase. Defining which habitat-linked cues are implicated in sessile invertebrate larval settlement has largely concentrated on chemical cues which are thought to signal optimal habitat. There has been less effort establishing physical settlement cues, including the role of surface microtopography. This laboratory based study tested whether surface microtopography alone (without chemical cues) plays an important contributing role in the settlement of larvae of coral reef sessile invertebrates. We measured settlement to tiles, engineered with surface microtopography (holes) that closely matched the sizes (width) of larvae of a range of corals and sponges, in addition to surfaces with holes that were markedly larger than larvae. Larvae from two species of scleractinian corals (Acropora millepora and Ctenactis crassa) and three species of coral reef sponges (Luffariella variabilis, Carteriospongia foliascens and Ircinia sp.,) were used in experiments. L. variabilis, A. millepora and C. crassa showed markedly higher settlement to surface microtopography that closely matched their larval width. C. foliascens and Ircinia sp., showed no specificity to surface microtopography, settling just as often to microtopography as to flat surfaces. The findings of this study question the sole reliance on chemical based larval settlement cues, previously established for some coral and sponge species, and demonstrate that specific physical cues (surface complexity) can also play an important role in larval settlement of coral reef sessile invertebrates.

Treatment of biorefractory organic compounds in wool scour effluent by hydroxyl radical oxidation.

Wool scouring effluent that had been treated with chemical flocculation and aerobic biological treatment (Sirolan CFB effluent) was tertiary treated by hydroxyl radical oxidation to remove residual organic compounds. These compounds impart a high chemical oxygen demand of 500-3000 mg/L and dark colour. However, a H2O2/UV process was found to effectively treat the majority of residual compounds, with up to 75% COD, 85% total organic carbon, and 100% removal of colour (T(480 nm)) achieved. This was despite the effluent being strongly absorbing in the UV region, with a film thickness of 0.21 mm reducing T(254 nm) by 50%. Treatment was unaffected by pH over the range 3-9. H2O2/UV treatment increased the biodegradability of the effluent (5-day biochemical oxygen demand increased from < 10 to 86 mg/L), but a combined chemical and biological process did not increase maximum COD removal or overall process efficiency. The tertiary treated effluent had a final COD in the range 125-750 mg/L, equating to a total COD removal from raw wool scour effluent of approximately 97.5%. This degree of treatment is sufficient for discharge in many, but not all, circumstances.

Treatment of strongflow wool scouring effluent by biological emulsion destabilisation.

The stable oil-in-water emulsion contained in wool scouring effluent was destabilised by aerobic biological treatment as the basis of a potential new effluent treatment process. The de-emulsified wool wax, which is recalcitrant to biodegradation, can then be readily removed by centrifugation. In 12-day batch experiments, 97% of wool wax and 87% of COD were removed after gentle centrifuging at 200 x g, compared to only 6% and 8%, respectively, for sterile controls. Steady-state chemostat experiments under optimum conditions gave essentially complete removal of wool wax and 90% removal of COD at less than 40 h retention time, and demonstrated that the mechanism of pollutant removal was by bioflocculation rather than aerobic degradation. At 100 L pilot scale, 95% of wool wax and 82% of COD were consistently removed over a period of 116 days of continuous operation at 38 h retention time and 30 degrees C, producing a spadable sludge of 5.7 mL/g. Variable influent concentration or filamentous bacteria did not disrupt this process and foaming was readily controlled using a mechanical foam breaker. After a shutdown period of 15 days the process could be restarted easily, achieving normal performance within one retention time. The successful operation of the pilot reactor suggests this process could be developed to full scale and incorporated into an overall treatment package.

Where to settle--settlement preferences of Mytilus galloprovincialis and choice of habitat at a micro spatial scale.

The global mussel aquaculture industry uses specialised spat catching and nursery culture ropes made of multi-filament synthetic and natural fibres to optimise settlement and retention of mussels for on-growing. However, the settlement ecology and preferences of mussels are poorly understood and only sparse information exists in a commercial context. This study quantified the settlement preferences of pediveligers and plantigrades of Mytilus galloprovincialis on increasingly complex surfaces and settlement locations at a micro spatial scale on and within ropes under commercial hatchery operating conditions using optical microscopy and X-ray micro-computed tomography (µCT). M. galloprovincialis has clear settlement preferences for more complex materials and high selectivity for settlement sites from the pediveliger through to the plantigrade stage. Pediveligers of M. galloprovincialis initially settle inside specialised culture ropes. Larger pediveligers were located close to the exterior of ropes as they increased in size over time. In contrast, smaller individuals were located deeper inside of the ropes over time. This study demonstrates that X-ray µCT is an excellent non-destructive technique for mapping settlement and attachment sites of individuals as early as one day post settlement, and quantifies the number and location of settled individuals on and within ropes as a tool to understand and optimise settlement in complex multi-dimensional materials and environments.