Pilot scale demonstration of a two-stage pretreatment and bioethanol fermentation process for cotton gin trash.

A two-stage dilute acid and steam explosion (SE) pretreatment process was developed and evaluated at pilot scale for ethanol production from cotton gin trash (CGT). Optimal conditions for CGT processing were defined as 1:6 solids to liquids ratio with 9% H2SO4 wt. on solids at 180 °C for 15 min. during stage 1 with ensuing pressed fibres successively exposed to SE at 200 °C for 5 min during stage 2. SE fibres were highly acquiescent to enzyme hydrolysis (76%) in the presence of PEG 6000, yielding 381 g glucose kg-1 fibre. Simultaneous saccharification and fermentation (SSF) trials validated the selected process option and additional fed-batch SSFs confirmed titres above the minimum 4% ww-1 benchmark for economically viable distillation. The practicality of converting CGT to ethanol was demonstrated at pilot scale with titres above 4% ww-1 and a conversion efficiency of 60% t-1 dry GCT.

Potential use of feedlot cattle manure for bioethanol production.

This paper reports on processing options for the conversion of feedlot cattle manures into composite sugars for ethanol fermentation. Small-scale anaerobic digestion trials revealed that the process significantly reduces the content of glucan and xylan (ca. 70%) without effecting lignin. Moreover, anaerobic digestate (AD) fibres were poor substrates for cellulase (Cellic® CTec 2) saccharification, generating a maximum combined sugar yield of ca. 12% per original dry weight. Dilute acid pretreatment and enzyme saccharification of raw manures significantly improved total sugar recoveries, totalling 264 mg/g (79% theoretical). This was attained when manures were pretreated with 2.5% H2SO4 for 90 min at 121°C and saccharified with 50 FPU CTec 2/g glucan. Saccharomyces cerevisiae efficiently fermented crude hydrolysates within 6 h, yielding 7.3 g/L ethanol, representing glucose to ethanol conversion rate of 70%. With further developments (i.e., fermentation of xylose), this process could deliver greater yields, reinforcing its potential as a biofuel feedstock.

Selectable in-vivo recombination to increase antibody library size--an improved phage display vector system.

Phage display technology permits the display of libraries of random combinations of light (LC) and heavy chain (HC) antibody genes. Maximizing the size of these libraries would enable the isolation of antibodies with high affinity and specificity. In this study, the loxP/Cre system of in-vivo recombination has been employed to construct an improved vector system for the display of antibodies. In this system, the chloramphenicol acetyl transferase (CAT) gene is linked to a HC library in a donor plasmid, pUX. This CAT gene is 'silent' before recombination but active after recombination. A second acceptor phagemid, pMOX, is used for cloning the LC repertoire. Following infection with a Cre producing phage, pMOX accepts the CAT/HC library from pUX via site-specific recombination at the loxP sites. Recombinants can then be selected via chloramphenicol resistance. Using this vector system, we have generated libraries of 4x109 recombinants. Restriction analysis and Fab expression confirmed that 100% of the colonies in the library were recombinants. This system provides a stable selectable mechanism for the generation of large libraries and avoids the isolation of non-recombinants encountered with earlier in-vivo recombination systems.

Ethanol production from cotton gin trash using optimised dilute acid pretreatment and whole slurry fermentation processes.

Cotton ginning trash (CGT) collected from Australian cotton gins was evaluated for bioethanol production. CGT composition varied between ginning operations and contained high levels of extractives (26-28%), acid-insoluble material (17-22%) and holocellulose (42-50%). Pretreatment conditions of time (4-20 min), temperature (160-220 °C) and sulfuric acid concentration (0-2%) were optimised using a central composite design. Response surface modelling revealed that CGT fibre pretreated at 180 °C in 0.8% H2SO4 for 12 min was optimal for maximising enzymatic glucose recoveries and achieved yields of 89% theoretical, whilst the total accumulated levels of furans and acetic acid remained relatively low at <1 and 2 g/L respectively. Response surface modelling also estimated maximum xylose recovery in pretreated liquors (87% theoretical) under the set conditions of 150 °C in 1.9% H2SO4 for 23.8 min. Yeast fermentations yielded high ethanol titres of 85%, 88% and 70% theoretical from glucose generated from: (a) enzymatic hydrolysis of washed pretreated fibres, (b) enzymatic hydrolysis of whole pretreated slurries and (c) simultaneous saccharification fermentations, respectively.

Ethanol production from Eucalyptus plantation thinnings.

Conditions for optimal pretreatment of eucalypt (Eucalyptus dunnii) and spotted gum (Corymbia citriodora) forestry thinning residues for bioethanol production were empirically determined using a 3(3) factorial design. Up to 161mg/g xylose (93% theoretical) was achieved at moderate combined severity factors (CSF) of 1.0-1.6. At CSF>2.0, xylose levels declined, owing to degradation. Moreover at high CSF, depolymerisation of cellulose was evident and corresponded to glucose (155mg/g, ∼33% cellulose) recovery in prehydrolysate. Likewise, efficient saccharification with Cellic® CTec 2 cellulase correlated well with increasing process severity. The best condition yielded 74% of the theoretical conversion and was attained at the height of severity (CSF of 2.48). Saccharomyces cerevisiae efficiently fermented crude E. dunnii hydrolysate within 30h, yielding 18g/L ethanol, representing a glucose to ethanol conversion rate of 0.475g/g (92%). Based on our findings, eucalyptus forest thinnings represent a potential feedstock option for the emerging Australian biofuel industry.

Enhanced enzyme saccharification of Sorghum bicolor straw using dilute alkali pretreatment.

The impacts of varying pretreatment parameters (temperature, time, and alkalinity) on enzymatic hydrolysis of sorghum straw were investigated. Following pretreatment, both solids and lignin content was found to be inversely proportional to the severity of the treatments. Higher temperatures and alkali strength were quintessential for maximising sugar recoveries from enzyme saccharifications. Total sugar release peaked when sorghum straw was pretreated in 2% NaOH at 121 degrees C for 60 min; representing a 5.6-fold higher yield compared to samples pretreated at 60 degrees C in the absence of alkali. Similarly, 4.3-fold increases in total sugars from samples treated with 2% NaOH at 60 degrees C for 90 min, confirmed the importance of alkali inclusion. Addition of beta-glucosidase and xylanase to saccharification mixtures enhanced reaction rates and final sugar yields, whilst reducing cellulase dosage 4-fold. Saccharification efficiency of pretreated solids approached 90% and 95% (w/w) with as little as 2.5 and 5.0 FPU cellulase/g, respectively.

Rapid isolation and high-throughput determination of cellulase and laminarinase activity in soils.

A new method for extracting soil enzymes is described and a microplate method for assaying soil beta-1,4-glucanases (cellulases) and beta-1,3-glucanases (laminarinases). Soil samples were mechanically disrupted to produce crude enzyme extracts, and diluted preps incubated in microplates containing either carboxymethyl cellulose (CMC) to determine cellulase activity or laminarin substrate to determine laminarinase activity. The resulting glucose was measured using the fluorometric Amplex Red glucose assay. The method was reproducible, could be completed in 1 day and measured twice as much enzyme activity than the standard passive soil enzyme extraction procedure. The method described herein facilitates the development of high-throughput soil multiplex enzymatic assays from several soil samples at one time, and is well suited to the study of functional microbial ecology.

Enhancing cell survival of atrazine degrading Rhodococcus erythropolis NI86/21 cells encapsulated in alginate beads.

AIMS: To develop a method to produce beads with encapsulated Rhodococcus erythropolis NI86/21 with high cell density, extended shelf life, ease of handling and good atrazine degradation capabilities in both liquid and in agricultural soil. METHODS AND RESULTS: Our findings show that the supplementary recovery step in nutrient broth media shortly after cell encapsulation facilitates cell survival in both wet and dry beads upon extended storage at 4 degrees C. Air drying has little or no impact on encapsulated R. erythropolis cell's ability to degrade atrazine in liquid or soil. Bead storage for periods extending up to 12 months at 4 degrees C did not affect the capacity of R. erythropolis encapsulated cells to degrade atrazine in either BMN or nonsterile soil extracts. Bentonite-amended beads formulated with 1% skim milk and exposed to the supplementary growth step, outperformed all other bead formats. These beads provided adequate numbers of vigorous R. erythropolis cells in either liquid or soil media to degrade atrazine. CONCLUSIONS: Supplementary growth in nutrient broth media immediately following cell encapsulation greatly enhances R. erythropolis cells survival in both wet and dry beads upon extended storage at 4 degrees C. Wet and dried beads have similar capacity for atrazine degradation, and their usefulness and appeal in agronomic practise will only be known after bioassay evaluation and successful demonstration at field scale using incurred residues. SIGNIFICANCE AND IMPACT OF THE STUDY: R. erythropolis NI86/21 encapsulated cells have the potential to reduce residual atrazine in soil, thereby minimizing the likelihood of off-site transport to ground or river water and reduce the loss of crops because of phytotoxicity of residual herbicide. Owing to their ease of handling, storage and possible compatibilities with pre-existing mechanical equipment, dried bead formats are ideally suited for agricultural and remediational applications.

Atrazine degradation by encapsulated Rhodococcus erythropolis NI86/21.

AIMS: To develop an encapsulation procedure for Rhodococcus erythropolis NI86/21 and demonstrate its use as a slow-release inoculant for reducing atrazine levels in aquatic and terrestrial environments. METHODS AND RESULTS: Alginate encapsulation procedures were developed for the atrazine-degrading bacteria R. erythropolis NI86/21. Several bead amendments, including bentonite, powdered activated carbon (PAC) and skimmed milk (SM), were evaluated for slow release of R. erythropolis NI86/21 and efficacy of atrazine degradation. All bead types demonstrated a capacity to degrade atrazine in basal minimal nutrient buffer whilst continually releasing viable bacterial cells. We found that the addition of bentonite hastened cell release whilst SM sustained cell viability in bead formulations. Reducing the percentage of SM to 1% (w/v) resulted in faster rates of atrazine degradation in both liquid and soil, and was found to prolong cell survival upon bead storage. Limited oxygen transfer affects the capacity of the encapsulated R. erythropolis cells to degrade atrazine. CONCLUSIONS: Degradation studies have demonstrated the efficacy of R. erythropolis encapsulated cells to degrade atrazine in amended liquid and soil. However, in their current formulation, the wet alginate-based beads are impractical for field application because of their poor cell viability during storage. SIGNIFICANCE AND IMPACT OF THE STUDY: R. erythropolis NI86/21-encapsulated cells have the potential to reduce atrazine residues in a number of soil and water environments, possibly ensuring the continued registration and use of atrazine in agriculture by minimizing or eliminating nontarget effects.

The relationship between concentration of a dual marker strain of Salmonella Typhimurium in bovine faeces and its probability of detection by immunomagnetic separation and culture.

AIMS: To modify a strain of Salmonella serotype Typhimurium to express unique marker traits and then define how the concentration of the marker in bovine faeces affects the probability of its detection by culture preceded by immunomagnetic separation (IMS). METHODS AND RESULTS: DNA encoding for the production of green fluorescent protein (gfp) and resistance to kanamycin was inserted into the bacterial chromosome of Salm. Typhimurium. Transposon insertion was demonstrated by Southern blot hybridization. Varying amounts of one electroporant (gfpSal-1) were inoculated into suspensions of bovine faeces and attempts made to isolate gfpSal-1 using a protocol based on pre-enrichment incubation, IMS and enrichment in selective media. Isolates of gfpSal-1 were differentiated from wild strains of Salmonella using fluorescence under u.v. light and expression of kanamycin resistance. A logistic and Gompertz function each derived from the dose-response data partially explained the observations with the fit of the Gompertz function judged to be superior. The 10, 50 and 90% limits of detection from the Gompertz function were estimated to be 1.92, 2.03 and 2.27 CFU g(-1) respectively. CONCLUSIONS: Reliance on the traditional concept of 'limit of detection' could introduce unacceptable errors in the interpretation of test findings when the concentration of Salm. Typhimurium in bovine faeces (pooled or individual) is below ca 3 CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The dose-response curve can be used to aid the design of protocols for detecting Salmonella in individual and pooled faecal specimens. The experiments demonstrate that both reporter genes in tandem are useful for studying the performance of culture-based methods for detecting pathogens in faeces.