Two-stage microbial conversion of crude glycerol to 1,3-propanediol and polyhydroxyalkanoates after pretreatment.

With increasing demand for biodiesel, crude glycerol as a by-product in biodiesel production has been generated and oversupplied. This study, therefore, explored the pretreatment and a subsequent two-stage microbial system to convert crude glycerol into high value-added products: 1,3-propanediol (1,3-PDO) and polyhydroxyalkanoates (PHAs). After pretreatment, long chain fatty acids (LCFAs) could be effectively removed from crude glycerol to eliminate the inhibition effects on subsequent microbial process. In the anaerobic fermentation, when fed treated crude glycerol increased from 20 g/L to 100 g/L, 1,3-PDO yield decreased from 0.438 g/g to 0.345 g/g and accompanied carboxylic acids shifted from acetate and lactate dominant to lactate overwhelmingly dominant. Meanwhile, the relative abundance of Clostridiales sustained around 50% but Enterobacteriales increased from 19% to 53%. Further fed glycerol increase to 140 g/L resulted in severe substrate inhibition, which could be relieved by intermittent feeding. In aerobic process, glycerol anaerobic digestion effluent (ADE) was fed to the consortium of Bacillus megaterium and Corynebacterium hydrocarbooxydans for selectively consumption of carboxylic acids and residual glycerol from 1,3-PDO to produce PHAs as a secondary high value-added product. The consortium accumulated maximum 8.0 g/L poly (3-hydroxybutyrate) (PHB), and 1,3-PDO purity increased from initial 27.7% to almost 100% when fed with 100 g/L glycerol ADE. Overall, this study provided comprehensive and insightful information on microbial conversion of crude glycerol to high value-added products after pretreatment.

Determination of monomeric composition in polyhydroxyalkanoates by liquid chromatography coupled with on-line mass spectrometry and off-line nuclear magnetic resonance.

Polyhydroxyalkanoates (PHAs) are commercially-valuable biocompatible and biodegradable polymers with many potential medical, pharmaceutical and other industrial applications. The analysis of PHA monomeric composition is especially challenging due to the broad chemical diversity of PHA monomers and lack of analytical standards to represent the chemically-diverse PHA monomer constituents. In this study, a novel strategy based on on-line liquid chromatography-mass spectrometry (LC-MS) and off-line liquid chromatography-nuclear magnetic resonance (LC-NMR) was established to quantify seven PHA monomers with available standards and used to elucidate the structures of unknown PHA monomers. The strategy was successfully applied for the determination of monomeric composition in bacterial PHAs isolated from Pseudomonads cultivated on different carbon sources after hydrolysis. The results of this work demonstrated that the newly-developed strategy was efficient, repeatable, and could have good potential to be employed for detailed analysis of PHA monomeric composition.

Bioconversion of styrene to poly(hydroxyalkanoate) (PHA) by the new bacterial strain Pseudomonas putida NBUS12.

Styrene is a toxic pollutant commonly found in waste effluents from plastic processing industries. We herein identified and characterized microorganisms for bioconversion of the organic eco-pollutant styrene into a valuable biopolymer medium-chain-length poly(hydroxyalkanoate) (mcl-PHA). Twelve newly-isolated styrene-degrading Pseudomonads were obtained and partial phaC genes were detected by PCR in these isolates. These isolates assimilated styrene to produce mcl-PHA, forming PHA contents between 0.05±0.00 and 23.10±3.25% cell dry mass (% CDM). The best-performing isolate was identified as Pseudomonas putida NBUS12. A genetic analysis of 16S rDNA and phaZ genes revealed P. putida NBUS12 as a genetically-distinct strain from existing phenotypically-similar bacterial strains. This bacterium achieved a final biomass of 1.28±0.10 g L(-1) and PHA content of 32.49±2.40% CDM. The extracted polymer was mainly comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanoate (C8 ), 3-hydroxydecanoate (C10 ), 3-hydroxydodecanoate (C12 ), and 3-hydroxytetradecanoate (C14 ) monomers at a ratio of 2:42:1257:17:1. These results collectively suggested that P. putida NBUS12 is a promising candidate for the biotechnological conversion of styrene into mcl-PHA.

Co-composting of horticultural waste with fruit peels, food waste, and soybean residues.

Horticultural waste was co-composted with fruit peels, food waste, and soybean residues individually to evaluate the effects of these easily available organic wastes in Singapore on the composting process and product quality. Each co-composting material was mixed with horticultural waste in the wet weight ratio of 1:1 and composted for 46 days. Results showed that all co-composting materials accelerated the degradation of total carbon and resulted in higher nutrients of nitrogen (N), phosphorous (P), and potassium (K) in the final product compared with horticultural waste alone. Mixture with fruit peels achieved the fastest total carbon loss; however, did not reach the minimum required temperature for pathogen destruction. The end product was found to be the best source for K and had a higher pH that could be used for the remediation of acidic soil. Food waste resulted in the highest available nitrate (NO3-N) content in the end product, but caused high salt content, total coliforms, and slower total carbon loss initially. Soybean residues were found to be the best co-composting material to produce compost with high N, P, and K when compared with other materials due to the highest temperature, fastest total carbon loss, fastest reduction in C/N ratio, and best conservation of nutrients.

Urban nutrient recovery from fresh human urine through cultivation of Chlorella sorokiniana.

High rate food consumption in urban cities causes vast amounts of nitrogen and phosphorus used in agriculture to end up in urban wastewaters. To substantially recover these nutrients, source-separated human urine should be targeted. The present study was to investigate the feasibility of recovering nitrogen and phosphorus in urine via microalgae cultivation. In concentrated urine, urea hydrolysis and precipitation occur rapidly, making microalgal growth difficult and nutrient recovery ineffective. However, when fresh urine was added as nutrient stock for 1-day growth requirement, biomass of Chlorella sorokiniana grew from 0.44 to 0.96 g L(-1) utilising 62.64 mg L(-1) of N and 10.64 mg L(-1) of P, achieving 80.4% and 96.6% recoveries, respectively in a 1-day non-sterile cultivation cycle. Overall, microalgae grown with urine added as nutrient supplement show no signs of inferiority as compared to those grown in recipe medium BG11 in terms of mass and chlorophyll a growth rates as well as resulting lipids (36.8%) and energy contents (21.0 kJ g(-1)).

Transformation of dissolved organic matters in swine, cow and chicken manures during composting.

The changes of dissolved organic matters (DOMs) extracted from swine, cow and chicken manures were assessed by Fourier transform infrared, ultraviolet, gel permeation chromatography (GPC), excitation-emission-matrix fluorescence (EEM-FL), Biolog Eco and (1)H NMR during 60-day composting. Pumice was adopted to eliminate the disturbing of common organic bulking agents. The results showed chicken manure had the highest DOC, DTN (dissolved total nitrogen) and lowest DOC/DTN among the three manures; cow manure had the highest volatile solids, lowest DTN, slowest DOMs hydrolysis rate and the fastest bio-stabilization rate. (1)H NMR showed the decrease rates of OC band and saturated carbon chain were distinctly faster than that of olefinic and aromatic structures. The molecular size distribution of DOMs in the three manures was in the range of 1-10 kDa detected by GPC. Microbial carbon utilization capacity decreased in cow manure with composting time, but the contrast was observed in the chicken and swine manures.

Enhanced styrene recovery from waste polystyrene pyrolysis using response surface methodology coupled with Box-Behnken design.

A work applied response surface methodology coupled with Box-Behnken design (RSM-BBD) has been developed to enhance styrene recovery from waste polystyrene (WPS) through pyrolysis. The relationship between styrene yield and three selected operating parameters (i.e., temperature, heating rate, and carrier gas flow rate) was investigated. A second order polynomial equation was successfully built to describe the process and predict styrene yield under the study conditions. The factors identified as statistically significant to styrene production were: temperature, with a quadratic effect; heating rate, with a linear effect; carrier gas flow rate, with a quadratic effect; interaction between temperature and carrier gas flow rate; and interaction between heating rate and carrier gas flow rate. The optimum conditions for the current system were determined to be at a temperature range of 470-505°C, a heating rate of 40°C/min, and a carrier gas flow rate range of 115-140mL/min. Under such conditions, 64.52% WPS was recovered as styrene, which was 12% more than the highest reported yield for reactors of similar size. It is concluded that RSM-BBD is an effective approach for yield optimization of styrene recovery from WPS pyrolysis.

Enhanced gas chromatography-mass spectrometry method for bacterial polyhydroxyalkanoates analysis.

A gas chromatography-mass spectrometry method for quantification of polyhydroxyalkanoates (PHAs), containing 4-carbon to 16-carbon monomers, even in the absence of standards, was developed. Strong linear correlations existed between PHA carbon number and retention time/response factor (R(2) ≥ 0.987). Based on the correlations, high recovery values, between 100.5% and 114.3%, were obtained for PHA polymers.

A strategy for urban outdoor production of high-concentration algal biomass for green biorefining.

The present study was to investigate the feasibility of carrying out effective microalgae cultivation and high-rate tertiary wastewater treatment simultaneously in a vertical sequencing batch photobioreactor with small areal footprint, suitable for sustainable urban microalgae production. For 15 consecutive days, Chlorella sorokiniana was cultivated in synthetic wastewater under various trophic conditions. A cycle of 12-h heterotrophic: 12-h mixotrophic condition produced 0.98 g l(-1) d(-1) of algal biomass in tandem with a 94.7% removal of 254.4 mg l(-1) C-acetate, a 100% removal of 84.7 mg l(-1) N-NH4 and a removal of 15.0 mg l(-1) P-PO4. The cells were harvested via cost-effective chitosan flocculation with multiple dosing (3 times) applying established chitosan:cell ratio (1:300 w/w) and pH control (6.3-6.8). Reproducible flocculation efficiencies of greater than 99% and high-concentration algal broths (>20% solids) were achieved.

Anaerobic co-digestion of source segregated brown water (feces-without-urine) and food waste: for Singapore context.

The objective of this study was to evaluate the feasibility of anaerobic co-digestion of brown water (BW) [feces-without-urine] and food waste (FW) in decentralized, source-separation-based sanitation concept. An effort has been made to separate the yellow water (urine) and brown water from the source (using no-mix toilet) primarily to facilitate further treatment, resource recovery and utilization. Batch assay analytical results indicated that anaerobic co-digestion [BW+FW] showed higher methane yield (0.54-0.59 L CH(4)/gVS(added)) than BW or FW as a sole substrate. Anaerobic co-digestion was performed in the semi-continuously fed laboratory scale reactors viz. two-phase continuous stirred-tank reactor (CSTR) and single-stage sequencing-batch operational mode reactor (SeqBR). Initial 120 d of operation shows that SeqBR performed better in terms of organic matter removal and maximum methane production. At steady-state, CODs, CODt, VS removals of 92.0±3.0, 76.7±5.1 and 75.7±6.6% were achieved for SeqBR at 16d HRT, respectively. This corresponds to an OLR of 2-3 gCOD/L d and methane yield of about 0.41 L CH(4)/gVS(added). Good buffering capacity did not lead to accumulation of VFA, showing better process stability of SeqBR at higher loading rates. The positive findings show the great potential of applying anaerobic co-digestion of BW+FW for energy production and waste management. In addition, daily flush water consumption is reduced up to 80%. Decentralized, source-separation-based sanitation concept is expected to provide a practical solution for those countries experiencing rapid urbanization and water shortage issues, for instance Singapore.

Microbial cocktail for bioconversion of green waste to reducing sugars.

Green waste has been identified as a sustainable resource to convert into reducing sugars and subsequently for production of ethanol. In this study, enhancement of reducing sugar production from green waste by the different combination of pure strains was investigated. The best-defined microbial cocktail for high reducing sugars production, consisting of one fungus (Pseudallescheria sp. D42) and three bacteria (Microbacterium sp. F28, Tsukamurella sp. C35, and Bacillus sp. F4), was successfully constructed. The maximum reducing sugars yield by this fungal-bacterial cocktail was 165.2 mg/g-green waste within 24 h, which is approximate 10 times higher than the selected individual microbial strains. Without extraction and purification of specific enzymes, whole-cell-bioconversion by a defined microbial cocktail is proven as a potential alternative process for lignocellulose hydrolysis and reducing sugars production.

Reducing sugar-producing bacteria from guts of Tenebrio molitor Linnaeus (yellow mealworm) for lignocellulosic waste minimization.

The guts of Tenebrio Molitor Linnaeus (yellow mealworm) were used as inocula to isolate reducing sugar-producing bacteria during bioconversion of lignocellulose to reducing sugars in this study. Three carbon sources, i.e., carboxymethyl cellulose (CMC), filter paper (FP), and lignocellulosic waste (LIG), were specifically selected; and two types of culturing media (M1 and M2) were used. After 6 months of sequential cultivation, lignocellulose (i.e., polysaccharides) degradation of enrichments M1-CMC (47.5%), M1-FP (73.3%), M1-LIG (70.4%), M2-CMC (55.7%), M2-FP (73.1%) and M2-LIG (71.7%) was achieved, respectively, with incubation for 48 h. Furthermore, seven bacterial strains were successfully isolated corresponding to most of the major bands detected by denaturing gradient gel electrophoresis analysis. The maximum reducing sugars yield by the combination of Agromyces sp. C42 and Stenotrophomonas sp. A10b was 56.7 mg g·LIG(-1) of 48 h, which is approximate 2-5 times higher than the original enrichments and individual microbial strains. These findings suggest that bioconversion by microorganisms from mealworm guts has great application potential for lignocellulose hydrolysis.

Multiple syntrophic interactions in a terephthalate-degrading methanogenic consortium.

Terephthalate (TA) is one of the top 50 chemicals produced worldwide. Its production results in a TA-containing wastewater that is treated by anaerobic processes through a poorly understood methanogenic syntrophy. Using metagenomics, we characterized the methanogenic consortium inside a hyper-mesophilic (that is, between mesophilic and thermophilic), TA-degrading bioreactor. We identified genes belonging to dominant Pelotomaculum species presumably involved in TA degradation through decarboxylation, dearomatization, and modified ß-oxidation to H(2)/CO(2) and acetate. These intermediates are converted to CH(4)/CO(2) by three novel hyper-mesophilic methanogens. Additional secondary syntrophic interactions were predicted in Thermotogae, Syntrophus and candidate phyla OP5 and WWE1 populations. The OP5 encodes genes capable of anaerobic autotrophic butyrate production and Thermotogae, Syntrophus and WWE1 have the genetic potential to oxidize butyrate to CO(2)/H(2) and acetate. These observations suggest that the TA-degrading consortium consists of additional syntrophic interactions beyond the standard H(2)-producing syntroph-methanogen partnership that may serve to improve community stability.

Characterization of active microbes in a full-scale anaerobic fluidized bed reactor treating phenolic wastewater.

This study investigated the active microbial community in a full-scale granular activated carbon-anaerobic fluidized bed (GAC-AFB) reactor treating wastewater from the manufacturing of phenolic resin, using 16S rRNA-based molecular analyses. The results of cDNA from 16S rRNA revealed that Methanosaeta-related (83.9% of archaeal clones) and Syntrophorhabdaceae (formerly named Deltaproteobacteria group TA)-related (68.9% of bacterial clones) microorganisms were as the most predominant populations in the phenol-degrading GAC-AFB reactor. The high abundance of Syntrophorhabdaceae was supported by a terminal restriction fragment length polymorphism (T-RFLP) analysis, which showed that a Syntrophorhabdaceae-like fragment of 119 bp (~80% of total fragments) was the most predominant phylotype. Furthermore, fluorescence in situ hybridization (FISH) analyses suggested that Syntrophus- and Chloroflexi-like cells were also in high abundance in the GAC biofilm. A non-layered structure of microorganisms was found in the GAC biofilm, where Methanosaeta (thick filamentous), Syntrophorhabdaceae (oval-shaped), Syntrophus (small rods) and Chloroflexi (thin-filamentous) were randomly distributed with high abundance. These findings greatly improve our understanding of the diversity and distribution of microbial populations in a full-scale mesophilic bioreactor treating an actual phenol-containing waste stream.

Identification of important microbial populations in the mesophilic and thermophilic phenol-degrading methanogenic consortia.

Active mesophilic and thermophilic phenol-degrading methanogenic consortia were obtained after an 18-month acclimation and enriching process in the serum bottles, and characterized using the rRNA-based molecular approach. As revealed by cloning, fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP), these two enrichments differed greatly in the community structures. The results for the first time suggest that group TA in the Deltaproteobacteria (88.0% of EUBmix FISH-detectable bacterial cell area) and Pelotomaculum spp. in the Desulfotomaculum family (81.2%) were the predominant fermentative bacteria under mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions, respectively. These populations closely associated with mesophilic and thermophilic members of Methanosaetaceae, Methanobacteriaceae and Methanomicrobiales to mineralize phenol as the sole carbon substrate to carbon dioxide and methane. Moreover, these two enrichments could mineralize terephthalate and benzoate. During benzoate degradation in the mesophilic enrichment, a shift in the predominant bacterial population from Deltaproteobacteria group TA to Syntrophus spp. was observed, suggesting Syntrophus-related spp. could have a higher substrate affinity for benzoate. FISH further revealed that member of the Deltaproteobacteria group TA represented more than 68.3% of EUBmix FISH-detectable bacterial cell area in a full-scale mesophilic bioreactor treating phenol-containing wastewaters.

Microbial community structure in a thermophilic anaerobic hybrid reactor degrading terephthalate.

A thermophilic terephthalate-degrading methanogenic consortium was successfully enriched for 272 days in an anaerobic hybrid reactor, and the microbial structure was characterized using terminal RFLPs, clone libraries and fluorescence in-situ hybridization with rRNA-targeted oligonucleotide probes. All the results suggested that Methanothrix thermophila-related methanogens, Desulfotomaculum-related bacterial populations in the Gram-positive low-G + C group, and OP5-related populations were the key members responsible for terephthalate degradation under thermophilic methanogenic conditions except during periods when the reactor experienced heat shock and pump failure. These perturbations caused a significant shift in bacterial population structure in sludge samples taken from the sludge bed but not from the surface of the packing materials. After system recovery, many other bacterial populations emerged, which belonged mainly to the Gram-positive low-G + C group and Cytophaga-Flexibacter-Bacteroides, as well as beta-Proteobacteria, Planctomycetes and Nitrospira. These newly emerged populations were probably also capable of degrading terephthalate in the hybrid system, but were out-competed by those bacterial populations before perturbations.