Ultra-rapid development of 'solid' aerobic granular sludge by stable transition/filling of inoculated 'hollow' mycelial pellets in hypersaline wastewater.

To overcome the long start-up period in cultivating aerobic granular sludge (AGS) under hypersaline environment, mycelial pellets (MPs) of halotolerant fungus Cladosporium tenuissimum NCSL-XY8 were inoculated to try to realize the ultra-rapid development of salt-tolerant AGS by stable transition of 'hollow' MPs into 'solid' AGS without apparent fragmentation. The granules directly met the standard of AGS after inoculating MPs (Day 0), and it basically satisfied relatively strict standards of AGS (SVI30 < 50 mL/g, D50 > 300 µm, D10 > 200 µm and SVI30/SVI5 > 0.9) under anaerobic/aerobic mode during whole cultivation processes. Microstructure of the granular cross section clarified that MPs with hollow/loose inner layer transitioned into solid/dense AGS under anaerobic/aerobic mode within 7 days, while formed skin-like floating pieces and unstable double-layer hollow granules under aerobic mode. Organics removal reached relatively stable within 13 days under anaerobic/aerobic mode, 6 days faster than aerobic mode. This study provided a strategy for ultra-rapid and stable development of AGS, which showed the shortest granulation period in various AGS-cultivation strategies.

Influence of aeration modes and DO on simultaneous nitrification and denitrification in treatment of hypersaline high-strength nitrogen wastewater using sequencing batch biofilm reactor (SBBR).

Sequencing batch biofilm reactor (SBBR) has the potential to treat hypersaline high-strength nitrogen wastewater by simultaneous nitrification-denitrification (SND). Dissolved oxygen (DO) and aeration modes are major factors affecting pollutant removal. Low DO (0.35-3.5 mg/L) and alternative anoxic/aerobic (A/O) mode are commonly used for municipal wastewater treatment, however, the appropriate DO concentration and operation mode are still unknown under hypersaline environment because of the restricted oxygen transfer in denser extracellular polymeric substances (EPS) barrier and the decreased carbon source consumption during the anoxic phase. Herein, two SBBRs (R1, fully aerobic mode; R2, A/O mode) were used for the treatment of hypersaline high-strength nitrogen wastewater (200 mg/L NH4+-N, COD/N of 3 and 3% salinity). The results showed that the relatively low DO (2 mg/L) could not realize effective nitrification, while high DO (4.5 mg/L) evidently increased nitrification efficiency by enhancing oxygen transfer in denser biofilm that was stimulated by high salinity. A stable SND was reached 16 days faster with a ∼10% increase of TN removal under A/O mode. Mechanism analysis found that denser biofilm with coccus and bacillus were present in A/O mode instead of filamentous microorganisms, with the secretion of more EPS. Corynebacterium and Halomonas were the dominant genera in both SBBRs, and HN-AD process might assist partial nitrification-denitrification (PND) for highly efficient TN removal in biofilm systems. By using the appropriate operation mode and parameters, the average NH4+-N and TN removal efficiency could respectively reach 100% and 70.8% under the NLR of 0.2 kg N·m-3·d-1 (COD/N of 3), which was the highest among the published works using SND-based SBBRs in treatment of saline high-strength ammonia nitrogen (low COD/N) wastewater. This study provided new insights in biofilm under hypersaline stress and provided a solution for the treatment of hypersaline high-strength nitrogen (low COD/N) water.

Third-generation D-lactic acid production using red macroalgae Gelidium amansii by co-fermentation of galactose, glucose and xylose.

Macroalgae biomass has been considered as a promising renewable feedstock for lactic acid production owing to its lignin-free, high carbohydrate content and high productivity. Herein, the D-lactic acid production from red macroalgae Gelidium amansii by Pediococcus acidilactici was investigated. The fermentable sugars in G. amansii acid-prehydrolysate were mainly galactose and glucose with a small amounts of xylose. P. acidilactici could simultaneously ferment the mixed sugars of galactose, glucose and xylose into D-lactic acid at high yield (0.90 g/g), without carbon catabolite repression (CCR). The assimilating pathways of these sugars in P. acidilactici were proposed based on the whole genome sequences. Simultaneous saccharification and co-fermentation (SSCF) of the pretreated and biodetoxified G. amansii was also conducted, a record high of D-lactic acid (41.4 g/L) from macroalgae biomass with the yield of 0.34 g/g dry feedstock was achieved. This study provided an important biorefinery strain for D-lactic acid production from macroalgae biomass.

Upgrading dry acid pretreatment by post-hydrolysis for carbon efficient conversion of lignocellulose.

Dry acid pretreatment (DAP) as a promising process for industrial biorefinery provide an efficient bioconversion of cellulose without free wastewater, although the partial xylan and lignin degrade to inhibitors or recondense. A biorefinery strategy for carbon efficient conversion of lignocellulose into bioethanol, xylose, and reactive lignin was developed by upgrading DAP with post-hydrolysis. The results showed that lignocellulose after mild DAP (175 °C, acid dosage of 15 mg/g dry material) obtained higher xylan recovery and lower inhibitors than that of general DAP. Subsequently, post-hydrolysis, simultaneous saccharification and ethanol fermentation were performed at solids loading of 20 wt% without detoxification and sterilization, resulting in xylose and ethanol yield of 71.8 % and 67.6 %. The fractionated lignin presented more reactive ß-aryl ether linkages and less condensation than that from DAP. 66 % of lignocellulose carbon was recovered as ethanol, xylose and reactive lignin. This upgrading biorefinery strategy provided an easy-to-operate process for integrated utilization of lignocellulose.

Rapid, stable, and highly-efficient development of salt-tolerant aerobic granular sludge by inoculating magnetite-assisted mycelial pellets.

Long cultivation time hinders the industrial applications of aerobic granular sludge (AGS) in treatment of hypersaline wastewater. Mycelial pellets (MPs) have been used to efficiently strengthen the flocculent sludge aggregation and accelerate the formation of AGS. However, the MPs-based AGS was easily crushed or fragmented into several small pieces/granules that brought the uncertainty and extended the transition process to form mature AGS. In this study, magnetite was used to strengthen MPs (halotolerant fungus Cladosporium tenuissimum NCSL-XY8), and co-culture and adsorption type of magnetite-assisted mycelial pellets (CMMPs and AMMPs) were prepared and used for acceleration of salt-tolerant aerobic granular sludge (SAGS) cultivation under 3% salinity conditions. Compared to inoculating MPs, the inoculation of either CMMPs or AMMPs could stably transition to mature SAGS without evident fragmentation, which obviously increased the certainty and stability of SAGS formation. Also, highly-efficient simultaneous nitrogen and carbon removal (∼98% TOC and ∼80% TN removal) could be reached in 8 days. Typically, the granules maintained perfect characteristics (D50 > 1300 µm, D10 > 350 µm, SVI30 < 45 mL/g, and SVI30/SVI5 = 1.0) during the whole cultivation/transition processes (Day 0-55) by using the inoculum of CMMPs. ITS rDNA sequencing revealed the inoculated fungus Cladosporium tenuissimum played key roles in the formation of SAGS. All the phenomena indicated the rapid, stable, and highly-efficient start-up of SAGS could be successfully realized by inoculating CMMPs.

A systemic evaluation of aerobic granular sludge among granulation, operation, storage, and reactivation processes in an SBR.

As a biological promising wastewater treatment technology, aerobic granular sludge (AGS) technology had been widely studied in sequencing batch reactors (SBRs) for the decades. Presently, the whole processes of its granulation, long-term operation, storage, and reactivation have not been thoroughly evaluated, and also the relationships among microbial diversity, granular size, and characteristics were still not that clear. Hence, they were systematically evaluated in an AGS-SBR in this work. The results demonstrated that Proteobacteria and Bacteroidetes were the dominant phyla, Flavobacterium, Acinetobacter, Azoarcus, and Chryseobacterium were the core genera with discrepant abundances in diverse stages or granular size. Microbial immigration was significant in various stages due to microbial diversity had a line relationship with COD/MLVSS ratio (R2 = 0.367). However, microbial diversity had no line relationship with granular size (R2 = 0.001), indicating the microbial diversity in different-sized AGS was similar, although granular size had a line relationship with settleability (R2 = 0.978). Overall, compared to sludge traits (e.g., sludge size, settleability), COD/MLVSS played a key role on microbial evolution. This study revealed the relationships between granule characteristics and microbial community, and contributed to the future AGS-related studies.

Potential of direct granulation and organic loading rate tolerance of aerobic granular sludge in ultra-hypersaline environment.

Salt-tolerant aerobic granular sludge (SAGS) technology has shown potentials in the treatment of ultra-hypersaline high-strength organic wastewater. However, the long granulation period and salt-tolerance acclimation period are still bottlenecks that hinder SAGS applications. In this study, "one-step" development strategy was used to try to directly cultivate SAGS under 9% salinity, and the fastest cultivation process was obtained under such high salinity compared to the previous papers with the inoculum of municipal activated sludge without bioaugmentation. Briefly, the inoculated municipal activated sludge was almost discharged on Day 1-10, then fungal pellets appeared and it gradually transitioned to mature SAGS (particle size of ∼4156 µm and SVI30 of 57.8 mL/g) from Day 11 to Day 47 without fragmentation. Metagenomic revealed that fungus Fusarium played key roles in the transition process probably because it functioned as structural backbone. RRNPP and AHL-mediated systems might be the main QS regulation systems of bacteria. TOC and NH4+-N removal efficiencies maintained at ∼93.9% (after Day 11) and ∼68.5% (after Day 33), respectively. Subsequently, the influent organic loading rate (OLR) was stepwise increased from 1.8 to 11.7 kg COD/m3·d. It was found that SAGS could maintain intact structure and low SVI30 (< 55 mL/g) under 9% salinity and the OLR of 1.8-9.9 kg COD/m3·d with adjustment of air velocity. TOC and NH4+-N (TN) removal efficiencies could maintain at ∼95.4% (below OLR of 8.1 kg COD/m3·d) and ∼84.1% (below nitrogen loading rate of 0.40 kg N/m3·d) in ultra-hypersaline environment. Halomonas dominated the SAGS under 9% salinity and varied OLR. This study confirmed the feasibility of direct aerobic granulation in ultra-hypersaline environment and verified the upper OLR boundary of SAGS in ultra-hypersaline high-strength organic wastewater treatment.

Aerobic granular sludge treating hypersaline wastewater: Impact of pH on granulation and long-term operation at different organic loading rates.

pH is one of the major parameters that influence the granulation and long-term operation of aerobic granular sludge (AGS). In hypersaline wastewater, the impact of pH on granulation and the extent of organic loading rate (OLR) that AGS can withstand under different pH are still not clear. In this study, AGS was cultivated at 3% salinity in three sequencing batch reactors with influent pH values of 5.0, 7.0, and 9.0, respectively, and the OLR was stepwise increased from 2.4 to 16.8 kg COD/m3·d after the granules maturation. The results showed the satisfactory granulation and organic removal under different influent pH conditions, in which the granulation was completed on day 43, 23, and 23, respectively. Neutral influent was the most appropriate for development of salt-tolerant aerobic granular sludge (SAGS), while acidic environment induced the formation of fluffy filamentous granules, and alkaline environment weakened the granule stability. Metagenomic analysis revealed the similar microbial community of neutral and alkaline conditions, with the predominance of genus Paracoccus_f__Rhodobacteraceae. While in acidic environment, fungus Fusarium formed the skeleton of filamentous granules and functioned as the carrier of bacteria including Azoarcus and Pararhodobacter. With the elevation of OLR, SAGSs were found to maintain the compact structure under OLRs of 2.4, 7.2, and 2.4 kg COD/m3·d, and obtain high TOC removal (>95.0%) under OLRs of 7.2, 14.4, and 14.4 kg COD/m3·d, respectively. For hypersaline high-strength organic wastewater, satisfactory TOC removal could also be obtained at broad pH ranges (5.0-9.0), in which neutral environment was the most suitable and acidic environment was the worst. This study contributed to a better understanding of SAGS granulation and treatment of hypersaline high-strength organic wastewater with different pH values.

Enhanced cellulosic d-lactic acid production from sugarcane bagasse by pre-fermentation of water-soluble carbohydrates before acid pretreatment.

After several rounds of milling process for sugars extraction from sugarcane, certain amounts of water-soluble carbohydrates (WSC) still remain in sugarcane bagasse. It is a bottleneck to utilize WSC in sugarcane bagasse biorefinery, since these sugars are easily degraded into inhibitors during pretreatment. Herein, a simple pre-fermentation step before pretreatment was conducted, and 98 % of WSC in bagasse was fermented into d-lactic acid. The obtained d-lactic acid was stably preserved in bagasse and 5-hydroxymethylfurfural (HMF) generation was sharply reduced from 46.0 mg/g to 6.2 mg/g of dry bagasse, after dilute acid pretreatment. Consequently, a higher d-lactic acid titer (57.0 g/L vs 33.2 g/L) was achieved from the whole slurry of the undetoxified and pretreated sugarcane bagasse by one-pot simultaneous saccharification and co-fermentation (SSCF), with the overall yield of 0.58 g/g dry bagasse. This study gave an efficient strategy for enhancing lactic acid production using the lignocellulosic waste from sugar industry.

One-pot d-lactic acid production using undetoxified acid-pretreated corncob slurry by an adapted Pediococcus acidilactici.

Inhibitor tolerance is still a bottleneck for lactic acid bacteria in lignocellulose biorefinery, while it is hard to obtain one engineered strain with strong tolerance to all inhibitors. Herein, a robust adapted d-lactic acid producing strain Pediococcus acidilactici XH11 was obtained by 111 days' long-term adaptive evolution in undetoxified corncob prehydrolysates. The adapted strain had higher inhibitors tolerance compared to the parental strain, primarily due to its increased conversion capacities of four typical aldehyde inhibitors (furfural, HMF, vanillin, and 4-hydroxybenzaldehyde). One-pot simultaneous saccharification and co-fermentation was successfully achieved using the whole slurry of acid-pretreated corncob without solid-liquid separation and detoxification, by applying the adapted P. acidilactici XH11. Finally, 61.9 g/L of d-lactic acid was generated after 96 h' fermentation (xylose conversion of 89.9 %) with the overall yield of 0.48 g/g dry corncob. This study gave an important option for screening of industrial strains in cellulosic lactic acid production processes.

Engineering Corynebacterium glutamicum for synthesis of poly(3-hydroxybutyrate) from lignocellulose biomass.

Lignocellulose is the only feasible carbohydrates feedstock for commercial scale and carbon neutral production of poly(3-hydroxybutyrate) (PHB) biopolymer by its great abundance and availability. Microbial cell factories for fermentative PHB synthesis are highly restricted by the growth suppression of inhibitors from lignocellulose pretreatment. This study targeted a potential PHB-producing cell factory Corynebacterium glutamicum owing to its strong inhibitors tolerance. A systematic metabolic engineering was conducted starting with the stable PHB synthesis pathway construction from glucose and xylose, followed by the enhancement of PHB synthesis on PHA synthase activity and stability, cell morphology modification, and growth factors regulation. The relocation of the PHA synthase on the cell membrane guided by secrete signal peptides and cell membrane display motifs increased the PHB content by 2.4 folds. Excessive nitrogen preferentially promoted the PHB synthesis capacity and resulted in the PHB content increased by 13.3 folds. Modification of the genes responsible for cell division changed the cell morphology but the cell size was not enlarged to a PHB accumulation favorable environment. The metabolic engineering of C. glutamicum resulted in a high fermentative production of PHB using wheat straw as feedstock. This study provided an important microbial cell factory choice for PHB production using lignocellulose feedstock.

Formulating a fully converged biorefining chain with zero wastewater generation by recycling stillage liquid to dry acid pretreatment operation.

Huge wastewater generation is the major challenge of biorefinery technology for production of cellulosic ethanol. This study designed and verified a method for completely recycling of wastewater stream (the stillage liquid from the beer column) in cellulosic ethanol production by dry biorefining processing. When the stillage liquid was directly recycled to dry acid pretreatment operation, ethanol production gradually reduced after two recycles primarily because the inorganic compounds accumulated by around 139%. To ultimately solve this technical barrier, the stillage liquid was evaporated and condensed into distillated water, then recycled to the pretreatment for complete dry biorefining process. This strategy supported a stable cellulosic ethanol production, and the overall mass and heat balance confirmed that only 65% of the lignin residue consumption was used for wastewater evaporation with 35% surplus for electricity generation. This study provided a fully converged biorefining process with a closed-loop wastewater recycling.

Acidic deep eutectic solvent assisted isolation of lignin containing nanocellulose from thermomechanical pulp.

Nanocellulose is a promising material but its isolation generally requires unrecyclable hazardous chemicals and high energy consumption and its overall yield is low due to the use of high purity cellulose as precursor. In order to overcome these shortcomings, in this study, thermomechanical pulp (TMP) was investigated as a precursor for isolating lignin containing nanocellulose (LNC) using an environmentally friendly acidic deep eutectic solvent (DES) pre-treatment. Flat "ribbon" like LNCs (around 7.1 nm wide, 3.7 nm thick) with uniformly distributed lignin nanoparticles of 20-50 nm in diameter were successfully obtained at 57 % yield under optimum pre-treatment conditions (90 °C, 6 h, 1:1 oxalic acid dihydrate to choline chloride ratio). The LNCs exhibit cellulose Iß structure, high lignin content (32.6 %), and high thermal stability (Tmax of 358 °C). In general, green acidic DES pre-treatment has shown high efficiency in converting high lignin content biomass into value-added LNC, which benefits both lignocellulose utilization and environmental protection.

Facilitation of l-Lactic Acid Fermentation by Lignocellulose Biomass Rich in Vitamin B Compounds.

Vitamins are important nutrients for many fermentations, but they are generally costly. Agricultural lignocellulose biomass contains considerable amounts of vitamin B compounds, but these water-soluble vitamins are easily lost into wastewater discharge during pretreatment or detoxification of lignocellulose in biorefinery processes. Here, we showed that the dry acid pretreatment and biodetoxification process allowed the preservation of significant amounts of vitamin B, which promoted l-lactic acid fermentation efficiency significantly. Supplementation with specific vitamin B compounds, VB3 and VB5, into corn stover hydrolysate led to further increases of cellulosic l-lactic acid yield and fermentation rates. This study provided a new solution for the enhancement of biorefinery fermentation efficiency by using vitamin B compounds in lignocellulose biomass.

Microbial extraction of biotin from lignocellulose biomass and its application on glutamic acid production.

Biotin (vitamin B7) is an important nutrient for various fermentations. It is abundant in agricultural lignocellulose biomass and maintains stable in biorefinery processing chain including acid pretreatment, biodetoxification and saccharification. Here we show a microbial extraction of biotin from biotin-rich corn leaves hydrolysate. Corynebacterium glutamicum was found to have the highest biotin uptake capacity among different biotin auxotrophic microorganisms, and it was further significantly increased by overexpressing the bioYMN gene cluster encoding biotin transporter. Finally 250 folds greater biotin was extracted by recombinant C. glutamicum (303.8 mg/kg dry cell) from virgin corn leaves (1.2 mg/kg), which was far higher than that in commonly used fermentation additives including yeast extract (∼2 mg/kg), molasses (∼1 mg/kg) and corn steep liquor (∼0.75 mg/kg). The biotin extracted from corn leaves was successfully applied to glutamic acid fermentation. This is the first report on microbial extraction of biotin from lignocellulose biomass and fermentation promotion application.

Improving cellulosic ethanol fermentation efficiency by converting endogenous water-soluble carbohydrates into citric acid before pretreatment.

Water-soluble carbohydrates in raw lignocellulosic biomass are converted into hydroxymethylfurfural (HMF) in the acid-based pretreatment, thus increasing the detoxification intensity and reducing the fermentation efficiency of cellulosic ethanol. Therefore, reducing water-soluble carbohydrates in raw corn stover is crucially important to reduce the inhibitors' generation and improve the ethanol fermentation efficiency. In this study, aerobic solid-state fermentation of corn stover by inoculating Aspergillus niger spores converted 83% of the endogenous water-soluble carbohydrates into citric acid, leading to the decrease of 41% of HMF generation and 8% of sulfuric acid usage during the dry acid pretreatment. The reduced inhibitor generation improved the ethanol fermentability by 11% more ethanol than that of the corn stover without water-soluble carbohydrates' removal. This suggests that the removal of the water-soluble carbohydrates before pretreatment significantly reduced the inhibitors' generation in pretreatment and improved the fermentation efficiency of cellulosic ethanol.

An Approach of Utilizing Water-Soluble Carbohydrates in Lignocellulose Feedstock for Promotion of Cellulosic l-Lactic Acid Production.

Agricultural lignocellulose biomass generally contains certain amounts of water-soluble carbohydrates (WSC) such as glucose, fructose, or sucrose. These sugars are generally degraded in pretreatment at high temperature or discharged with wastewater in a detoxification process. This study proposed an approach of utilizing frequently ignored water-soluble carbohydrates for promotion of cellulosic l-lactic acid production. A simple solid state fermentation was performed during a corn stover storage period to convert the sugars into l-lactic acid and then a dry biorefining technology was applied to convert cellulose and hemicellulose fractions into the same l-lactic acid product. The 5-hydroxymethylfurfural (HMF) formation in pretreatment was significantly reduced and the consequent biodetoxification time was shortened. l-Lactic acid production was increased from 130.2 g/L to 139.0 g/L, and the minimum l-lactic acid selling price was reduced by 5.9%. This study provided an important option of biorefinery processing technology for production of value added biochemicals.

Oxidative production of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth by Gluconobacter oxydans.

An oxidative production process of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth was designed, experimentally investigated, and evaluated. Dry dilute acid pretreated and biodetoxified corn stover was simultaneously saccharified and fermented into 59.80g/L of ethanol (no xylose utilization). 65.39g/L of xylose was obtained in the distillation stillage without any concentrating step after ethanol was distillated. Then the xylose was completely converted into 66.42g/L of xylonic acid by Gluconobacter oxydans. The rigorous Aspen Plus modeling shows that the wastewater generation and energy consumption was significantly reduced comparing to the previous xylonic acid production process using xylose in pretreatment liquid. This study provided a practical process option for xylonic acid production from lignocellulose feedstock with significant reduction of wastewater and energy consumption.