Insoluble solids at high concentrations repress yeast's response against stress and increase intracellular ROS levels.

Lignocellulosic ethanol production requires high substrate concentrations for its cost-competitiveness. This implies the presence of high concentrations of insoluble solids (IS) at the initial stages of the process, which may limit the fermentation performance of the corresponding microorganism. The presence of 40-60% IS (w/w) resulted in lower glucose consumption rates and reduced ethanol volumetric productivities of Saccharomyces cerevisiae F12. Yeast cells exposed to IS exhibited a wrinkled cell surface and a reduced mean cell size due to cavity formation. In addition, the intracellular levels of reactive oxygen species (ROS) increased up to 40%. These ROS levels increased up to 70% when both lignocellulose-derived inhibitors and IS were simultaneously present. The general stress response mechanisms (e.g. DDR2, TPS1 or ZWF1 genes, trehalose and glycogen biosynthesis, and DNA repair mechanisms) were found repressed, and ROS formation could not be counteracted by the induction of the genes involved in repairing the oxidative damage such as glutathione, thioredoxin and methionine scavenging systems (e.g. CTA1, GRX4, MXR1, and TSA1; and the repression of cell cycle progression, CLN3). Overall, these results clearly show the role of IS as an important microbial stress factor that affect yeast cells at physical, physiological, and molecular levels.

Lactobacillus pentosus CECT 4023 T co-utilizes glucose and xylose to produce lactic acid from wheat straw hydrolysate: Anaerobiosis as a key factor.

Lactic acid is a versatile chemical that can be produced via fermentation of lignocellulosic materials. The heterolactic strain Lactobacillus pentosus CECT 4023 T, that can consume glucose and xylose, was studied to produce lactic acid from steam exploded wheat straw prehydrolysate. The effect of temperature and pH on bacterial growth was analyzed. Besides, the effect of oxygen on lactic acid production was tested and fermentation yields were compared in different scenarios. This strain showed very high tolerance to the inhibitors contained in the wheat straw prehydrolysate. The highest lactic acid yields based on present sugar, around 0.80 g g-1 , were obtained from glucose in presence of 25%, 50%, and 75% v v-1 of prehydrolysate in strict anaerobiosis. Lactic fermentation of wheat straw hydrolysate obtained after enzymatic hydrolysis of the prehydrolysate yielded 0.39 g of lactic acid per gram of released sugars, which demonstrated the high potential of L. pentosus to produce lactic acid from hemicellulosic hydrolysates. Results presented herein not only corroborated the ability of L. pentosus to grow using mixtures of sugars, but also demonstrated the suitability of this strain to be applied as an efficient lactic acid producer in a lignocellulosic biorefinery approach. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2739, 2019.

Volatile fatty acids production from protease pretreated Chlorella biomass via anaerobic digestion.

Volatile fatty acids (VFAs) produced via anaerobic digestion (AD) are regarded as a low cost production process of building blocks of interest for the chemical industry. In this study, VFAs and methane production were assessed in batch reactors at different temperature ranges (psychrophilic 25°C, mesophilic 35°C, thermophilic 50°C) and different pH values (5.5 and 7.5) using protease pretreated Chlorella sp. biomass as substrate. Acetic acid and propionic acid were the most abundant products (up to 73% of the total VFAs) during the first days independently of the conditions. VFAs concentration decreased over time as methane was produced after a lag phase of 7-10 days. Results showed that best conditions for VFAs production were mesophilic temperature ranges (35°C) at neutral initial pH values (7.5), and psychrophilic temperature ranges (25°C) at low initial pH values (5.5) which resulted in a conversion of the initial COD into VFAs of 48%, respectively. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1363-1369, 2018.

Biochemical methane potential of microalgae biomass using different microbial inocula.

BACKGROUND: Microalgae biomass is regarded as a potential feedstock for bioenergy purposes through anaerobic digestion (AD). Even though AD is a well-proven technology, the use of new feedstocks requires in-depth studies. A lot of research has been conducted assessing methane yield without paying attention to the anaerobic microbiome and their activities. For such a goal, the present investigation was designed to link methane yield to those two later sludge characteristics. In this sense, different anaerobic sources were tested, namely adapted to microalgae biomass and adapted to sewage sludge. RESULTS: Despite the registered differences for the anaerobic microbiome analysis and specific methane activities towards model substrates, sludge adapted to digest sewage sludge did not affect the methane yield of Chlorella sorokiniana and Scenedesmus sp. Opposite to that, sludge samples adapted to digest microalgae exhibited a concomitant increase in methane yield together with increasing digestion temperatures. More specifically, the values attained were 63.4 ± 1.5, 79.2 ± 3.1 and 108.2 ± 1.9 mL CH4 g COD in-1 for psychrophilic, mesophilic and thermophilic digestions, respectively. While psycro- and mesophilic digestion supported similar yields (most probably linked to their anaerobic microbiome resemblance), the values attained for thermophilic digestion evidenced the usefulness of having a highly specific microbiome. The relative abundance of Firmicutes, particularly Clostridia, and Proteobacteria together with an important abundance of hydrogenotrophic methanogens was highlighted in this inoculum. CONCLUSION: Overall, this study showed that working with tailored anaerobic microbiome could help avoiding pretreatments devoted to methane yield enhancement.

Integrated production of second generation ethanol and lactic acid from steam-exploded elephant grass.

Elephant grass was subjected to steam explosion to enhance cellulose accessibility and convert it into ethanol. After catalyzed pretreatment at 190 °C for 5 min, enzymatic hydrolysis was carried out using high rate of solid loading combined with different enzyme dosages. Assays employing 20% (w/v) solids loading and an enzyme dosage of 20 FPU g-1 substrate led to a yield of 86.02 g glucose released per 100 g potential glucose in the water insoluble solids. This condition was selected to carry out the simultaneous saccharification and fermentation procedure through S. cerevisiae CAT-1, producing 42.25 g L-1 ethanol with a yield of 74.57% regard to the maximum theoretical. The liquor containing C5 and C6-sugars was successfully converted into lactic acid using L. buchneri NRRL B-30929, resulting in 13.35 g L-1 with a yield of 68.21% in relation to the maximum theoretical.

Efficient Anaerobic Digestion of Microalgae Biomass: Proteins as a Key Macromolecule.

Biogas generation is the least complex technology to transform microalgae biomass into bioenergy. Since hydrolysis has been pointed out as the rate limiting stage of anaerobic digestion, the main challenge for an efficient biogas production is the optimization of cell wall disruption/hydrolysis. Among all tested pretreatments, enzymatic treatments were demonstrated not only very effective in disruption/hydrolysis but they also revealed the impact of microalgae macromolecular composition in the anaerobic process. Although carbohydrates have been traditionally recognized as the polymers responsible for the low microalgae digestibility, protease addition resulted in the highest organic matter solubilization and the highest methane production. However, protein solubilization during the pretreatment can result in anaerobic digestion inhibition due to the release of large amounts of ammonium nitrogen. The possible solutions to overcome these negative effects include the reduction of protein biomass levels by culturing the microalgae in low nitrogen media and the use of ammonia tolerant anaerobic inocula. Overall, this review is intended to evidence the relevance of microalgae proteins in different stages of anaerobic digestion, namely hydrolysis and methanogenesis.

Effect of microalgae storage conditions on methane yields.

During the last decade, a lot of research has been focused on identifying the methane yields achievable when using microalgae biomass (fresh and pretreated) as a substrate in anaerobic digestion. Encountered differences are frequently attributed to the different microalgae strains (cell walls and macromolecular profiles) or the different metabolic activities of anaerobic sludge used as inoculum. Nevertheless, under the hypothesis that the state of microalgae upon biomass storage may also play a significant role, this study was designed to evaluate the effect of biomass processing and storage on methane yields and hydrolysis kinetics in batch mode assays. Slight changes in the macromolecular profile distribution of the different tested biomass were observed. Regardless of the time that the biomass was stored, results revealed that frozen biomass doubled the hydrolysis constant and enhanced methane yield by 1.56-fold compared to fresh microalgae biomass (82.4 mL CH4 g COD in-1). Similar enhancement was obtained with the freeze-dried biomass, and slightly lower values were obtained (1.34-fold) for the biomass kept at 4 °C longer than a week. Likewise, the semi-continuously operated reactor fed with microalgae biomass stored for 28 days at 4 °C did not show any effect in terms of methane production, although nitrogen mineralization was higher than expected. Remarkably, the initial stage of the biomass should be carefully considered for comparison purposes with the available literature on batch mode assays. This study highlights the importance of considering how the biomass is stored before the anaerobic digestion process to avoid misleading conclusions.

Acclimation to extremely high ammonia levels in continuous biomethanation process and the associated microbial community dynamics.

Acclimatized anaerobic communities to high ammonia levels can offer a solution to the ammonia toxicity problem in biogas reactors. In the current study, a stepwise acclimation strategy up to 10g NH4+-N L-1, was performed in mesophilic (37±1°C) continuously stirred tank reactors. The reactors were co-digesting (20/80 based on volatile solid) cattle slurry and microalgae, a protein-rich, 3rd generation biomass. Throughout the acclimation period, methane production was stable with more than 95% of the uninhibited yield. Next generation 16S rRNA gene sequencing revealed a dramatic microbiome change throughout the ammonia acclimation process. Clostridium ultunense, a syntrophic acetate oxidizing bacteria, increased significantly alongside with hydrogenotrophic methanogen Methanoculleus spp., indicating strong hydrogenotrophic methanogenic activity at extreme ammonia levels (>7g NH4+-N L-1). Overall, this study demonstrated for the first time that acclimation of methanogenic communities to extreme ammonia levels in continuous AD process is possible, by developing a specialised acclimation AD microbiome.

Microbial communities of biomethanization digesters fed with raw and heat pre-treated microalgae biomasses.

Microalgae biomasses are considered promising feedstocks for biofuel and methane productions. Two Continuously Stirred Tank Reactors (CSTR), fed with fresh (CSTR-C) and heat pre-treated (CSTR-T) Chlorella biomass were run in parallel in order to determine methane productions. The methane yield was 1.5 times higher in CSTR-T with regard to CSTR-C. Aiming to understand the microorganism roles within of the reactors, the sludge used as an inoculum (I), plus raw (CSTR-C) and heat pre-treated (CSTR-T) samples were analyzed by high-throughput pyrosequencing. The bacterial communities were dominated by Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes. Spirochaetae and Actinobacteria were only detected in sample I. Proteobacteria, mainly Alfaproteobacteria, were by far the dominant phylum within of the CSTR-C bioreactor. Many of the sequences retrieved were related to bacteria present in activated sludge treatment plants and they were absent after thermal pre-treatment. Most of the sequences affiliated to the Bacteroidetes were related to uncultured groups. Anaerolineaceae was the sole family found of the Chloroflexi phylum. All of the genera identified of the Firmicutes phylum carried out macromolecule hydrolysis and by-product fermentation. The proteolytic bacteria were prevalent over the saccharolytic microbes. The percentage of the proteolytic genera increased from the inoculum to the CSTR-T sample in a parallel fashion with an available protein increase owing to the high protein content of Chlorella. To relate the taxa identified by high-throughput sequencing to their functional roles remains a future challenge.

Ammonia tolerant inocula provide a good base for anaerobic digestion of microalgae in third generation biogas process.

This study investigated the ability of an ammonia-acclimatized inoculum to digest efficiently protein-rich microalgae for continuous 3rd generation biogas production. Moreover, we investigated whether increased C/N ratio could alleviate ammonia toxicity. The biochemical methane potential (BMP) of five different algae (Chlorella vulgaris)/manure (cattle) mixtures showed that the mixture of 80/20 (on VS basis) resulted in the highest BMP value (431mLCH4 gVS-1), while the BMP of microalgae alone (100/0) was 415mLCH4 gVS-1. Subsequently, anaerobic digestion of those two substrates was tested in continuous stirred tank reactors (CSTR). Despite of the high ammonium levels (3.7-4.2g NH4+-NL-1), CSTR reactors using ammonia tolerant inoculum resulted in relatively high methane yields (i.e. 77.5% and 84% of the maximum expected, respectively). These results demonstrated that ammonia tolerant inocula could be a promising approach to successfully digest protein-rich microalgae and achieve a 3rd generation biogas production.

Comparison of Chlorella vulgaris and cyanobacterial biomass: cultivation in urban wastewater and methane production.

Anaerobic digestion of microalgae is hampered by its complex cell wall. Against this background, cyanobacteria cell walls render this biomass as an ideal substrate for overcoming this drawback. The aim of the present study was to compare the growth of two cyanobacteria (Aphanizomenon ovalisporum and Anabaena planctonica) and a microalga (Chlorella vulgaris) in urban wastewater when varying the temperature (22, 27 and 32 °C). Cyanobacterial optimal growth for both strains was attained at 22 °C, while C. vulgaris did not show remarkable differences among temperatures. For all the microorganisms, ammonium removal was higher than phosphate. Biomass collected was subjected to anaerobic digestion. Methane yield of C. vulgaris was 184.8 mL CH4 g COD in(-1) while with A. ovalisporum and A. planctonica the methane production was 1.2- and 1.4-fold higher. This study showed that cyanobacteria growth rates could be comparable to microalgae while presenting the additional benefit of an increased anaerobic digestibility.

Enzymatic pretreatment of Chlorella vulgaris for biogas production: Influence of urban wastewater as a sole nutrient source on macromolecular profile and biocatalyst efficiency.

Two biocatalysts, namely carbohydrases and proteases, were assessed for organic matter solubilisation and methane yield enhancement of microalgae biomass. This study evidenced Chlorella vulgaris carbohydrate accumulation (40% on VSS basis) when grown in urban wastewater. Despite of the carbohydrate prevailing fraction, protease pretreatment showed higher organic matter hydrolysis efficiency (54%). Microscopic observation revealed that carbohydrases affected slightly the cell wall while protease was not selective to wall constituents. Raw and pretreated biomass was digested at 1.5 kg tCOD m(-3) day(-1) organic loading rate (OLR1) and 20 days hydraulic retention time (HRT). The highest methane yield (137 mL CH4 g COD in(-1)) was achieved in the reactor fed with protease pretreated C. vulgaris. Additionally, anaerobic digestion was conducted at OLR2 (3 kg tCOD m(-3) day(-1)) and HRT (15 days). When compared to raw biomass, methane yield increased 5- and 6.3-fold at OLR1 and OLR2, respectively. No inhibitors were detected during the anaerobic digestion.

Phenols and lignin: Key players in reducing enzymatic hydrolysis yields of steam-pretreated biomass in presence of laccase.

Phenols are known as inhibitors for cellulases and fermentative microorganisms in bioethanol production processes. The addition of laccases removes the phenolic compounds and subsequently reduces the lag phase of the fermentative microorganism. However, the application of laccases diminishes glucose release during the enzymatic hydrolysis. In this study a model cellulosic substrate (Sigmacell) together with lignin extract, whole steam-pretreated wheat straw (slurry) and its water insoluble solid fraction (WIS) were subjected to enzymatic hydrolysis to evaluate the effects of laccase treatment in presence of lignin and phenols. The presence of laccase in enzymatic hydrolysis of Sigmacell with lignin extract reduced glucose yield by 37% compared with assays without laccase. Furthermore, this reduction was even more marked in presence of phenols (55% reduction). Interestingly, when hydrolyzing WIS, the addition of phenols coupled with laccase treatment did not show a reduction when compared with only laccase addition. This fact suggests the key role of lignin in the hydrolysis inhibition since in WIS the ratio cellulase per gram of lignin was much lower than in Sigmacell experiments. Finally, the lower cellobiose and xylose recoveries point out that phenolic oligomers formed by laccase oxidation play important roles in the inhibition of endoglucanases, cellobiohydrolases and xylanases. To conclude, the proportion of lignin and the composition of phenols are key players in the inhibition of cellulases when the enzymatic hydrolysis is combined with laccases detoxification.

Algicidal microorganisms and secreted algicides: New tools to induce microalgal cell disruption.

Cell disruption is one of the most critical steps affecting the economy and yields of biotechnological processes for producing biofuels from microalgae. Enzymatic cell disruption has shown competitive results compared to mechanical or chemical methods. However, the addition of enzymes implies an associated cost in the overall production process. Recent studies have employed algicidal microorganisms to perform enzymatic cell disruption and degradation of microalgae biomass in order to reduce this associated cost. Algicidal microorganisms induce microalgae growth inhibition, death and subsequent lysis. Secreted algicidal molecules and enzymes produced by bacteria, cyanobacteria, viruses and the microalga themselves that are capable of inducing algal death are classified, and the known modes of action are described along with insights into cell-to-cell interaction and communication. This review aims to provide information regarding microalgae degradation by microorganisms and secreted algicidal substances that would be useful for microalgae cell breakdown in biofuels production processes. A better understanding of algae-to-algae communication and the specific mechanisms of algal cell lysis is expected to be an important breakthrough for the broader application of algicidal microorganisms in biological cell disruption and the production of biofuels from microalgae biomass.

Optimization of uncatalyzed steam explosion pretreatment of rapeseed straw for biofuel production.

Rapeseed straw constitutes an agricultural residue with great potential as feedstock for ethanol production. In this work, uncatalyzed steam explosion was carried out as a pretreatment to increase the enzymatic digestibility of rapeseed straw. Experimental statistical design and response surface methodology were used to evaluate the influence of the temperature (185-215°C) and the process time (2.5-7.5min). According to the rotatable central composite design applied, 215°C and 7.5min were confirmed to be the optimal conditions, considering the maximization of enzymatic hydrolysis yield as optimization criterion. These conditions led to a maximum yield of 72.3%, equivalent to 81% of potential glucose in pretreated solid. Different configurations for bioethanol production from steam exploded rapeseed straw were investigated using the pretreated solid obtained under optimal conditions as a substrate. As a relevant result, concentrations of ethanol as high as 43.6g/L (5.5% by volume) were obtained as a consequence of using 20% (w/v) solid loading, equivalent to 12.4g ethanol/100g biomass.

Enzymatic cell disruption of microalgae biomass in biorefinery processes.

When employing biotechnological processes for the procurement of biofuels and bio-products from microalgae, one of the most critical steps affecting economy and yields is the "cell disruption" stage. Currently, enzymatic cell disruption has delivered effective and cost competitive results when compared to mechanical and chemical cell disruption methods. However, the introduction of enzymes implies additional associated cost within the overall process. In order to reduce this cost, autolysis of microalgae is proposed as alternative enzymatic cell disruption method. This review aims to provide the state of the art of enzymatic cell disruption treatments employed in biorefinery processes and highlights the use of endopeptidases. During the enzymatic processes of microalgae life cycle, some lytic enzymes involved in cell division and programmed cell death have been proven useful in performing cell lysis. In this context, the role of endopeptidases is emphasized. Mirroring these natural events, an alternative cell disruption approach is proposed and described with the potential to induce the autolysis process using intrinsic cell enzymes. Integrating induced autolysis within biofuel production processes offers a promising approach to reduce overall global costs and energetic input associated with those of current cell disruption methods. A number of options for further inquiry are also discussed.

Inhibition of cellulose enzymatic hydrolysis by laccase-derived compounds from phenols.

The presence of inhibitors compounds after pretreatment of lignocellulosic materials affects the saccharification and fermentation steps in bioethanol production processes. Even though, external addition of laccases selectively removes the phenolic compounds from lignocellulosic prehydrolysates, when it is coupled to saccharification step, lower hydrolysis yields are attained. Vanillin, syringaldehyde and ferulic acid are phenolic compounds commonly found in wheat-straw prehydrolysate after steam-explosion pretreatment. These three phenolic compounds were used in this study to elucidate the inhibitory mechanisms of laccase-derived compounds after laccase treatment. Reaction products derived from laccase oxidation of vanillin and syringaldehyde showed to be the strongest inhibitors. The presence of these products causes a decrement on enzymatic hydrolysis yield of a model cellulosic substrate (Sigmacell) of 46.6 and 32.6%, respectively at 24 h. Moreover, a decrease in more than 50% of cellulase and ß-glucosidase activities was observed in presence of laccase and vanillin. This effect was attributed to coupling reactions between phenoxyl radicals and enzymes. On the other hand, when the hydrolysis of Sigmacell was performed in presence of prehydrolysate from steam-exploded wheat straw a significant inhibition on enzymatic hydrolysis was observed independently of laccase treatment. This result pointed out that the other components of wheat-straw prehydrolysate are affecting the enzymatic hydrolysis to a higher extent than the possible laccase-derived products.

Unraveling the effects of laccase treatment on enzymatic hydrolysis of steam-exploded wheat straw.

Laccase enzymes are promising detoxifying agents during lignocellulosic bioethanol production from wheat straw. However, they affect the enzymatic hydrolysis of this material by lowering the glucose recovery yields. This work aimed at explaining the negative effects of laccase on enzymatic hydrolysis. Relative glucose recovery in presence of laccase (10IU/g substrate) with model cellulosic substrate (Sigmacell) at 10% (w/v) was almost 10% points lower (P<0.01) than in the absence of laccase. This fact could be due to an increase in the competition of cellulose binding sites between the enzymes and a slight inhibition of ß-glucosidase activity. However, enzymatic hydrolysis and infrared spectra of laccase-treated and untreated wheat straw filtered pretreated residue (WS-FPR), revealed that a grafting process of phenoxy radicals onto the lignin fiber could be the cause of diminished accessibility of cellulases to cellulose in pretreated wheat straw.

Algaculture integration in conventional wastewater treatment plants: anaerobic digestion comparison of primary and secondary sludge with microalgae biomass.

This study evaluated the feasibility of using microalgae biomass as feedstock for anaerobic digestion together with other biomasses (primary and secondary sludge) normally generated in WWTP. Raw microalgae biomass anaerobic biodegradability (33%) was higher than that of secondary sludge (23%). Thermal pretreatment enhanced 62% and 16% methane yield for Chlorellavulgaris and secondary sludge, respectively. When both substrates were codigested, methane yields remained low. On the other hand, primary sludge supported the highest anaerobic biodegradability (97%) and when combined with thermally pretreated C. vulgaris, methane yields were higher (13-17%) than the ones expected theoretically. Despite the high protein content of those substrates and the high nitrogen mineralization, no ammonia inhibition was detected. Thereby, this study showed that algae biomass is a potential cosubstrate for biogas production together with municipal wastewater sludge.