Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

Lignocellulose‐derived hydrolyzates typically display a high degree of variation depending on applied biomass source material as well as process conditions. Consequently, this typically results in variable composition such as different sugar concentrations as well as degree and the presence of inhibitors formed during hydrolysis. These key obstacles commonly limit its efficient use as a carbon source for biotechnological conversion. The gram‐negative soil bacterium Pseudomonas putida KT2440 is a promising candidate for a future lignocellulose‐based biotechnology process due to its robustness and versatile metabolism. Recently, P. putida KT2440_xylAB which was able to metabolize the hemicellulose (HC) sugars, xylose and arabinose, was developed and characterized. Building on this, the intent of the study was to evaluate different lignocellulose hydrolyzates as platform substrates for P. putida KT2440 as a model organism for a bio‐based economy. Firstly, hydrolyzates of different origins were evaluated as potential carbon sources by cultivation experiments and determination of cell growth and sugar consumption. Secondly, the content of major toxic substances in cellulose and HC hydrolyzates was determined and their inhibitory effect on bacterial growth was characterized. Thirdly, fed‐batch bioreactor cultivations with hydrolyzate as the carbon source were characterized and a diauxic‐like growth behavior with regard to different sugars was revealed. In this context, a feeding strategy to overcome the diauxic‐like growth behavior preventing accumulation of sugars is proposed and presented. Results obtained in this study represent a first step and proof‐of‐concept toward establishing lignocellulose hydrolyzates as platform substrates for a bio‐based economy.     

Co‐hydrolysis of lignocellulosic biomass for microbial lipid accumulation

The herbaceous perennial energy crops miscanthus, giant reed, and switchgrass, along with the annual crop residue corn stover, were evaluated for their bioconversion potential. A co‐hydrolysis process, which applied dilute acid pretreatment, directly followed by enzymatic saccharification without detoxification and liquid–solid separation between these two steps was implemented to convert lignocellulose into monomeric sugars (glucose and xylose). A factorial experiment in a randomized block design was employed to optimize the co‐hydrolysis process. Under the optimal reaction conditions, corn stover exhibited the greatest total sugar yield (glucose + xylose) at 0.545 g g^?1 dry biomass at 83.3% of the theoretical yield, followed by switch grass (0.44 g g^?1 dry biomass, 65.8% of theoretical yield), giant reed (0.355 g g^?1 dry biomass, 64.7% of theoretical yield), and miscanthus (0.349 g g^?1 dry biomass, 58.1% of theoretical yield). The influence of combined severity factor on the susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernible, showing that co‐hydrolysis is a technically feasible approach to release sugars from lignocellulosic biomass. The oleaginous fungus Mortierella isabellina was selected and applied to the co‐hydrolysate mediums to accumulate fungal lipids due to its capability of utilizing both C5 and C6 sugars. Fungal cultivations grown on the co‐hydrolysates exhibited comparable cell mass and lipid production to the synthetic medium with pure glucose and xylose. These results elucidated that combining fungal fermentation and co‐hydrolysis to accumulate lipids could have the potential to enhance the utilization efficiency of lignocellulosic biomass for advanced biofuels production. Biotechnol. Bioeng. 2013; 110: 1039–1049. © 2012 Wiley Periodicals, Inc.     

Evaluation of small organic acids present in fast pyrolysis bio‐oil from lignocellulose as feedstocks for bacterial bioconversion

Small organic acids derived from fast pyrolysis of lignocellulosic biomass represent a significant proportion of microbially accessible carbon in bio‐oil. However, using bio‐oil for microbial cultivation is a highly challenging task due to its strong adverse effects on microbial growth as well as its complex composition. In this study, the main small organic acids present in bio‐oil as acetate, formate and propionate were evaluated with respect to their suitability as feedstocks for bacterial growth. For this purpose, the growth behavior of four biotechnological production hosts—Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Corynebacterium glutamicum—was quantified and compared. The bacteria were cultivated on single acids and mixtures of acids in different concentrations and evaluated using common biotechnological efficiency parameters. In addition, cultivation experiments on pretreated fast pyrolysis‐derived bio‐oil fractions were performed with respect to the suitability of the bacterial strains to tolerate inhibitory substances. Results suggest that both P. putida and C. glutamicum metabolize acetate—the major small organic acid generated during fast pyrolysis of lignocellulosic biomass—as sole carbon source over a wide concentration range, are able to grow on mixtures of small organic acids present in bio‐oil and can, to a limited extent, tolerate the highly toxic inhibitory substances within bio‐oil. This work provides an important step in search of suitable bacterial strains for bioconversion of lignocellulosic‐based feedstocks and thus contributes to establishing efficient bioprocesses within a future bioeconomy.     

Hydrothermal liquefaction of Malaysia's algal biomass for high‐quality bio‐oil production

Currently, fossil materials form the majority of our energy and chemical source. Many global concerns force us to rethink about our current dependence on the fossil energy. Limiting the use of these energy sources is a key priority for most countries that pledge to reduce greenhouse gas emissions. The application of biomass, as substitute fossil resources for producing biofuels, plastics and chemicals, is a widely accepted strategy for sustainable development. Aquatic plants including algae possess competitive advantages as biomass resources compared to the terrestrial plants in this current global situation. Bio‐oil production from algal biomass is technically and economically viable, cost competitive, requires no capacious lands and minimal water use and reduces atmospheric carbon dioxide. The aim of this paper is to review the potential of converting algal biomass, as an aquatic plant, into high‐quality crude bio‐oil through applicable processes in Malaysia. In particular, bio‐based materials and fuels from algal biomass are considered as one of the reliable alternatives for clean energy. Currently, pyrolysis and hydrothermal liquefaction (HTL) are two foremost processes for bio‐oil production from biomass. HTL can directly convert high‐moisture algal biomass into bio‐oil, whereas pyrolysis requires feedstock drying to reduce the energy consumption during the process. Microwave‐assisted HTL, which can be conducted in aqueous environment, is suitable for aquatic plants and wet biomass such as algae.     

A review of thermochemical upgrading of pyrolysis bio‐oil: Techno‐economic analysis,life cycle assessment,and technology readiness

Technologies for upgrading fast pyrolysis bio‐oil to drop‐in fuels and coproducts are under development and show promise for decarbonizing energy supply for transportation and chemicals markets. The successful commercialization of these fuels and the technologies deployed to produce them depend on production costs, scalability, and yield. To meet environmental regulations, pyrolysis‐based biofuels need to adhere to life cycle greenhouse gas intensity standards relative to their petroleum‐based counterparts. We review literature on fast pyrolysis bio‐oil upgrading and explore key metrics that influence their commercial viability through life cycle assessment (LCA) and techno‐economic analysis (TEA) methods together with technology readiness level (TRL) evaluation. We investigate the trade‐offs among economic, environmental, and technological metrics derived from these methods for individual technologies as a means of understanding their nearness to commercialization. Although the technologies reviewed have not attained commercial investment, some have been pilot tested. Predicting the projected performance at scale‐up through models can, with industrial experience, guide decision‐making to competitively meet energy policy goals. LCA and TEA methods that ensure consistent and reproducible models at a given TRL are needed to compare alternative technologies. This study highlights the importance of integrated analysis of multiple economic, environmental, and technological metrics for understanding performance prospects and barriers among early stage technologies.     

High‐solids biphasic CO2–H2O pretreatment of lignocellulosic biomass

A high pressure (200 bar) CO₂–H₂O process was developed for pretreating lignocellulosic biomass at high‐solid contents, while minimizing chemical inputs. Hardwood was pretreated at 20 and 40 (wt.%) solids. Switchgrass, corn stover, big bluestem, and mixed perennial grasses (a co‐culture of big bluestem and switchgrass) were pretreated at 40 (wt.%) solids. Operating temperatures ranged from 150 to 250°C, and residence times from 20 s to 60 min. At these conditions a biphasic mixture of an H₂O‐rich liquid (hydrothermal) phase and a CO₂‐rich supercritical phase coexist. Following pretreatment, samples were then enzymatically hydrolyzed. Total yields, defined as the fraction of the theoretical maximum, were determined for glucose, hemicellulose sugars, and two degradation products: furfural and 5‐hydroxymethylfurfural. Response surfaces of yield as a function of temperature and residence time were compared for different moisture contents and biomass species. Pretreatment at 170°C for 60 min gave glucose yields of 77%, 73%, and 68% for 20 and 40 (wt.%) solids mixed hardwood and mixed perennial grasses, respectively. Pretreatment at 160°C for 60 min gave glucan to glucose yields of 81% for switchgrass and 85% for corn stover. Biotechnol. Bioeng. 2010;107: 451–460. © 2010 Wiley Periodicals, Inc.     

Linking a farm model and a location optimization model for evaluating energetic and material straw valorization pathways—A case study in Baden‐Wuerttemberg

Diminishing fossil carbon resources, global warming, and increasing material and energy needs urge for the rapid development of a bioeconomy. Biomass feedstock from agro‐industrial value chains provides opportunities for energy and material production, potentially leading to competition with traditional food and feed production. Simulation and optimization models can support the evaluation of biomass value chains and identify bioeconomy development paths, potentials, opportunities, and risks. This study presents the linkage of a farm model (EFEM) and a techno‐economic location optimization model (BIOLOCATE) for evaluating the straw‐to‐energy and the innovative straw‐to‐chemical value chains in the German federal state of Baden‐Wuerttemberg taking into account the spatially distributed and price‐sensitive nature of straw supply. The general results reveal the basic trade‐off between economies of scale of the energy production plants and the biorefineries on the one hand and the feedstock supply costs on the other hand. The results of the farm model highlight the competition for land between traditional agricultural biomass utilization such as food and feed and innovative biomass‐to‐energy and biomass‐to‐chemical value chains. Additionally, farm‐modeling scenarios illustrate the effect of farm specialization and regional differences on straw supply for biomass value chains as well as the effect of high straw prices on crop choices. The technological modeling results show that straw combustion could cover approximately 2% of Baden‐Wuerttemberg's gross electricity consumption and approximately 35% of the district heating consumption. The lignocellulose biorefinery location and size are affected by the price sensitivity of the straw supply and are only profitable for high output prices of organosolv lignin. The location optimization results illustrate that economic and political framework conditions affect the regional distribution of biomass straw conversion plants, thus favoring decentralized value chain structures in contrast to technological economies of scale.     

Strategies for the production of cell wall‐deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

Microbial cell wall‐deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall‐deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall‐deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yields and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue‐specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. We also summarize methods that have been used to access and maintain activity of plant‐generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.     

Potential trade‐offs of employing perennial biomass crops for the bioeconomy in the EU by 2050: Impacts on agricultural markets in the EU and the world

Perennial biomass crops (PBC) are considered a crucial feedstock for sustainable biomass supply to the bioeconomy that compete less with food production compared to traditional crops. However, large‐scale development of PBC as a means to reach greenhouse gas (GHG) mitigation targets would require not only the production on land previously not used for agriculture, but also the use of land that is currently used for agricultural production. This study aims to evaluate agricultural market impacts with biomass demand for food, feed, and PBC in four bioeconomy scenarios (“Business as usual,” “Improved relevance of bioeconomy,” “Extensive transformation to a bioeconomy,” “Extensive transformation to a bioeconomy with diet change”) to achieve a 75% GHG reduction target in the emission trading sector of the EU until 2050. We simulated bioeconomy scenarios in the energy system model TIMES‐PanEU and the agricultural sector model ESIM and conducted a sensitivity analysis considering crop yields, PBC yields, and land use options of PBC. Our results show that all bioeconomy scenarios except the one with diet change lead to increasing food prices (the average food price index increases by about 11% in the EU and 2.5%–3.0% in world markets). A combination of the transformation to a bioeconomy combined with diet change toward less animal protein in the EU is the only scenario that results in only moderately increasing food prices within the EU (+3.0%) and even falling global food prices (–6.4%). In addition, crop yield improvement and cultivation of PBC on marginal land help to reduce increases in food prices, but higher land prices are inevitable because those measures have only small effects on sparing agricultural land for PBC. For a transition to a bioeconomy that acknowledges climate mitigation targets, counter‐measures for those substantial direct and indirect impacts on agricultural markets should be taken into account.     

Co‐utilization of acidified glycerol pretreated‐sugarcane bagasse for microbial oil production by a novel Rhodosporidium strain

Acidified glycerol pretreatment is very effective to deconstruct lignocellulosics for producing glucose. Co‐utilization of pretreated biomass and residual glycerol to bioproducts could reduce the costs associated with biomass wash and solvent recovery. In this study, a novel strain Rhodosporidium toruloides RP 15, isolated from sugarcane bagasse, was selected and tested for coconversion of pretreated biomass and residual glycerol to microbial oils. In the screening trails, Rh. toruloides RP 15 demonstrated the highest oil production capacity on glucose, xylose, and glycerol among the 10 strains. At the optimal C:N molar ratio of 140:1, this strain accumulated 56.7, 38.3, and 54.7% microbial oils based on dry cell biomass with 30 g/L glucose, xylose, and glycerol, respectively. Furthermore, sugarcane bagasse medium containing 32.6 g/L glucose from glycerol‐pretreated bagasse and 23.4 g/L glycerol from pretreatment hydrolysate were used to produce microbial oils by Rh. toruloides RP 15. Under the preliminary conditions without pH control, this strain produced 7.7 g/L oil with an oil content of 59.8%, which was comparable or better than those achieved with a synthetic medium. In addition, this strain also produced 3.5 mg/L carotenoid as a by‐product. It is expected that microbial oil production can be significantly improved through process optimization.     

Assessment of microbial diversity in the rhizosphere of Pinus roxburghii (Sarg.) and bio‐inoculant potential of selected pine bacterial isolates for wheat varieties based on cultureindependent and culture‐dependent techniques

• Evidence is lacking regarding compatibility of pine bacteria as bio‐inoculants for crops. The diversity and abundance of rhizosphere bacteria of Pinus roxburghii has never been investigated with simultaneous application of culture‐dependent and culture‐independent techniques. The present study was aimed to isolate, characterise, check the bio‐inoculant potential of pine bacteria and assess rhizosphere bacterial diversity using culture‐independent advanced approaches.
• Forty bacteria isolated from the rhizoplane of P. roxburghii growing in a cold climate at high altitude in Murree, were morphologically characterised; nine were identified by 16S rRNA sequence analyses and used in experiments. Diversity and abundance of the 16S rRNA gene and nif H gene in the rhizosphere was assessed by cloning, RFLP analysis, 454‐amplicon pyrosequencing and qPCR.
• The bacterial isolates significantly improved dry weight of shoot, root, root area, IAA and GA₃ content, number of grains plant^?1, weight of grains plant^?1 in wheat varieties Chakwal‐50 and Fareed‐06 under axenic and field conditions. The number of 16S rRNA sequences (2979) identified by pyrosequencing shared similarity with 13 phyla of bacteria and archaea.
• The results confirm the existence of diverse bacteria of agricultural and industrial importance in the rhizosphere and compatibility of rhizoplane bacteria as bio‐inoculants for wheat varieties.