Our group has demonstrated the applicability of this process by generating hydrolysates with high concentrations of monomeric sugars and organic acids from several feed stocks like grasses, hardwoods, and softwoods, and converting them to terpene-based jet-fuel molecules using engineered strains of the yeast Rhodosporidium toruloides . Nevertheless, it is important to expand the range of lignocellulosic feedstocks used in this process to evaluate its versatility to advance towards the goal of developing a truly lignocellulosic feed stock-agnostic bio-refinery. Hemp is an attractive crop due to its fast growth, bio-remediation potential, and diverse agricultural applications, including the production of natural fibers, grains, essential oils, and other commodities. This biomass is composed of an outer fiber that represents approximately 30% of the weight and an inner core known as hurd that accounts for the remaining 70% . The hemp fiber is utilized in the textile industry as insulation material and for the production of bio-plastics in the automotive industry, while hemp hurd is used for low value applications such as animal bedding, concrete additives, or disposed of by combustion and landfill accumulation. This indicates that approximately 70 wt% of hemp biomass has the potential to be valorized into higher-value products and applications, indoor weed growing accessories which would improve the economics of the hemp industry and increase its sustainability footprint to promote a green economy. Mycelium-based composites are emerging as cheap and environmentally sustainable materials generated by fungal growth on a scaffold made of agricultural waste materials.
The mycelium composite can replace foams, timber, and plastics for applications like insulation, packaging, flooring, and other furnishings. For example, the company Ecovative Design LLC produces a foamlike packaging material made of hemp hurd and fungal mycelia, which is fully compostable. Anticipating the possibility of an increased demand of eco-friendly packaging materials in the near future, we are interested in evaluating the feasibility of diverting this used packing material away from landfills or composting facilities towards higher value applications, such as feedstock for bio-fuels. It is known that fungal enzymes can reduce the recalcitrance of the biomass to deconstruction, likely through modification of polysaccharides and lignin in plant biomass. Therefore, we hypothesized that the mycelium composite material could be more easily deconstructed and converted into higher value fuels and chemicals than the raw hemp hurd. In this study, hemp hurd and the mycelium-based packaging material were tested as biomass feedstocks for the production of the jet-fuel precursor bisabolene, using a one-pot ionic liquid technology and microbial conversion. First, we examined the deconstruction efficiency of the packaging material compared to hemp hurd, when subjugated to a onepot ionic liquid pretreatment process. Second, the influence of the pretreatment process parameters on the sugar yields was investigated by using a Box–Behnken statistical design. Finally, the generated hydrolysates were fermented to evaluate the bio-conversion of the depolymerized components by a bisabolene-producing R. toruloides strain. The composition of the hemp hurd and packaging material was determined as shown in Table 1. The total extractives of the hemp hurd and packaging material comprised 8.3 and 14.7% of the biomass, respectively.
The higher extractive content of the packaging material may be a result of the fungal growth stage in the packaging construction process. For the polysaccharide content, hemp hurd had higher glucan and xylan contents than the glucan and xylan content of the packaging material. Combining glucan and xylan content, the total fermentable sugars of the hemp hurd and packaging material was 43.7% and 40.4% of the hemp hurd biomass, respectively.This indicates that a small fraction of the polysaccharides may have been consumed and converted into extractives during mycelial growth. However, both types of biomass contain a substantial amount of polymeric carbohydrates that can be depolymerized into simple sugars for fermentation. The lignin content for both materials was the same ; however, it is possible that the mycelial growth in the packaging material could have altered the structure of lignin and made the polysaccharides more accessible to hydrolysis. We used the one-pot ionic liquid process on hemp hurd and package materials to test this hypothesis. One of the bottlenecks for the efficient conversion of lignocellulosic hydrolysates is the presence of compounds generated during the pretreatment and enzymatic hydrolysis stages that are toxic to bio-fuel-producing microbes. The degree of toxicity mainly depends on the type of biomass, pretreatment conditions, and the identity of the microorganism that will be used for fermenting the depolymerized substrates. Therefore, we performed a bio-compatibility test with the hydrolysates prepared from hemp hurd and packaging materials, using an engineered strain of the yeast R. toruloides known to be tolerant to ILs and biomass-derived compounds, and convert glucose and xylose to the jet fuel precursor bisabolene.
When the strain was inoculated directly in concentrated hydrolysates, negligible sugar consumption and very little growth was observed, as shown in Figure 2. Therefore, we prepared 50% diluted hydrolysates for further testing. Under these conditions, more than 90% of glucose and xylose conversion was observed in both hydrolysates, and the cells were able to grow and produce bisabolene . The utilization of hydrolysate with higher concentrations is beneficial for the economically feasible biorefinery development. Therefore, other strategies such as hydrolysate culture adaptation or detoxification may be required to improve bio-compatibility.The optimum levels of parameters for glucose and xylose yields from packaging materials recommended by the model were: reaction temperature of 126 and 128 ◦C, reaction time of 2.1 and 2.0 h, and ionic liquid loading of 7.3% and 7.9%, corresponding to a predicted glucose and xylose yield of 74.6% and 81.7%. However, this optimal condition did not significantly improve the yields compared to the center point, rolling benches even though the reaction conditions required a 4% higher temperature than the center point, a rather small difference in temperature. This result suggests that other process parameters such as agitation and biomass solid loading percentage should be tested for further improvement in the yield. The model for hemp hurd found a saddle point instead of optimum levels, which means that the optimum process condition was not aligned within the current experimental conditions. Further investigation into the different range of reaction conditions such as higher reaction temperature is required to optimize the reaction condition for hemp hurd. If operating with a limited budget and time, the reaction condition having the highest glucose and xylose yield can be chosen. The highest glucose yield in the current reaction condition was obtained from hemp hurd at 140 ◦C, 1 h reaction time and 7.5% ionic liquid loading, which has higher severity in reaction condition than the optimized reaction condition of packaging materials. This result indicates that the reaction parameter affects the sugar yield differently according to the biomass type, implying that the biomass properties change by mycelium growth. Regarding the packaging materials, the combined effects of reaction temperature, reaction time and ionic liquid loading on glucose yields are illustrated in Figure 4 and xylose yields in Figure 4. Response surface plots show that the glucose yield increased with the reaction temperature up to 133 ◦C with subsequent decrease in yield at a higher temperature. The xylose yields showed a similar trend. Additionally, the glucose and xylose yield increased with the reaction time up to 2 h and 7.5% ionic liquid loading.
After those points, the glucose and xylose yield decreased, probably due to the loss of enzyme activity caused by the higher ionic liquid concentration. Additionally, the longer reaction time and the higher ionic liquid concentration might facilitate the production of other compounds such as furan derivatives or organic acids, which inhibits the enzyme activity during the pretreatment. Moreover, the production of other components probably led to a decrease in accessible carbohydrates to the enzyme . Further tests may be necessary to improve the sugar yield. ANOVA results shown in Table S5 indicate that reaction temperature and reaction time has statistically significant effects on glucose yield , while ionic liquid loading was not significant . Additionally, the statistically significant interaction effects of reaction temperature with reaction time and ionic liquid loadings were confirmed. ANOVA results associated with xylose yield show that reaction temperature had a significant effect on the yield , while reaction time and ionic liquids had no effect . Additionally, the interaction effect of reaction temperature with reaction time and ionic liquid loading was not significant, while the interaction effects of reaction time with ionic liquid were significant .This work demonstrates the feasibility of hemp hurd and packaging materials made of mycelium grown on hemp hurd to be used as feedstocks for bio-conversion to a jet-fuel precursor using a one-pot ionic liquid technology. During the initial test , the packaging materials produced higher sugar concentrations and yield than the hemp hurd . However, the Box–Behnken experimental design showed that the reaction conditions for the maximum sugar yields from each material was different and that the significance of the process parameter effect on the fermentable sugar yield was dependent on the biomass properties, suggesting that the mycelial growth affected the deconstructability of the hemp hurd. Furthermore, the fermentation test to convert fermentable sugar into bisabolene showed that hydrolysates fromthe packaging material resulted in a higher bisabolene titer than hydrolysates from the hemp hurd, probably due to the higher sugar concentrations generated form the packaging material. To fully take advantage of these packaging materials to produce bio-fuels after they are used and discarded, a more detailed correlation study between the fermentable sugar yield and physicochemical properties of biomass and packaging materials or packaging process parameters is required by testing different hemp material sources. In addition, methods to overcome hydrolysate toxicity will need to be employed to enable utilization of concentrated hydrolysate for increased product titers and a reduction in water consumption. Finally, further investigation into other process parameters such as agitation and biomass loadings are merited to fully optimize the pretreatment conditions, as well as performing pilot scale tests to generate data that can help assess the economic feasibility of this new conceptual process. Overall, this study indicates that it is possible to produce lignocellulosic supply chains for production of bio-fuels and biochemicals that include both raw biomass and biomass that has been first processed and valorized as commercial products, such as packaging materials, enabling the carbon in these lignocellulosic products to generate value multiple times in their life cycle. Understanding momentum, heat, and scalar mass exchanges between vegetation and the atmosphere is necessary for the quantification of evaporation and sensible heat flux for hydrologic budgets, ozone deposition on urban forests, nonmethane hydrocarbon emissions from natural vegetation, carbon storage in ecosystems, etc. Such exchanges are governed by a turbulent mixing process that appears to exhibit a number of universal characteristics . Early attempts to predict these universal characteristics made use of rough-wall boundary layer analogies but limited success was reported . A basic distinction between canopy and rough-wall boundary layer turbulence is that the ‘‘for-est–atmosphere’’ system is a porous medium permitting finite velocity and velocity perturbations well within the canopy. Hence, the canopy–atmosphere interface cannot impose a severe constraint on fluid continuity as an impervious boundary, as discussed in Raupach and Thom , Raupach , and Raupach et al. . Raupach et al. and Raupach et al. proposed a mixing layer analogy to model the universal characteristics of turbulence close to the canopy atmosphere interface in uniform and extensive canopies. Their analogy is based on solutions to the linearized perturbed two-dimensional inviscid momentum equations using hydrodynamic stability theory . For such a system of equations, HST predicts the unstable mode generation of two-dimensional transverse Kelvin–Helmholz waves with stream wise wavelength if the longitudinal velocity profile has an inflection point . Such instabilities are the origins of organized eddy motion in plane mixing layers; however, a KH eddy motion cannot be produced or sustained in boundary layers due to the absence of such an inflection point in the velocity profile. A plane mixing layer is a ‘‘boundary-free shear flow’’ formed in a region between two coflowing fluid streams of different velocity but same density . Raupach et al. recently argued that a strong inflection point in the mean velocity profile at the canopy–atmosphere interface results in a flow regime resembling a mixing layer rather than a boundary layer neighboring this interface. Raupach et al.’s ML analogy is the first theoretical advancement to analyzing the structure of turbulence close to the canopy– atmosphere interface of a horizontally extensive uniform forest.