On the rise: Development of a multi-tiered, indoor duckweed cultivation system.

Duckweed species (Lemnaceae) are suitable for remediation and valorization of agri-feed industry wastewaters and therefore can contribute to a more sustainable, circular economy where waste is a resource. Industrial applications will, however, require space efficient cultivation methods that are not affected by prevailing weather conditions. Here, the development and operation of a multi-tiered duckweed bioreactor is described. The developed prototype bioreactor depicted in this paper is composed of four cultivation layers (1 m2 each) with integrated LED lighting (generating up to 150 µmol m-2  s-1 ), a system of pumps and valves to manage the recirculatory flow (2.5 L min-1 ) of wastewater, and an automatic harvesting system. Using a nutrient poor medium, good growth of the duckweed species Lemna minor was achieved in the bioreactor, and this was matched by strong nutrient depletion from the medium, especially for phosphorus (45-mg total phosphorus [TP] removed per m-2  day-1 ). A fully automatic harvesting arm reliably captured similar amounts of duckweed biomass across multiple harvesting cycles, revealing a future scenario whereby labor and interventions by human operators are minimized. Further developments to advance the system towards fully automated operation will include, for example, the use of specific nutrient sensors to monitor and control medium composition. It is envisaged that multi-tiered, indoor bioreactors can be employed in the agri-feed industry where wastewaters are, in many cases, continuously generated throughout the year and need remediating immediately to avoid costly storage. Given the extensive use of automation technology in conventional wastewater treatment plants, multi-tiered duckweed bioreactors can be realistically integrated within the operating environment of such treatment plants. PRACTITIONER POINTS: Duckweed is suitable for remediation and valorization of agri-feed wastewater. Industrial duckweed applications require space efficient cultivation methods. Development and operation of a multi-tiered duckweed bioreactor is detailed. Flow dynamics and automatic harvesting in the bioreactor are optimized. It is concluded that a multi-tiered bioreactor can be used in industry.

Acclimation capability inferred by metabolic performance in two sea cucumber species from different latitudes.

The notion that thermal specialists from tropical regions live closer to their temperature limits than temperate eurytherms, seems too generalized. Species specific differences in physiological and biochemical stress reactions are linked to key components of organism fitness, like metabolic capacity, which indicates that acclimation potential across latitudes might be highly diverse rather than simplistic. In this study the exposure of a tropical (Holothuria scabra) and a temperate (Holothuria forskali) sea cucumber species to identical cold- and warm-acclimation stress was compared using the key metabolic parameters, respiration rate, enzyme activity (ETS, LDH, IDH), and energy reserve fractions (lipid, carbohydrate and protein). Results show much broader respiratory adjustments, as response to temperature change, in H. scabra (2-30 µgO2*gww-1*h-1) compared to H. forskali (1.5-6.6 µgO2*gww-1*h-1). Moreover, the tropical species showed clearly pronounced up and down regulation of metabolic enzymes and shifts in energy reserves, due to thermal acclimation, while the same metabolic indicators remained consistent in the temperate species. In summary, these findings indicate enhanced metabolic plasticity in H. scabra at the cost of elevated energy expenditures, which seems to favor the tropical stenotherm in terms of thermal acclimation capacity. The comparison of such holistic metabolic analyses between conspecifics and congeners, may help to predict the heterogeneous effects of global temperature changes across latitudinal gradients.

Temperature-induced aerobic scope and Hsp70 expression in the sea cucumber Holothuria scabra.

The Aerobic Scope (AS), which reflects the functional capacity for biological fitness, is a highly relevant proxy to determine thermal tolerance in various taxa. Despite the importance of this method, its implementation is often hindered, due to lacking techniques to accurately measure standard- (SMR) and maximal- (MMR) metabolic rates, especially in sluggish marine invertebrates with low oxygen consumption rates, such as sea cucumbers. In this study the AS concept was modified to define a Temperature-induced Aerobic Scope (TAS), based on metabolic rate changes due to temperature adjustments rather than traditionally used physical activity patterns. Consequentially, temperature dependent peak and bottom O2 consumption rates, defined as Temperature-induced Maximal- (TMMR) and Standard Metabolic Rates (TSMR), respectively, served as MMR and SMR alternatives for the sea cucumber Holothuria scabra. TMMR and TSMR were induced through acute temperature change (2°C per hour; 17-41°C) until critical warm (WTcrit) and cold (CTcrit) temperatures were reached, respectively. In addition, Hsp70 gene expression linked to respiration rates served as synergistic markers to confirm critical threshold temperatures. O2 consumption of H. scabra peaked distinctly at WTcrit of 38°C (TMMR = 33.2 ± 4.7 µgO2 g-1 h-1). A clear metabolic bottom line was reached at CTcrit of 22°C (TSMR = 2.2 ± 1.4 µgO2 g-1 h-1). Within the thermal window of 22-38°C H. scabra sustained positive aerobic capacity, with assumed optimal performance range between 29-31.5°C (13.85-18.7 µgO2 g-1 h-1). Between 39-41°C H. scabra decreased respiration progressively, while gene expression levels of Hsp70 increased significantly at 41°C, indicating prioritization of heat shock response (HSR) and homeostatic disruption. At the cold end (17-22°C) homeostatic disruption was visible through incrementally increasing energetic expenses to fuel basal maintenance costs, but no Hsp70 overexpression occurred. TMMR, TSMR and TAS proved to be reliable metrics, similar to the traditional energetic key parameters MMR, SMR and AS, to determine a specific aerobic performance window for the sluggish bottom dwelling species H. scabra. In addition, the linkage between respiration physiology and molecular defense mechanisms showed valuable analytical synergies in terms of mechanistic prioritization as response to thermal stress. Overall, this study will help to define lethal temperatures for aquaculture and to predict the effects of environmental stress, such as ocean warming, in H. scabra.

Effects of thermal stress on the immune and oxidative stress responses of juvenile sea cucumber Holothuria scabra.

Holothuria scabra is the most valued and cultured tropical sea cucumber, given the great demand of this species for human consumption. However, despite its ecological and economic relevance, little is known regarding its immune responses under thermal stress. Here, the main goal was to study the response of sea cucumbers to temperature stress, assessing sub-organismal alterations and acclimation capacities of juveniles to temperature changes. After changing temperature (1 °C/day) for 6 days, organisms were exposed to temperature conditions of 21 °C (cold), 27 °C (control), and 33 °C (warm) over a 30 day period. At each 15-day interval (T0, T15, and T30), six replicates per condition were killed for biochemical analysis. Immune responses were addressed by studying the activity of phenoloxidase (PO) and prophenoloxidase (ProPO) in the coelomic fluid. Antioxidant defence responses-catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR) enzymatic activities-were measured in the muscle and respiratory tree tissues, whereas oxidative damage was evaluated by measuring levels of superoxide radicals (ROS), DNA-strand breaks and lipid peroxidation (LPO). Juvenile H. scabra increased SOD and PO activities when temperature was elevated, and revealed low levels of ROS and damage in both cold and warm treatments throughout the experiment, confirming the organism's moderate thermal stress. After the short acclimation period, the immune and antioxidant responses prevented damage and maintained homeostasis. This multi-biomarker approach highlights its usefulness to monitor the health of H. scabra and to gain insight concerning the use of this high-valued species in global-scale aquaculture from different temperature regions.